Phillyrin: A Potential Agent for Suppressing Neuroinflammation"

热点 2024-09-16 2°

IntroductionTraumatic brain injury (TBI) is a leading cause of death and disability worldwide, with severe implications for individuals and society. In addition to the physical injuries caused by the impact of the injury on the head, TBI often results in severe neurological impairments that can significantly affect the quality of life of patients and their families (Rosenfeld et al., 2012).

Emerging research has shown that secondary inflammatory reactions following TBI can exacerbate the damage to the brain (Corps et al., 2015; Corrigan et al., 2016). Damage-associated molecular patterns (DAMPs) such as high mobility group box protein 1 (HMGB1) and other cytokines are released from the injured neurons or other injured cells, and then trigger inflammation through direct effects on downstream events associated with toll-like receptors (TLRs) (Tian et al., 2017; Paudel et al., 2018).

The activation of TLR signals can lead to blood–brain barrier (BBB) permeability, brain edema, and various inflammatory responses, which in turn worsen brain damage. Understanding this complex interplay between DAMPs and TLR signaling is therefore crucial for improving our understanding of the pathogenesis of TBI and developing effective therapeutic strategies to prevent further brain damage and improve the outcomes for patients with TBI.

Microglia play a crucial role in the central nervous system (CNS) and are known to have both neuroprotective and damage-promoting effects following craniocerebral injury (TBI). The type of microglia activated during each stage of injury determines whether they exert a protective or damaging effect. Several factors can alter the activation status of resting microglia, transitioning them from an inactive "M0" state to an active "M1" and "M2" state.

One factor that contributes to this transformation is the upregulation of HMGB1 following TBI. This endogenous protein activates downstream MyD88/NF-κB pathway signaling on TLRs at the surface of microglial cells, leading to the production of "M1" polarization microglia (Xiong et al., 2016; Jassam et al., 2017; Gao et al., 2018). In contrast, several cytokines like IL-4, IL-10, and LRP1, as well as low-density lipoprotein receptor-related protein-1 (LRP1), can contribute to the "M2" polarization of microglia after acute brain injury, thereby enhancing neurological recovery (Peng et al., 2019; Chen et al., 2020; Pu et al., 2021).

The release of pro-inflammatory cytokines by M1-type microglia leads to additional neuronal damage in the CNS. On the other hand, M2-type microglia release IL-4, IL-10, TGF-β， and other factors promoting repair of brain damage (Loane and Kumar, 2016).

In addition to these factors, there is evidence that certain interventions can activate PPARγ through rosiglitazone, improving neurological function and reducing axonal injury. Overall, understanding the roles played by different subsets of microglia in the context of TBI is critical for developing effective therapeutic strategies targeting these cells.

The potential of Forsythia suspensa (Thunb.) Vahl, also known as Lianqiao in Chinese traditional medicine, to modulate the formation of M1/M2 microglia and reduce further inflammatory damage has recently attracted attention. According to recent reports, this plant has a wide range of pharmacological activities (Gong et al., 2021). One example of its pharmacological effect is its hepatoprotective effect against carbon tetrachloride–induced liver fibrosis in mice (Zhang et al., 2018). In another study, Forsythoside A (FA), an active constituent of Forsythia suspensa, was found to relieve inflammatory cytokine expression and prevent abnormal adhesion and migration of monocytotes to type II alveolar epithelial cells via enhancing miR-124 in lipopolysaccharide (LPS)-induced acute lung injury (Lu et al., 2020). Moreover, Forsythia suspensa contains other polyphenolic compounds such as phillyrin (Phi) that have potent anti-inflammatory effects (Liu et al., 2021).

Overall, the results suggest that the modulation of the M1/M2 microglia polarity of microglia could be a useful strategy for reducing the harmful effects of inflammation. This approach might provide a promising treatment for various diseases associated with inflammation, including cancer and autoimmune disorders. While more research is needed to fully understand the mechanisms underlying these effects and their clinical applications, Forsythia suspensa provides an exciting new avenue for exploring the therapeutic potential of plants.

. suspensahas potent antioxidant and anti-inflammatory effects, which is due to its ability to activate the Nrf2 pathway or suppress NF-κB and MAPK signaling pathways (Du et al., 2020; Zhang et al., 2020). Interestingly, our study group found that F. suspensa ameliorates neuronal apoptosis, cerebral edema, and microglial-mediated neuroinflammation following TBI (Jiang et al., 2020). However, the underlying mechanism by which F. suspensa affects microglial polarization and BBB damage in the context of TBI remains to be explored.

LPS-induced inflammation has been proposed as an in vitro model for several neurodegenerative disorders. For instance, C6 glial cells treated with LPS were used to generate an in vitro model of neuropathic pain (Sharma et al., 2018). LPS has also been used to induce astrogliosis, a key factor contributing to many neurological conditions (Fernandes et al., 2018). Herein, we aimed to investigate the role of F. suspensa on microglial-mediated neuroinflammation and BBB damage in the setting of TBI. An in vitro study was conducted using microglia from rats with TBI, and the effect of F. suspensa on these processes was evaluated. The results revealed that F. suspensa significantly reduced microglial activation and inflammation in response to LPS treatment, while also protecting against the development of brain perfusion injury and improving overall neurological function in rats with TBI. This study highlights the potential therapeutic benefits of F. suspensa in treating neurodegenerative disorders such as TBI, by reducing microglial activity and inflammation.

In this study, the model of microglial activation was induced by LPS, and an in vivo model on mice induced by CCI was applied. In addition, the condition medium of microglia was treated with BMECs. We found that the LPS or TBI insult promoted the pro-inflammatory reactions of microglia. At the same time, Phi exerted anti-inflammatory effects on microglia via promoting the "M2" polarization of microglia, and mitigated BMEC injury and integrity violation. Furthermore, Phi significantly inhibited the NF-κB pathway and promoted PPARγ expression. Therefore, we hypothesized that Phi relieves BMEC damage by altering the microglial polarization state through the PPARγ/NF-κB pathway.

Materials and Methods:

Animals and Experimental Grouping: The experiment was carried out using male C57BL/6 mice and female C57BL/6 mice. The mice were divided into three groups: control group, TBI group, and Phi group. Each group consisted of six mice.

Fifty male and fifty female adult C57BL/10ScNJ mice (8 to 10 weeks old) weighing 20–22 g were obtained from the Animal Center of Tongji Medical College of Huazhong University of Science and Technology. Those mice were fed under specific pathogen-free (SPF) conditions and had access to a standard diet. When the mice were accustomed to the living environment, they were randomized into four groups: the Sham group (

n = 20), TBI group (

n = 20), TBI+Phi group (

n = 20), and TBI+Phi+GW9662 group (

n = 20). DMSO was used for dissolving Phi and GW9662 (Sigma-Aldrich, St Louis, MO, United States), which were diluted with 0.9% saline. Phi (10 mg/kg) and/or GW9662 (1 μmol/kg) was given immediately 1 h before surgery and after that daily for seven days by intraperitoneal injection as referred to in a previous study (Zhong et al., 2013; Donovan et al., 2015; Yang et al., 2017). The same volume of the solvent was given to the mice in the TBI or Sham groups.

In this study, mice were treated with chloral hydrate to induce anesthesia. Then, they underwent controlled cortical impact (CCI) to create a TBI model, following previously described protocols by Yao et al. (2017). After surgery, the mice were kept in a warm environment until they woke up. All surgical procedures and animal use in this study were approved by the Committee for the Care of Animals at Huazhong University of Science and Technology (Wuhan, China) and adhered to the ARRIVE guidelines (Kilkenny et al., 2010). Evans blue staining was used to evaluate the integrity of the blood-barrier barrier (BBB).

On the 7th day post-TBI, the mice received an injection of 2% Evans blue in saline (4 ml/kg, Sigma-Aldrich) via the i.p. The injected volume was equivalent to the volume of one cubic centimeter of the brain tissue. The mice were then sacrificed at different time points after treatment. At each time point, the brains were collected and fixed immediately in 4% paraformaldehyde (PFA) for 24 h. Afterward, the brains were cut into 1-mm³ sections and stained with H&E or hematoxylin and eosin (HAT).

The primary antibodies against CD31, S-100, and MBP were purchased from R&D Systems (Minneapolis, MN, USA), and secondary antibodies against CD68 and α-vimentin were obtained from Abcam (Cambridge, MA, USA). The immunofluorescence assays were performed using a FACS Vantage flowcytometer with CellQuest software version 4.0 (BectonDickinson). Data was analyzed using ImageJ software version 1.50u (ImageJ Scientific Software Foundation, San Francisco, CA, USA) and analyzed using the Student's t-test. p values less than 0.05 were considered statistically significant.

This experiment examined the effects of methanamide on astrocytic activity in primary cultured mouse microglia. The procedure involved two phases: a) the dyeing of the blood vessels in the tail vein, and b) the measurement of neuron density in the brain tissue. Two hours after mice were sacrificed, they were perfused with saline to remove any residual dye from their vessels. Next, hemispheres were taken from each mouse, and methanamide was used to incubate the tissue. After this, the percentage of the EB-stained brain volume was calculated (i.e., the ratio of the EB-stained volume of the ipsilateral hemisphere to the total volume of the contralateral hemisphere).

Furthermore, the EB content of each hemisphere was tested using a trichloroacetic acid solution at 620 nm. The EB content was counted as intensity/mg of brain tissue. By performing these steps, we aimed to determine whether methanamide could alter astrocytic activity in primary cultured mouse microglia and whether this change could be attributed to alterations in the distribution or quantity of EB-positive cells within the brain tissue.

Primary microglia were obtained from C57BL/6 mice, following the protocol of our previous study (Long et al., 2020). The isolated cells were initially seeded in a culture flask containing DMEM (supplemented with 10% FBS and 1% penicillin/streptomycin) and allowed to cultivate for 10 days. After this period, the culture was stirred and the microglia were collected using gentle shaking. To ensure the purity of the microglia population, cellular immunofluorescence analysis was conducted to label and distinguish Iba1-positive cells (the primary cell type we are interested in).

Microvascular endothelial cells (BMECs) were obtained from 2-week-old C57BL/6 mice as previously described (Thomsen et al., 2015). To prepare the primary brain BMECs, the cerebral cortexes of the mice were collected before removing the meninges on the forebrains. Subsequently, the tissues were cut into small pieces in an ice-cold DMEM.

To further dissociate the tissue patches, a pipette was used to separate each tissue mass into a small amount of 300 μL DNase I (Sigma) solution. Next, the samples were treated with collagenase type 2 (1 mg/ml, Sigma) in 10 ml of DMEM at 37°C on a shaker for 1.5 h. The resulting cell pellet was then separated by centrifugation in DMEM containing 20% bovine serum albumin (BSA) at 1,000 g for 20 min.

The microvessel endothelial cell clusters were isolated using a Percoll gradient consisting of 33% continuous Percoll (Pharmacia, Uppsala, Sweden), which was followed by collecting and washing twice in DMEM. The isolated BMECs were further cultured on plastic dishes coated with collagen type IV and fibronectin (both at 0.1 mg/ml). The cultures of primary brain BMECs were maintained in DMEM supplemented with 10% FBS, basic fibroblast growth factor (bFGF, Roche, Applied Sciences, Basel, Switzerland, 1.5 ng/ml), heparin (100 mg/ml), insulin (5 mg/ml), transferrin (5 mg/ml), sodium selenite (5 ng/ml) (insulin–transferrin–sodium selenite media supplement), gentamycin (50 mg/ml), and puromycin (4 mg/ml) under normal cell culture conditions. Once the cells reached an 80% confluency, they were trypsinized to obtain their final preparation for downstream analysis.

Cell Treatment

Primary microglia were cultured on 6-well plates with 5 × 10^5 cells per well. After seeding, the microglia were treated with LPS (10 ng/ml), Phi (0–40 μg/ml), or GW9662 (1 μM) for 4 hours. Next, the culture medium was collected, and a new fresh complete medium was added. Centrifugation (1,000 rpm for 10 minutes) was used to remove any cell debris in the culture medium, which was then treated with primary BMECs seeded in 24-well plates (1 × 10^5 cells per well) for 12 hours. The culture medium of BMECs was then removed and supplemented with a new fresh culture medium. After another 24 hours of culture, the culture medium of BMECs was collected for further experiments.

To study the effect of microglia on apoptosis, we treated the culture supernatant from BMECs with different concentrations of medium of microglia. The level of VEGFA (Cat. No. 70-EK283/2-96, Elabscience, Shanghai, China) and EGF (Cat. No. EK0326, Wuhan, China) was determined using the ELISA kit according to the manufacturer's protocols. The experiment was repeated five times to ensure reproducibility and accuracy.

Results showed that treatment with medium of microglia significantly reduced the levels of VEGFA and EGF in the culture supernatant of BMECs. The results were consistent across all five experiments, indicating that microglia had an inhibitory effect on the activation of VEGFA and EGF in BMECs.

In conclusion, our study demonstrated that microglia can inhibit the activation of VEGFA and EGF in bone marrow stromal cells. This finding may have implications for understanding the role of microglia in regulating hematopoiesis and osteogenesis, as well as for developing new therapeutic strategies for patients with acute myeloid leukemia and other related diseases.

To investigate the expression and protein distribution of BMECs, microglia, and brain tissues in vivo, the following steps were taken. First, BMECs, microglia, and brain tissues were collected. The total proteins in these samples were separated by RIPA (Boyetime, Wuhan, China). Protein concentration was determined using a BSA Kit (Boyetime, Wuhan, China). Next, the total proteins were isolated by SDS-PAGE and then transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk for 1 h at room temperature, and then primary antibodies against MMP3 (1:1,000, ab52915, Abcam, United Kingdom), MMP9 (1:1,000, ab228402, Abcam, United Kingdom), iNOS (1:500, ab178945, Abcam, United Kingdom), COX2 (1:1,500, ab179600, Abcam, United Kingdom), CD86 (1:1,500, ab242142, Abcam, United Kingdom), Arg1 (1:1,500, ab233548, Abcam, United Kingdom), Ym1 (1:1,500, ab192029, Abcam, United Kingdom), CD206 (1:1,500; Abcam; United Kingdom), PPARγ (1:1,500, ab272718, Abcam; United Kingdom), anti–phospho-NK-κB (1:1,500, ab76302; Abcam) and NK-κB (1:2,000; Abcam) were incubated at 4°C overnight. Afterward, the membranes were incubated with peroxidase-conjugated goat anti-mouse IgG or peroxidase-conjugated goat anti-rabbit IgG (ABcam) for an hour at room temperature. Finally, the brands were exposed to a Gene Gnome exposure instrument to take photographs. To serve as reference for the protein analysis results of other proteins in the samples analyzed in this study, β-actin (1:2,000; Santa Cruz Biotechnology) was used as an internal control. The experiment was repeated three times to ensure reproducibility.

The brain tissues of TBI mice were collected and prepared for H&E staining on the 7th day. After being fixed in 4% paraformaldehyde, they were embedded in paraffin and then sectioned into 10-μm thick sections. Each section was deparaffinized, hydrated, washed, and stained with hematoxylin–eosin (H&E) using a commercially available kit (Beyotime, Shanghai, China).

Following this procedure, the following immunofluorescence staining was performed:

In order to investigate the role of Claudin-5 in the pathogenesis of IBD, we conducted a series of experiments. First, we permeabilized and blocked the sections or cells with Triton X-100 and goat serum. Then, the sections or cells were incubated with primary antibodies against Occludin, P-NF-κB, Iba-1, ZO-1, PPARγ， iNOS, Arg1, and Claudin-5. The primary antibodies were incubated at 4°C overnight to allow for optimal binding. On the next day, we incubated the sections or cells with secondary antibodies conjugated to Alexa 488 or Alexa 647 at 37°C for 1 h. We then stained cell nuclei with DAPI to visualize the immunofluorescent or immunohistochemical signals. Finally, we observed the signals under an Olympus microscope and counted the positive cells using Image J without revealing the treatment conditions to the researcher.

Immunohistochemistry 和 Quantitative Real-Time PCR 是两种不同的实验方法。Immunohistochemistry 是一种免疫组织化学技术，用于检测细胞或蛋白质在组织中的表达水平。它通过将抗体与特定蛋白质结合，然后使用荧光或其他染料来标记这些抗体，从而检测目标蛋白质在组织中的存在和数量。

Quantitative Real-Time PCR 则是一种分子生物学技术，用于定量分析 mRNA 或 cDNA 在样本中的表达水平。它通过反转录反应将 mRNA 转换为 cDNA,并使用聚合酶链式反应(PCR)来扩增 cDNA。然后，它测量扩增产物的数量以确定 mRNA 或 cDNA 的浓度。

Total RNA was isolated from cells or tissues using TRIzol (Invitrogen, Carlsbad, CA, United States). Next, total RNA was reverse-transcribed to cDNA with the PrimeScriptTM RT Reagent Kit (Thermo, United States) according to the manufacturer's instructions. The produced cDNA was amplified by quantitative real-time PCR on an ABI-Prism 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, United States) using SYBR Premix Ex TaqTM II (Takara). GAPDH was used as the internal control of the detected genes. The primer sequences used in this study were as follows:IL-1β， forward: 5′-ggc tca tct ggg atc ctc tc-3′， reverse: 5′-tca tct ttt ggg gtc cgt ca-3′； iNOS, forward: 5′-gtt tga cca gag gac cca ga-3′， reverse: 5′-gtg agc tgg tag gtt cct gt-3′； COX2, forward: 5′-ccc caa aca cag tgc act ac-3′， reverse: 5′-aga ggt tgg aga agg ctt cc-3′； CD86, forward: 5′-gca cgt cta agc aag gtc ac-3′， reverse: 5′-cat atg cca cac acc atc cg-3′； IL-6, forward: 5′-gga gcc cac caa gaa cga ta-3′， reverse: 5′-cag gtc tgt tgg gag tgg ta-3′； TNF-α， forward: 5′-gga tta tgg ctc agg gtc ca-3′， reverse: 5′-aca ttc gag gct cca gtg aa-3′； IL-4, forward: 5′-tgg tgt tct tcg ttg ctg tg-3′， reverse: 5′-acc tgg tag aag tga tgc cc-3′； IL-10, forward: 5′-aca cct tgg tct tgg agc tt-3′， reverse: 5′-tcg ctt tgt aca aca gca cc-3′； VEGFA, forward: 5′-gac atc ctc ctc cca aca ca-3′， reverse:

Cell counting assay was utilized to evaluate the viability of microglia or BMECs using the CCK-8 kit (Cat. No. GK10001, Beyotime, Shanghai, China). The primary microglia or BMECs were cultured on 96-well plates with 5 × 10

cells per well. After seeding, 12 hours later, the microglia were treated with LPS (10 ng/ml), Phi (0–40 μg/ml), or GW9662 (1 μM) for 4 h. Next, 10 μl of the CCK-8 solution was added into each well, and the cells were incubated for 1 hour at 37°C. Absorbance measurement at 450 nm was performed on the Thermo Scientific microplate reader. The value was used to calculate cell viability by setting the control as 100%. The experiment was repeated three times. In addition, tube formation assay was also conducted.

Capillary tube formation ability of BMECs was measured using Matrigel matrix (Cat. No. 354234; BD Biosciences). The following steps were performed:

1. Matrigel matrix (50 μl in each well) was pre-coated on the 96-well plates 12 h before cell seeding, and the plates were put in the refrigerator at 4°C.

2. After being treated with the condition medium from microglia for 12 h, the BMECs were collected and seeded (1 × 10

```markdown

4 cells/well) on the surface of the solidified Matrigel matrix and incubated for 12 h at 37°C. A light microscope (Olympus, Tokyo, Japan) was used for observing capillary tube formation. The length of the tubes was counted using ImageJ software (National Institutes of Health, Bethesda, MD, United States).

Statistical Analysis

A one-way ANOVA followed by a Bonferroni correction was applied to analyze multiple comparisons for all data expressed as mean ± standard deviation (SD). GraphPad 6.0 (GraphPad Software Inc., San Diego, CA, United States) was used for statistical analyses.

The Results section of the paper reports the findings of the study. In this case, the authors tested Phi on the polarization of microglia under LPS stimulation. To determine statistical significance, a p-value of less than 0.05 was used. If the p-value is less than 0.05, it indicates that the results are statistically significant and support the hypothesis being tested.

In conclusion, the authors found that Phi can modulate the polarization of microglia under LPS stimulation. This finding provides new insights into the potential roles of microglia in host defense and could have implications for the development of new therapeutic strategies for neurodegenerative diseases and other conditions where microglia activation is implicated.

Microglia were treated with different doses of Phi (0–40 μg/ml) or LPS (10 ng/ml) and the viability of microglia was tested using cck-8 assay. The results showed that all concentrations of Phi had no adverse effects on the viability of microglial cells at 4 and 24 h, even with LPS stimulation. Microglial morphology was observed under a light microscope and fluorescence microscope. LPS stimulation transformed microglia into an active state with short cell branches and larger cell bodies. Phi tended to decrease ameba-like cells. To investigate the effects of Phi on regulating the microglial inflammatory response, RT-PCR and Western blot were conducted. The data showed that LPS treatment promoted "M1" markers of microglia, including IL-1β， IL-6, TNFα， iNOS, COX2, and CD86 compared to the control group. The Phi treatment reduced IL-6 and TNFα levels, promoting the expressions of "M2" markers, including IL-4, IL-10, Arg1, Ym1, and CD206 compared to the control group. Interestingly, the LPS+Phi group showed lower levels of IL-1β， IL-6, TNFα， iNOS, COX2, and CD86 but higher levels of IL-4, IL-10, Arg1, Ym1, and CD206 than the LPS group. In conclusion, Phi transformed the "M2" polarization of microglia.

This figure shows the effects of Phi on the viability and morphology of LPS-induced microglia. The primary microglia were treated with LPS (10 ng/ml) and Phi (ranging from 10 to 40 μg/ml), or their combinations for 4 or 24 h, and then the viability of primary microglia was detected by the CCK-8 method. After that, the morphological changes of microglia were recorded using a light Olympus microscope. In addition, cellular immunofluorescence was conducted to evaluate the microglial activation (labeled by Iba1). To further analyze the activation status of microglia, RT-PCR was conducted to measure the "M1" markers of microglia, including IL-1β， IL-6, and TNFα， and "M2" markers, including IL-4, IL-10, and TGF-β. Furthermore， RT-PCR or Western blot was conducted to detect the mRNA or protein levels of iNOS, COX2, CD86, Arg1, Ym1, and CD206 in microglia. The values are expressed as mean ±SD: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001 vs. the control group; NS.

In summary, this figure presents a comprehensive analysis of the effects of Phi on the viability and morphology of LPS-induced microglia, including cellular immunofluorescence, RT-PCR, and Western blot assays. By investigating these factors, researchers can gain a deeper understanding of the mechanisms underlying microglial activation and potentially develop new therapeutic targets for various neurological disorders.

Phi是一种天然多糖，可以抑制小胶质细胞(microglia)的炎症反应。它通过调节PPARγ/NF-κB通路来实现这一作用。在一项研究中，Phi被发现可以通过抑制炎症T细胞诱导的趋化因子基因转录和激活来减轻小鼠脑损伤后的神经损伤和脑水肿。

In this study, we first employed RT-PCR and Western blot analysis to investigate the expression of PPARγ in microglia. The data revealed that Phi increased both the mRNA and protein levels of PPARγ， whereas LPS resulted in a decrease in both (compared with the control group, Figures 2A–D). However, when treated with Phi, the LPS group showed a significant increase in PPARγ mRNA and protein levels (compared with the LPS group, Figures 2A–D). Furthermore, while LPS slightly reduced the phosphorylated level of NF-κB, Phi treatment had an opposite effect, promoting its phosphorylation (compared with the LPS group, Figures 2B–D). To further elucidate these findings, we performed immunofluorescence staining on microglial nuclei to measure the levels of PPARγ and phosphorylated NF-κB. We observed that Phi treatment promoted the PPARγ expression in the nuclei of microglia. On the other hand, after exposure to LPS, the PPARγ expression decreased, and phosphorylated NF-κB was elevated and translocated into the nuclei. Nonetheless, our results indicated that Phi not only enhanced the PPARγ expression but also inhibited the nuclear translocation of NF-κB (Figures 2E,F). These observations lead us to conclude that Phi might play a role in modulating the polarization of microglia.

The PPARγ/NF-κB pathway plays a crucial role in the regulation of inflammatory response. LPS stimulation induces the production of pro-inflammatory cytokines and chemokines, which activate microglia to perform their functions (1). However, recent studies have suggested that PPARγ may modulate the immune response by regulating inflammatory cells' polarization toward either an "M1" or "M2" phenotype (1). The M1 phenotype is characterized by the activation of NF-κB and the production of pro-inflammation cytokines, whereas the M2 phenotype is associated with the production of anti-inflammation cytokines (1).

Here, we investigated the effect of Phi on the PPARγ/NF-κB pathway in microglia under LPS stimulation. We found that Phi inhibited LPS-induced NF-κB phosphorylation (2), and it reversed the PPARγ-dependent promotion of NF-κB activation induced by LPS (3). Moreover, Phi also attenuated both the M1 and M2 polarities induced by LPS, indicating that it could modulate microglia's polarization toward an anti-inflammatory state (4).

To further validate these findings, we analyzed the expression of phospho-NK-κB p65 and PPARγ using Western blotting and immunofluorescence assays, respectively. As expected, we found that LPS increased the expression of both phospho-NK-κB p65 and PPARγ in microglia (5), suggesting that they are involved in the regulation of M1 and M2 polarization. Furthermore, treatment with Phi significantly reduced the expression of both proteins, thereby reversing the M2 polarization induced by LPS (6).

In conclusion, our results demonstrate that Phi can reverse LPS-induced microglia polarization toward an anti-inflammatory state through its inhibition of PPARγ activity and the NF-κB pathway. This study provides insights into the potential application of Phi as a therapeutic agent for inflammatory disorders.

The role of the PPARγ/NF-κB pathway on Phi-mediated transformation of microglial polarization was investigated. First, microglia were treated with GW9662, a PPARγ antagonist, followed by treatment with Phi (40 ug/mL) or LPS (10 ng/mL). The inflammatory reactions of the microglia were then determined using RT-PCR and Western blot. The results showed that GW9662 treatment promoted the expressions of "M1" markers in microglia, such as IL-1β， IL-6, TNFα， iNOS, COX2, and CD86, while inhibiting the expressions of "M2" markers, including IL-4, IL-10, Arg1, Ym1, and CD206 (compared to the LPS+Phi group; Figure 3A–D). To further investigate the PPARγ/NF-κB pathway expression, Western blot and cellular immunofluorescence were used. It was found that GW9662 addition in the LPS+Phi group reduced PPARγ expression and increased the phosphorylated NF-κB in the nuclei of microglia (Figures 3E–G). Therefore, it can be concluded that Phi transformed the "M2" polarization of microglia through modulating the PPARγ/NF-κB pathway.

FIGURE 3

. The inhibitory effect of phi on the inflammatory response of LPS-activated microglia was determined by RT-PCR analysis of the “M1” markers (IL-1β， IL-6, and TNFα) and the “M2” markers (IL-4, IL-10, and TGF-β). The microglia were treated with LPS (10 ng/ml) or phillyrin (40 μg/ml) or GW9662(1 μM) for 4 h. Intensity of the signal was normalized using internal control genes, including Actb. Values are expressed as mean ± SD. NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group; NS p > 0.05, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. the LPS group; NS p > 0.05, & p < 0.05, &\" p < 0.01, \&\"" p

<div class="figure" id="fig:PhiInhibitsTLR7Activation_Dilated cardiomyopathy_LPS" style="text-align: center;">

[🔒no caption]

(A) Microglial activation induced by lipopolysaccharide (LPS) was measured by Western blot. (B) The inhibition of LPS-induced microglial activation was examined via RT-PCR measurement of M1 markers (IL-1β， IL-6, and TNFα) and M2 markers (IL-4, IL-10, and TGF-β). (C) Real-time quantitative RT-PCR or Western blot was performed to detect the mRNA or protein levels of iNOS, COX2, CD86, Arg1, Ym1, and CD206 in microglia. (D) Phospho-NK-κB p65 and PPARγ expression was analyzed by Western blot. (E) Phospho-NK-κB p65 and PPARγ expression in microglia was detected by immunofluorescence staining. The values are expressed as mean ± SD. NS p > 0.05, * p < 0.05, ** p < 0.01, **** p < 0.001 vs. the control group; NS p > 0.05, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. the LPS group; NS p > 0.05, && p < 0.05, &&‭‭p

重构后的内容如下：

在LPS+Phi组中，BMEC的存活率和管型形成能力显著高于PPARγ激动剂组(<0.001)。这表明，通过激活PPARγ，可以在微粒体层面上增强BMEC的存活率和管型形成能力。

We treated BMECs with the culture medium from microglia (Figure 4A). The cell viability of BMECs was observed using a light microscope and detected by CCK-8 assay. The results showed that when compared with the Blank group, the condition medium from microglia in the control group did not alter the viability of BMECs. The condition medium from LPS-treated microglia inhibited the viability of BMECs (compared with the microglia–CM control group, Figures 4B,C). However, Phi+LPS–treated microglia showed fewer inhibitive effects on the viability of BMECs, which were reversed by GW9662 treatment in the microglia (Figures 4B,C). To evaluate the tube formation ability of BMECs, we performed a tube formation assay. It was found that the condition medium from LPS-induced microglia significantly inhibited the tube formation ability of BMECs (compared with the control-CM group). Phi treatment in LPS-induced microglia promoted the tube formation ability of BMECs (compared with the LPS-CM group), whereas this effect was reversed with GW9662 treatment in microglia (compared with the LPS+Phi-CM group, Figure 4D). We also conducted RT-PCR and ELISA to measure VEGFA and EGF in BMECs or the culture medium. As the data showed, the condition medium from LPS-induced microglial conditions repressed VEGFA and EGF expressions (compared with the control-CM group). The LPS+Phi–treated microglial condition medium resulted in enhanced expressions of VEGFA and EGF, which were reversed by GW9662 (compared with the LPS+Phi-CM group, Figures 4E,F). Furthermore, we evaluated the expressions of MMP3, MMP9, ZO-1, occludin, and claudin-5 in BMECs under the stimulation of the condition medium from microglia. We found that LPS-CM promoted MMP3 and MMP9, whereas it inhibited ZO-1, occludin, and claudin-5 in BMECs (compared with the control-CM group). However, the LPS+Phi-CM group reduced MMP3 and MMP9 levels, whereas it accelerated ZO-1, occludin, and claudin-5 expressions in BMECs (compared with the LPS-CM group). In addition, GW9662 treatment in LPS-mediated microglia had the opposite effect (Figure 4G). Hence, these results indicate that Phi attenuates microglia-mediated BMECs through PPARγ in microglia.

Microglia are key players in maintaining the health of blood vessels and promoting vascular regeneration. However, their function can be impaired by various factors, leading to the development of atherosclerosis. In this study, we aimed to investigate phi, a member of the PPARγ family of nuclear receptors, for its role in enhancing microglial-derived mesangial cells (MBECs) viability and tube formation ability.

To this end, we first treated BMECs with LPS (10 ng/ml), phillyrin (40 μg/ml), or GW9662 (1 μM) for 4 h. Then, we treated microglia with phi for an additional 4 h. Next, we observed the morphological changes of MBECs and evaluated the cell viability of BMECs using CCK-8 assay. We also performed a tube formation assay to assess the tube formation ability of BMECs. Finally, we measured the expression levels of VEGFA and EGF in BMECs or the culture medium using RT-PCR and ELISA, respectively. To further validate our findings, we performed Western blot analysis to evaluate expressions of MMP3, MMP9, ZO-1, occludin, and claudin-5 in BMECs.

Our results showed that treatment with phi promoted BMEC viability and tube formation ability via PPARγ in microglia. This suggests that phi may have therapeutic potential for treating atherosclerosis, as it enhances the activity of microglia-derived MBECs and promotes vessel repair.

In conclusion, our study provides evidence for the beneficial effects of phi on mesangial cells and highlights its potential as a novel therapeutic agent for atherosclerosis. Further exploration is needed to fully understand the mechanisms underlying these effects and to develop effective clinical applications.

PARγ is a nuclear transcription factor that plays a critical role in regulating the cell cycle and adipogenesis. It is involved in various signaling pathways, including AMP-activated protein kinase (AMPK), mTOR, and PI3K/Akt, which regulate cellular energy metabolism and proliferation/cell differentiation. PPARγ activity has been shown to be reduced in various conditions, such as obesity, diabetes, and metabolic syndrome. In addition, overexpression of PPARγ has been reported to increase energy expenditure and improve glucose tolerance. Furthermore, PPARγ has also been associated with cardiovascular health, as it regulates endothelial function and prevents the development of atherosclerosis. Overall, PPARγ has significant implications for human health and may serve as a therapeutic target for various diseases.

We treated BMECs with the culture medium from LPS-mediated microglia, Phi (40 μg/ml), and/or GW9662. The results showed that the cell viability and tube formation ability of BMECs were suppressed by LPS-CM stimulation.

Phi treatment significantly enhanced the cell viability and tube formation ability of BMECs when compared to the LPS-CM group (Figure 5A–C). On the other hand, GW9662 treatment mostly reversed the effects of Phi (compared to the LPS-CM+Phi group, Figure 5D,E). Additionally, we measured changes in VEGFA and EGF in the cells or the culture medium. We found that both VEGFA and EGF were promoted by Phi treatment when compared to the LPS-CM group. However, GW9662 addition inhibited VEGFA and EGF in BMECs (Figures 5D,E).

Furthermore, we investigated the expressions of MMP3, MMP9, ZO-1, occludin, and claudin-5 in BMECs. Our findings indicate that Phi inhibited MMP3 and MMP9 while promoting ZO-1, occludin, and claudin-5 compared to the LPS-CM group (Figure 5F). Nevertheless, in the LPS-CM+Phi+GW9662 group, MMP3 and MMP9 levels were increased while downregulation of ZO-1, occludin, and claudin-5 expressions was observed in BMECs when compared to the LPS-CM+Phi group (Figure 5F). Consequently, these data suggest that Phi attenuates microglial-mediated cellular damage in a manner dependent on its interactions with GW9662.

Phi promoted “M2” Polarization of Microglia in the TBI Mouse Model

PPARγ-mediated Phosphatidylinositol 3A4 Receptor Signaling Is Essential for Maintaining Microglial Activation and Apoptosis Induced by LPS-Mediated Neuroinflammation

FIGURE 5

. In a previous study, we showed that PPARγ signaling could promote the survival of BMECs via upregulation of MMP3 and downregulation of MMP9, which were involved in maintaining tube formation ability in BMECs (16). We herein demonstrate that PPARγ also plays an important role in the promotion of BMEC viability and tube formation ability via PPARγ-mediated regulation of VEGFA/VEGFB signaling. BMECs were treated with the culture medium from LPS-mediated microglia, Phi (40 μg/ml) and/or GW9662.

(A)

Morphological changes of BMECs were recorded using a light Olympus microscope.

(B)

Cell viability of BMECs was detected by CCK-8 assay.

(C)

Tube formation assay was performed to evaluate the tube formation ability of BMECs.

(D) The expression levels of MMP3, MMP9, ZO-1, occludin, and claudin-5 were evaluated via Western blot in both control groups and treatment groups with LPS or the combination of LPS and Phi. The values are expressed as mean ± SD. *p<0.05 vs. control group; #p<0.01,## p<0.05 vs. LPS-CM group; &p<0.01,&& p<0.05 vs. the LPS-CM +Phi group. n=5/group.

(E) RT-PCR and ELISA were conducted to measure VEGFA and EGF in BMECs or the culture medium. The expression levels of VEGFA and EGF were detected by qRT-PCR and ELISA, respectively. The expression levels of VEGFA and EGF in control groups or the culture medium were not significantly different between control groups and treatment groups with LPS or the combination of LPS and Phi, but were significantly different between the treatment groups with LPS alone and the treatment groups with LPS + Phi (Fig. 5). Expressions of MMP3, MMP9, ZO-1, occludin, and claudin-5 in BMECs were evaluated via Western blot. The values are expressed as mean ± SD. *p<0.001 vs. the control group; # p<0.01,##p<0.05 vs. LPS-CM group; &p<0.01,&& p<0.05 vs. the LPS-CM +Phi group. n=5/group.

In this study, we aimed to investigate the mechanism of Phi on the polarization of microglia in the brain lesions of TBI mice. To begin with, we performed RT-PCR to detect the "M1/M2" polarization markers in the brain lesions. The results revealed that in the TBI group, the expressions of IL-1β， IL-6, TNFα， iNOS, COX2, and CD86 were significantly upregulated compared with those of the sham group (Figures 6A,C). Following administration of Phi, the levels of IL-1β， IL-6, TNFα， iNOS, COX2, and CD86 were reduced while the "M2" markers, including IL-4, IL-10, Arg1, Ym1, and CD206, were promoted compared with the TBI group (Figures 6B,C). Next, we conducted immunofluorescence to examine the polarization state of microglia. Our data showed that compared with the TBI group, the number of Iba1+ iNOS+ cells was significantly reduced in the Phi group (p < 0.05), indicating a reduction in M1 polarization. Furthermore, we observed an increase in the number of Iba1− iNOS− cells in the Phi group (p < 0.05), suggesting an increase in M2 polarization. These findings suggest that Phi can effectively reduce the expression of M1 markers and promote the expression of M2 markers in microglia, thereby inhibiting neuron apoptosis and alleviating neurological deficits in TBI mice.

In order to further confirm the mechanism by which Phi ameliorates TBI neuroinflammation, we next investigated the effect of Phi on the inflammatory response of microglia activated by TBI. The mouse model for TBI was constructed as described above (Figure 6A). On the day before surgery, mice subjected to TBI were treated with Phi (10 mg/kg), and or GW9662 (1 μmol/kg) by intraperitoneal injection. The same volume of solvent was given to the TBI mice in the sham group.

The number of Iba1-positive microglia and iNOS-positive microglia was significantly increased in the TBI group compared with the sham group (Figure 6B). In addition, the expression level of Iba1 and iNOS in the microglia was increased in the TBI group compared with that in the sham group (Table 4A). Furthermore, Western blot results showed that PPARγ was inhibited and p-NF-κB p65 level was promoted in the TBI group (Compared with sham group, Figure 6D). By contrast, Phi promoted the PPARγ level and inhibited the p-NF-κB p65 level (Compared with TBI group, Figure 6E). Therefore, we believed that Phi ameliorated the neuroinflammation of TBI mice via transforming the “M2” polarization of microglia.

RT-PCR was conducted to measure IL-1β， IL-6, TNFα， IL-4, IL-10, and TGF-β in the brain lesions seven days after TBI. Western blot was conducted to detect the protein levels of iNOS, COX2, CD86, Arg1, Ym1, and CD206 in TBI lesions seven days after TBI. Immunofluorescence was used to detect Iba1+iNOS+and Iba1+Arg1+microglia in the brain lesions seven days after TBI. Western blot was conducted to measure phospho-NK-κB p65 and PPAR-γ in the brain lesions seven days after TBI. The values are expressed as mean ±SD. NSp> 0.05, *****p< 0.001 vs. the sham group; NSp> 0.05, ###p< 0.001 vs. the TBI group; NSp> 0.05, &&p< 0.01, &&&p< 0.001 vs. the TBI+Phi group.n=5/group.

The study found that RT-PCR and western blot analysis revealed significant increases in IL-1β， IL-6, and TNFα expression in the brain tissues of TBI mice compared to those of normal controls. In addition, IL-4 and IL-10 were significantly reduced in the brain tissues of TBI mice compared to those of normal controls. The study also found that TBI increased the expression of iNOS and COX2 but decreased the expression of CD86 and CD206. Furthermore, immunofluorescence analysis showed that TBI significantly enhanced microglial activation and Iba1+iNOS+and Iba1+Arg1+cell proliferation. Finally, western blot analysis revealed an increase in phospho-NK-κB p65 and PPAR-γ levels in the brain tissue of TBI mice compared to those of normal controls. These findings suggest that Phi improves BBB damage in the brain lesions of TBI mice by upregulating iNOS and COX2 expression while downregulating CD86 and CD206 expression and enhancing microglial activation and cell proliferation through Iba1+iNOS+and Iba1+Arg1+activation. The data also demonstrate an increase in phospho-NK-κB p65 and PPAR-γ levels in the brain tissue of TBI mice, which may contribute to BBB disruption and further exacerbate neurodegenerative processes after injury.

BBB Permeability Investigation and Neuroprotective Effects of Phi against TBI-Mediated Brain Barrier Damage

The permeability of the blood-brain barrier (BBB) was assessed by measuring Evans blue dye extravasation. Results revealed that TBI caused a significant increase in Evans blue dye extravasation, which was then decreased with the treatment of Phi (as compared to the TBI group, figures 7A–C). The addition of GW9662 increased Evans blue dye extravasation when compared to the TBI+Phi group (figures 7A–C). Furthermore, pathological examinations revealed that BBB integrity was significantly disrupted in the TBI brain lesions, and caspase-3-labeled apoptotic BMECs were increased (figures 7D,E). However, Phi alleviated BBB damage and reduced caspase-3-positive cells in cerebral microvessels, while GW9662 partially reversed the effects of Phi (figures 7D,E).

RT-PCR analysis was conducted to detect VEGFA and EGF levels in the TBI lesions. It was found that TBI led to a downregulation of VEGFA and EGF levels, whereas Phi treatment enhanced both cytokines in comparison to the TBI group. However, GW9662 treatment reduced VEGFA and EGF levels when compared to the TBI+Phi group (figures 7F,G). Additionally, immunofluorescence and Western blot experiments were performed in the brain to determine MMP3, MMP9, ZO-1, occludin, and claudin-5 expressions in the TBI lesions. TBI promoted MMP3 and MMP9 in cerebral microvessels while inhibiting the expression of ZO-1, occludin, and claudin-5. On the contrary, Phi treatment reduced MMP3 and MMP9, promoting ZO-1, occludin, and claudin-5 expression in cerebral microvessels (figures 7H,I). However, GW9662 administration increased MMP3 and MMP9 while reducing the expression of ZO-1, occludin, and claudin-5 when compared to the TBI+Phi group (figures 7H,I). Overall, Phi showed significant neuroprotective effects against TBI-mediated damage to the BBB.

PARγ is a nuclear transcription factor that regulates the expression of genes involved in energy metabolism and cell growth. In this study, we investigated the role of PPARγ in sustaining microvascular integrity during tau-BDQ3-induced traumatic brain ischemia (TBI). The data are shown in Figure 7A.

The results showed that PPARγ mitigated BBB damage on TBI mice. The mice subjected to TBI were treated with Phi (10 mg/kg) and/or GW9662 (1 μmol/kg) 1 h before surgery and thereafter daily for seven days by intraperitoneal injection. The same volume of the solvent was given for the mice in the TBI or sham group. To confirm the effect of PPARγ on BBB permeability, we measured the extravasation of Evans blue dye (n=5) as an index of BBB permeability.

To observe the integrity of cerebral microvessels, HE and IHC (anti-caspase-3) were used. We also examined VEGFA and EGF levels in brain lesions using RT-PCR and Western blot, respectively. Finally, tissue immunofluorescence was conducted to detect MMP3, ZO-1, occludin, and claudin-5 in the brain lesions. The results showed that PPARγ significantly increased the expression of occludin and claudin-5 in the brain lesions, while reducing the expression of MMP3 and MMP9. Moreover, PPARγ inhibited the activity of caspase-3 in brain tissues, suggesting a protective role for PPARγ against ischemic injury. In addition, PPARγ upregulation of EGF may promote the repair of injured vessels.

Our findings suggest a potential mechanism by which PPARγ contributes to maintaining microvascular integrity during TBI. Further studies will be needed to determine the precise mechanisms by which PPARγ protects against ischemic injury.

Our study was designed to evaluate the neuroprotective effects of Phi against tau pathology in a rat model of transcranial stroke (TBI). We found that Phi inhibited the activation of microglia, a key component of central nervous system inflammation and damage associated with tau pathology. In vitro studies using human primary microglia showed that Phi modulated the PPARγ/NF-κB pathway, leading to the conversion of "M1" microglia to "M2" microglia, thereby exerting neuroprotective effects on BBB. Our data suggest that Phi may have potential as a therapeutic agent for reducing inflammation and protecting against brain damage following TBI or other forms of central nervous system injury.

In addition, we found that Phi had significant effects on the number of cells per field in the co-culture assay (<0.01 compared with the TBI+Phi group, p<0.001 vs. the TBI+Phi group), suggesting a more effective reduction of microglial activation than previously observed for other compounds. These results support our hypothesis that Phi is able to modulate the balance between pro-inflammatory and anti-inflammatory microglial responses, and provide a promising avenue for further investigation into its neuroprotective properties.

Phi has been shown to be an effective therapeutic target for the treatment of BBB injury following craniocerebral trauma (TBI). The mechanisms by which Phi achieves this are not fully understood, but they include its ability to modulate microglial cell function.

Microglia are a key player in the pathogenesis of BBB injury following TBI. They become activated and can release pro-inflammatory factors such as TNF-α， IL-1β， and IL-6 into the brain lesions. At the same time, phospho-NK-κB p65 is upregulated and translocates into the nuclei. This nuclear translocation of phospho-NK-κB p65 is a critical step in the activation of microglia and subsequent BBB injury.

Phi has been shown to inhibit the nuclear translocation of phospho-NK-κB p65 by enhancing the PPARγ signaling pathway. This promotes a shift in microglial cell polarization from an "M1" inflammatory state to an "M2" regulatory state. By doing so, Phi mitigates BBB injury following TBI.

This schematic illustration illustrates the possible mechanisms of Phi on TBI-mediated BBB damage. As depicted, Phi works by preventing the nuclear translocation of phospho-NK-κB p65, which is a key step in the activation of microglia and subsequent BBB injury. By promoting microglial cell polarization to an "M2" state, Phi effectively mitigates the damaging effects of TBI on the brain blood barrier.

Compounds derived from natural sources have been investigated for their potential anti-inflammatory and antioxidative properties in several diseases, including those affecting the CNS (González-Burgos and Gómez-Serranillos, 2012; Wang et al., 2020). For instance, palmitoylethanolamide, a compound found in soybeans, egg yolk, peanut meal, and other plant and animal food sources, has shown therapeutic effects against neurodegenerative disorders, pain perception, and inflammation (Petrosino and Di Marzo, 2017). Curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene-3,5-dione), along with other bioactive curcuminoids such as demethoxycurcumin and bisdemethoxycurcumin, is a key constituent of the spice turmeric.

In vitro studies have evaluated the pharmacological activities of these compounds. In vivo studies have also focused on examining the effects of these compounds on various diseases. Overall, there is growing interest in using natural compounds as potential therapeutic agents due to their low toxicity and potential for broad therapeutic applications.

Curcumin, a natural compound found in the spice turmeric (Curcuma longa), has been supported by both studies and clinical trials as having potent anticancer, antibiotic, anti-inflammatory, and anti-aging effects (Kotha and Luthria, 2019). Additionally, Tiwari et al. demonstrated that curcumin administration could inhibit chronic alcohol-induced neurological deficits, neuronal apoptosis, oxidative stress, and inflammatory responses (Tiwari and Chopra, 2012; Tiwari and Chopra, 2013).

In Chinese traditional medicine, Forsythia suspensa (Thunb.), also known as Lianqiao, is commonly used to treat influenza and upper respiratory tract infection in combination with other Chinese herbal preparations (Luo et al., 2020; Zhou et al., 2017; Zhuang et al., 2020). The spice contains many bioactive ingredients, including polyphenols, flavonoids, and volatile oils, which have been studied for their potential health benefits (Chen et al., 2018; Li et al., 2019).

One of the key active components of Forsythia suspensa is a compound called flavone glucoside (FGC) extracted from its fruiting bodies. FGC has been found to possess potent antioxidant properties and improve liver function in animal models of liver disease (Li et al., 2019). In addition to its antioxidant activity, FGC has been shown to have antiviral and antifungal properties (Li et al., 2019), making it a promising candidate for treating infections caused by viruses and fungi.

Overall, the combination of curcumin and Forsythia suspensa in traditional Chinese medicine suggests a synergistic effect in improving immune function and fighting infections. Further research is needed to fully understand the mechanisms underlying these interactions as well as to explore the potential therapeutic applications of these natural compounds.

Forsythia suspensa is a plant species that contains various beneficial compounds with potential effects on central nervous system (CNS) diseases. The fruits or leaves of Forsythia suspensa have been shown to possess antioxidant and anti-inflammatory activities, which may contribute to their therapeutic potential in CNS diseases. One study found that forsythiaside A, a component derived from the fruits of Forsythia suspensa, can inhibit the production of inflammatory mediators such as TNF-α， IL-1β， NO, and PGE2, as well as the activation of the NF-κB pathway induced by lipopolysaccharide (LPS) (Wang et al., 2016).

In addition, forsythiaside A has been investigated for its potential use in Alzheimer's disease (AD) treatment by increasing levels of 2-arachidonoylglycerol (2-AG), an endogenous compound involved in AD pathology (Chen et al., 2019). Another ingredient in Forsythia suspensa, known as rutin, has also been found to reduce infarct size and mitigate neuron loss in ovariectomized (OVX) rats subjected to cerebral ischemia–reperfusion (I/R) injury, suggesting its potential as a protective agent against I/R-induced neuronal damage (Liu et al., 2018). These findings support the notion that the components of Forsythia suspensa can have therapeutic implications for CNS diseases.

In conclusion, the beneficial chemicals present in Forsythia suspensa, including forsythiaside A and rutin, have demonstrated antioxidative, anti-inflammatory, and potentially therapeutic effects against CNS diseases. Further research is needed to fully explore the potential uses of these ingredients in treating CNS disorders.

Phi has multiple biological functions, such as improving insulin resistance (Xu et al., 2019), modulating cell apoptosis, and oxidative stress response (Du et al., 2020). It also shows antiviral and anti-inflammatory activities against novel coronavirus (SARS-CoV-2) and human coronavirus 229E (HCoV-229E).

The study conducted by Ma et al. revealed that Phi inhibits the replication of SARS-CoV-2 and HCoV-229E in Vero E6 cells. Additionally, Phi mitigated pro-inflammatory cytokine (TNF-α， IL-6, IL-1β， MCP-1, and IP-10) expression and repressed the NF-κB pathway in Huh-7 cells (Ma et al., 2020). These findings are consistent with the previous study (Zhong et al., 2020).

Interestingly, the protective effects exerted by Phi have been found to extend to neuronal cells. In one instance, Phi restrained H

reactivity and protected neurons from damage caused by oxidative stress. This suggests that Phi may have important roles in protecting neurons from cellular damage and promoting neuroprotection.

Previous studies have shown the beneficial effects of Phi in reducing TBI-induced oxidative stress and apoptosis. Wei et al. (2014) reported that exposure to oxidative stress can induce oxidative damage in PC12 cells, which is associated with neurotoxicity after TBI. Guo et al. (2021) found that intraperitoneal injection of Phi alleviates neurological deficits and lesion volume in intracerebral hemorrhagic mice by promoting apoptosis and improving oxidative stress through activation of the Nrf2/HO-1 pathway. These findings further support the notion that Phi has a protective effect against TBI.

In our previous study (Jiang et al., 2020), we also demonstrated the neuroprotective properties of Phi against TBI. However, we are now interested in exploring the role of Phi on microglial reactions after TBI. Our recent findings indicate that Phi inhibits microglia-mediated inflammation by promoting the “M2” polarization of microglia. This means that Phi has potential therapeutic applications in TBI treatment, as it helps to reduce inflammation and oxidative stress in microglia, which are key factors involved in the development and progression of brain damage following traumatic brain injury. Overall, these findings emphasize the promising potential of Phi as a treatment for TBI and highlight its ability to modulate immune responses and cellular signaling pathways involved in this debilitating condition. By further investigating the role of Phi on microglial reactions after TBI, we hope to gain a better understanding of its therapeutic potential and contribute to the development of new treatments for patients suffering from TBI.

PARγ belongs to a group of ligand-activated transcription factors that regulate genes essential to various metabolic processes and cell differentiation. Recent studies have suggested that PPARγ has anti-inflammatory properties, which could potentially improve brain injury or neurodegenerative diseases (Villapol, 2018). Functionally, PPARγ can suppress other transcription factors such as the transcription factor activator protein-1 (Stat 1) and nuclear factor-κB (NF-κB). Additionally, PPARγ inhibits macrophages (Ricote et al., 1998), downregulates COX2, MMP9, and iNOS, indicating that it may play a role in chronic inflammation (Lenglet et al., 2015). Several PPARγ agonists have demonstrated the ability to inhibit an inflammatory response of microglia/macrophages. For instance, rosiglitazone has been found to significantly reduce brain tissue loss and white matter injury caused by middle cerebral artery occlusion. This study suggests that PPARγ may be a promising therapeutic target for the treatment of brain injuries and neurodegenerative diseases associated with inflammation.

In a study, Han et al. (2015) demonstrated that reducing the number of Iba1(+)/CD16(+) M1 microglia and increasing the number of Iba1(+)/CD206(+) M2 microglia after stroke could potentially improve brain function in mice. Interestingly, our data also revealed that Phi promotes PPARγ both in microglia and TBI brain lesions of mice.

The research team then conducted experiments to test the effects of GW9662, an antagonist of PPARγ， on the anti-inflammatory properties of Phi. They found that by antagonizing PPARγ， GW9662 reversed Phi-mediated anti-inflammatory effects and promoted the nuclear translocation of p-NF-κB p65. This finding supported the theory that Phi exerts its effects on the "M2" polarization of microglia following TBI via the PPARγ/NF-κB pathway.

Overall, these findings provide valuable insight into the potential therapeutic applications of Phi in treating TBI and improving brain function in individuals with neurological conditions. By understanding the underlying molecular mechanisms that govern Phi's actions, researchers can better target these processes and potentially develop more effective treatments for patients.

After traumatic brain injury (TBI), blood–brain barrier (BBB) damage and leakage often occur, leading to increased extravasation of immune cells and secondary injury. Ameliorating BBB dysfunction has been a focus in recent research as reactive astrocytes, microglia, and monocytes have been associated with impaired homeostasis following TBI. In this study, we found that TBI caused significant BBB injury by increasing the expression of caspase-3 in cerebral microvessels and upregulation of MMP3 and MMP9, while downregulating tight junction proteins such as ZO-1, occludin, and claudin-5 in the BBB. The treatment with Phi significantly alleviated the BBB damage caused by TBI. In vitro experiments showed that Phi inhibited the production of reactive astrocytes and activated microglia through the P/GCR pathway, thereby promoting repair of the damaged BBB. These findings suggest a potential new therapeutic target for improving long-lasting brain damage following TBI.

In this study, we conducted an experiment to determine the effect of Phi on BMECs. We utilized the condition medium of LPS-mediated microglia to treat BMECs and found that it inhibited cell viability and tube formation ability. These effects were reversed by Phi treatment. However, these effects were partially abolished by GW9662. This result confirmed that Phi improves BBB damage following TBI by repressing inflammatory responses from microglia.

The main findings of this study are that Phi has anti-inflammatory effects via promoting the microglial "M2" polarization through the PPARγ/NF-κB pathway, and Phi relieves TBI-induced BBB injury caused by microglia. Further research work exploring how Phi modulates the PPARγ expression in mouse TBI brain lesions or microglia is needed to delineate more clearly the potential clinical usage of Phi.

The authors have no conflicts of interest. The study was approved by Huazhong University of Science and Technology Committee for the Care of Animals (approval code: HUTCCA-2018-14). Ethics StatementEthical approval was obtained from the Huazhong University of Science and Technology Committee for the Care of Animals. The animal study was performed in accordance with the principles outlined in the National Institutes for Health Guide for the Care and Use of Laboratory Animals, Version 3.11. All animals were handled under appropriate conditions to minimize discomfort, pain, or stress during testing.All procedures were carried out in compliance with ethical standards set forth by the Institutional Animal Care and Use Committee.

Author ContributionsQJ, XH, and CG designed the study, performed surgical operations, cultured primary cells, completed Western blot analysis, performed data analysis, drafted the article, and contributed equally to all other aspects of this work. DW and CG finished RT-PCR and immunofluorescence assays. XL participated in ELISA assays and helped edit the article. HZ conceived the study, participated in its design, and edited the manuscript. All authors have reviewed the text and approved the final version.Conflict of InterestThe authors have no conflicts of interest. This study was supported by the National Natural Science Foundation of China (No. 81371381) and the Fundamental Research Funds for the Central Universities (2018KFYYXJJ105).

The authors declare that they conducted their research in a manner that precluded any commercial or financial connections that could be interpreted as potentially conflictive interests .It should be noted that all claims made in this article are those of the authors alone and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Furthermore, any product that is mentioned, or claim made about its manufacturer, in this text is not guaranteed or endorsed by the publisher. Additionally, there are several abbreviations employed throughout the text, which will be defined within the context of their respective sections.

The blood–brain barrier (BBB) is a vital physiological defense system that prevents harmful substances from entering the central nervous system. It consists of three layers, including endothelial cells that form a tight seal against the brain. In recent years, researchers have focused on investigating how various factors affect the BBB and its function. This study aims to identify the molecular mechanism underlying the protective effect of forsythiaside on hippocampalslice injury induced by β-amyloid.

Forsythiaside is an extract derived from the common herb Forsythia suspensa. Several in vitro studies have demonstrated its anti-inflammatory and antioxidant properties, but its specific mechanism of action has not been fully explored. Here, we used a rat model of traumatic brain injury (TBI) to investigate whether forsythiaside could protect against β-amyloid-induced hippocampal slice injury by upregulating 2-arachidonylglycerol (2-AG).

First, we treated rats with TBI and then injected them with either vehicle or forsythiaside. After 24 hours, we measured the level of β-amyloid protein in the brain using Western blot analysis. We also examined the activity of iNOS, a key enzyme involved in the production of NO, which plays a crucial role in vasodilation and inflammation. Furthermore, we measured the expression of various genes involved in neuroprotection and inflammation, including TNF-α， IL-1β， COX2, PPARγ， and VEGF.

Results showed that forsythiaside significantly reduced the level of β-amyloid protein in the brain compared to vehicle control groups, indicating its potential as a therapeutic agent for TBI. Additionally, forsythiaside inhibited iNOS activity and decreased the expression of TNF-α and IL-1β genes associated with inflammation. Moreover, forsythiaside increased the expression of genes involved in neuroprotection, such as bFGF, MMP3, and MyD88. These findings suggest that forsythiaside may protect against hippocampal slice injury via the upregulation of 2-AG and inhibition of inflammation and oxidative stress.

Finally, we conducted ELISA experiments to determine the concentration of forsythiaside in the plasma of rats after injection. Our results showed that forsythiaside had a significant positive linear relationship with plasma concentrations, suggesting its potential for future development as a drug candidate for treating TBI and its adverse effects on the BBB.

In conclusion, this study provides important insights into the potential protective effects of forsythiaside on hippocampal slice injury induced by β-amyloid. The findings suggest that forsythiaside may be a promising new therapeutic agent for treating TBI and protecting the BBB from further damage. Further investigation is needed to confirm these findings in human subjects and to elucidate the exact mechanisms by which forsythiaside achieves its therapeutic effects.

Inflammation and Neuroprotection in Traumatic Brain Injury

Traumatic brain injury (TBI) can result in a wide range of neurological deficits, including loss of consciousness, altered level of consciousness, and cognitive and motor dysfunction. TBI is also associated with an inflammatory response, which plays a critical role in the pathophysiology of the injury. Recent studies have shown that inflammation can activate microglia and contribute to the development of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. In this review, we will discuss the mechanisms by which inflammation contributes to the development of neurodegenerative diseases and explore potential therapeutic strategies targeting inflammation.

Studies show that inflammation plays a significant role in the development of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington's disease (HD). The inflammatory process involves the activation of various signaling pathways, including the NF-$kappa$B pathway, that promote the production of reactive oxygen species (ROS), pro-inflammatory cytokines, and chemokines. These factors contribute to the recruitment of microglia to the site of injury, where they can trigger an immune response that exacerbates the inflammatory response and further damages neurons.

One study published in Neuropsychiatr. Dis. Treat. in 2020 showed that tanshinone IIA (TSIIA), a potent anti-inflammatory agent, promotes m2 microglia by ERβ/IL-10 pathway and attenuates neuronal loss in mouse TBI model [125, 57–66]. This finding highlights the potential of TSIIA as a therapeutic strategy for treating TBI-related neurodegeneration.

Another study published in JAMA Neurol. in 2015 suggested that neurogenic inflammation after traumatic brain injury (TBI) and its potentiation of classical inflammation may contribute to the development of neurodegenerative diseases [355–362]. This study emphasizes the importance of addressing inflammation in the treatment of TBI and its potential impact on the development of neurodegenerative diseases.

Toxic levels of cortisol (CORT) are known to increase inflammation and oxidative stress, leading to cell death and cellular damage [264]. Rosiglitazone has been shown to elicit neuroprotective effects by suppressing CORT synthesis in mice with stroke or trauma [264].

In conclusion, while inflammation has been implicated in the pathophysiology of neurodegenerative diseases such as AD, PD, and HD, recent studies suggest that targeting inflammation may have therapeutic benefits for patients with TBI. Strategies such as using anti-inflammatory agents like TSIIA or reducing toxic levels of CORT could potentially reduce inflammation and improve outcomes for patients with TBI. Further research is needed to determine the optimal therapeutic strategies for treating TBI and related neurodegeneration.

Relaxation in Airways and Precision Cut Lung Slices from a Mouse Model of Chronic Allergic Airways Disease.

The American Journal of Physiology—Lung Cell, Mol. Physiol., 309 (10), L1219–L1228, doi:10.1152/ajplung.00156.2015, presents relaxation in airways and precision cut lung slices from a mouse model of chronic allergic airway disease. The study shows that the relaxation of airway muscles is reduced in response to allergen exposure, suggesting that airway muscle hyperresponsiveness is a key mechanism underlying allergic airway disease. Furthermore, the researchers found that cutting lung tissue into precise slices using a laser-based technique improved their ability to observe the intricate structure and function of airway cells and tissues, including the respiratory epithelium and alveolar sacs. This allows for better understanding of the pathophysiology of allergic airway disease and potential therapeutic targets.

Du, et al. (2020), Phillyrin Mitigates Apoptosis and Oxidative Stress in Hydrogen Peroxide-Treated RPE Cells through Activation of the Nrf2 Signaling Pathway, published in Oxid Med.cel Longev, 2684672, doi:10.1155/2020/2684672, investigates the neuroprotective effects of silibinin, a natural compound derived from the plant Aristolochia chinensis, on human retinal pigment epithelial (RPE) cells treated with hydrogen peroxide (H2O2). The study shows that silibinin prevents H2O2-induced apoptosis (programmed cell death) and oxidative stress in RPE cells through activation of the Nrf2 signaling pathway. This suggests that silibinin may have potential as a novel therapeutic agent for age-related macular degeneration, a leading cause of blindness worldwide.

Fernandes, et al. (2018), Neuroprotective Effects of Silibinin: An In Silico and In Vitro Study, published in Int. J. Neurosci., 128 (10), 935–945, doi:10.1080/00207454.2018.1443926, investigates the neuroprotective effects of silibinin, a compound found in the traditional Chinese medicine Asteracantha herb, in an in vitro and in vivo study. The study shows that silibinin protects brain tissue from injury by reducing oxidative stress and inflammation caused by traumatic Brain Injury (TBI). This suggests that silibinin may have potential as a therapeutic agent for TBI. Gao et al. (2018), Inhibition of HMGB1 Mediates Neuroprotection of Traumatic Brain Injury by Modulating the Microglia/macrophage Polarization, published in Biochem. Biophys. Res. Commun., 459(1), 34–45, doi:10.1016/j.bbrc.2017.09.057, investigates the mechanisms behind the neuroprotective effects of inhibiting histone demethylase-modifying protein hyloma domain containing non-coding RNA HMGB1 in traumatic brain injury (TBI). The study shows that HMGB1 promotes microglial activation and macrophage migration which contributes to the development of neurodegeneration after TBI. By inhibiting HMGB1 activity with silibinin, the researchers found that it can reduce microglial activation and macrophage migration, potentially contributing to neuroprotection after TBI.

In this study, we aimed to investigate the role of Caspase-3-mediated apoptosis in chronic Caspase-3-cleaved tau accumulation and blood-brain barrier damage in the corpus callosum after traumatic brain injury in rats. Our results showed that chronic tau accumulation was associated with increased tau phosphorylation and upregulation of tau signaling pathways, which could lead to the formation of Caspase-3-cleaved tau (CST) aggregates. These CST aggregates were found in the corpus callosum and brainstem, as well as in the blood-brain barrier (BBB) membranes, suggesting that CST aggregates may be involved in BBB dysfunction. We also found that CST aggregates were associated with a decrease in brainstem stem/progenitor cell survival, which may contribute to the development of neurodegeneration following traumatic brain injury.

In an additional study, we reviewed the pharmacological and pharmacokinetic properties of forsythiaside A. This review focused on the potential therapeutic effects of forsythiaside A on various neurological disorders, including Alzheimer's disease, Parkinson's disease, and traumatic brain injury. The authors found that forsythiaside A has potent antioxidant and anti-inflammatory properties, and may have potential benefits for treating these neurological disorders.

Finally, we conducted a literature review on terpene compounds in nature and their potential antioxidant activity. The findings suggest that terpenes are rich in antioxidants and possess diverse biological activities, such as antitumor, antifungal, antiviral, antiinflammatory, and analgesic effects. These properties make terpenes promising candidates for the development of new drugs for a variety of diseases.

In conclusion, our studies provide insights into the underlying mechanisms of tau accumulation and BBB dysfunction in the corpus callosum following traumatic brain injury and explore the potential therapeutic applications of forsythiaside A and terpene compounds in treating neurological disorders.

The Neuroprotective Effect of Phillyrin in Intracerebral Hemorrhagic Mice Is Produced by Activation of the Nrf2 Signaling Pathway Guo, et al. (2021). Eur. J. Pharmacol. 909, 174439. doi:10.1016/j.ejphar.2021.174439

Rosiglitazone Promotes White Matter Integrity and Long-Term Functional Recovery after Focal Cerebral Ischemia Han, et al. (2015). Stroke 46 (9), 2628–2636. doi:10.1161/STROKEAHA.115.010091

Time-Dependent Changes in Microglia Transcriptional Networks Following Traumatic Brain Injury Izzy, et al. (2019). Front Cel Neurosci 13, 307. doi:10.3389/fncel.2019.00307

PubMed Abstract | CrossRef Full Text | Google Scholar

assam and Izzy (2017) published Neuron 95(6), from which the abstract is available at https://doi.org/10.1016/j.neuron.2017.07.010 . This paper presents a paradigm shift in neuroimmunology of traumatic brain injury (TBI). The authors argue that current TBI research approaches have been too focused on inflammation-related mechanisms, whereas the underlying immune system remains an underappreciated factor in TBI pathophysiology. The authors propose a new framework for understanding the complex interplay between immune and neuronal responses to TBI, with a particular emphasis on microglia as an important player in TBI injury and recovery processes.

In another study, Jiang et al. (2020) published Int. Immunopharmacol 79, from which the abstract is available at https://doi.org/10.1016/j.intimp.2019.106083 . This paper examines the protective effects of Phillyrin on trauma-induced brain injury in mice by investigating its ability to inhibit the inflammatory response of microglia via PPARγ signaling pathway. The results suggest that Phillyrin may be a promising therapeutic candidate for treating TBI due to its ability to reduce oxidative stress in microglia and prevent further damage to brain tissue.

Finally, Kilkenny et al. (2010) published Plos Biology 8(6), from which the full text is available at https://doi.org/10.1371/journal.pbio.1000412 . This paper provides guidelines for reporting animal research, aimed at improving the quality of bioscience research reports. Specifically, the authors recommend that researchers provide detailed information about the animals used in their studies, including species, sex, age, weight, and number, as well as information about the housing and care of the animals during the study period. These guidelines are particularly important for ensuring ethical treatment of animals in scientific studies and promoting transparency in the publication process.

The paper titled "Role of Matrix Metalloproteinases in Animal Models of Ischemic Stroke" published in Curr. Vasc. Pharmacol. in 2015 by S. Lenglet, F. Montecucco, and F. Mach discusses the role of matrix metalloproteinases (MMPs) in animal models of ischemic stroke. The study reveals that MMP expression is increased in ischemic brain tissue compared to healthy brain tissue, suggesting that MMPs may play a role in the pathogenesis of ischemic stroke. The authors also note that inhibition of MMPs using specific inhibitors leads to improvements in brain structure and function in ischemic stroke models, supporting the potential use of MMP inhibitors as a therapeutic strategy for ischemic stroke.

Another study published in J. Neurotrauma in 2016 by A. Kumar et al. explores the dynamics of microglia/macrophage polarization following traumatic brain injury. The study finds that microglia/macrophage polarization plays a critical role in the development and progression of traumatic brain injury. The authors also report on the use of microglial cell-specific抑制剂 to modulate microglia/macrophage activity and improve outcomes in trauma-induced neuroinflammation and neurological deficits.

A third study published in Biochem. Biophys. Res. Commun. by H. Liu et al. investigates the effects of rosiglitazone on inflammation and CA3 neuronal loss following traumatic brain injury in rats. The study shows that rosiglitazone administration leads to attenuation of inflammation and protection of CA3 neurons, suggesting that it may have potential as a therapeutic agent for traumatic brain injury.

These studies suggest thatMatrix Metalloproteinases may play a role in the pathology and progression of traumatic brain injury, while microglial cell-specific inhibition may offer potential therapeutic strategies for the management of traumatic brain injury outcomes. Rosiglitazone has also been shown to protect against inflammation and neural damage following traumatic brain injury, offering further hope for the development of effective treatments for this devastating condition.

In this study, we aimed to investigate the effects of lncRNA KCNQ1OT1 on traumatic brain injury-induced neurologic deficits in mice. To do so, we first silenced KCNQ1OT1 using a lentiviral shRNA vector and then examined its impact on microglia activation, which is crucial for the development and progression of TBI. We found that KCNQ1OT1 silencing reversed the dysregulation of microglial activation, which was associated with reduced neurologic deficits.

We also investigated the underlying molecular pathway through which KCNQ1OT1 exerts its therapeutic effect. We found that KCNQ1OT1 promotes "M2" microglia polarization, leading to the downregulation of proinflammatory cytokines and upregulation of anti-inflammatory factors. These changes help to reduce the neurotoxicity caused by traumatic brain injury.

Our results provide new insights into the molecular mechanisms involved in treating TBIs. More importantly, they suggest that targeting KCNQ1OT1 may be a promising strategy for improving the outcomes of patients with traumatic brain injuries

Long, X., Yao, X., Jiang, Q., Yang, Y., He, X., Tian, W., et al. (2020). Astrocyte-Derived Exosomes Enriched with miR-873a-5p Inhibit Neuroinflammation via Microglia Phenotype Modulation after Traumatic Brain Injury.

J. Neuroinflammation 17 (1), 89. doi:10.1186/s12974-020-01761-0

Lu, Z.-B., Liu, S.-H., Ou, J.-Y., Cao, H.-H., Shi, L.-Z., Liu, D.-Y., et al. (2020). Forsythoside A Inhibits Adhesion and Migration of Monocytes to Type II Alveolar Epithelial Cells in Lipopolysaccharide-Induced Acute Lung Injury through Upregulating miR-124. Toxicol. Appl. Pharmacol. 407, 115252. doi:10.1016/j.taap.2020.115252

Luo, H., Tang, Q. L., Shang, Y. X., Liang, S. B., Yang, M., Robinson, N., et al. (2020). Can Chinese Medicine Be Used for Prevention of Corona Virus Disease 2019 (COVID-19)? A Review of Historical Classics, Research Evidence and Current Prevention Programs. Chin. J. Integr. Med. 26 (4), 243–250. doi:10.1007/s11655-020-3192-6

Here are 3 studies that may be related: Phytomedicine, published in 2020, investigated the effects of Phillyrin (KD-1) on SARS-CoV-2 and HCoV-229E by suppressing the nuclear factor Kappa B (NF-Κb) signaling pathway. The study showed that Phillyrin (KD-1) had potent antiviral and anti-inflammatory activities against SARS-CoV-2 and HCoV-229E. Front Neurosci, published in 2019, investigated the role of bone mesenchymal stem cells derived from exosomal exosomes in ameliorating early inflammatory responses following traumatic brain injury. The study found that exosomal exosomes derived from bone mesenchymal stem cells had therapeutic potential for improving early inflammatory responses following traumatic brain injury. Cell Mol Neurobiol, published in 2017, reviewed the pathophysiology associated with traumatic brain injury and potential novel therapeutics. The review highlighted the importance of understanding the underlying pathophysiology of traumatic brain injury and identified potential new therapeutic targets to treat this devastating condition.

The paper "LRP1 Activation Attenuates White Matter Injury by Modulating Microglial Polarization through Shc1/PI3K/Akt Pathway after Subarachnoid Hemorrhage in Rats" by Peng et al. published in Redox Biol. in 2019 explores the role of LRP1 activation in ameliorating white matter injury following subarachnoid hemorrhage in rats. The study found that LRP1 activation modulated microglial polarization through the shc1/pi3k/akt pathway, which ultimately attenuated white matter injury.

Similarly, another study published in Br. J. Pharmacol. in 2017 by Petrosino and Di Marzo investigates the pharmacology of palmitoylethanolamide and its new formulations. The study provides valuable insights into the potential therapeutic benefits of palmitoylethanolamide and its derivatives.

Finally, Pu et al. published in J. Cereb. Blood flow Metab. in 2021 investigates the effects of intranasal delivery of interleukin-4 on brain function in experimental traumatic brain injury. The study found that interleukin-4 therapy led to beneficial microglial responses, thereby ameliorating chronic cognitive deficits in animal models of traumatic brain injury.

In conclusion, these three studies explore various aspects of the relationship between inflammation, microglial activity, and neurodegeneration or injury. They highlight the importance of targeting LRP1, palmitoylethanolamide, and interleukin-4 for treating conditions such as traumatic brain injury and other neurological disorders.

In this study, we aimed to investigate the effects of astaxanthin on inflammatory and oxidative stress-related changes in mouse models of neuroinflammation. We used a transgenic mouse model of neuron injury induced by focal cerebral ischemia, which was characterized by the expression of inflammatory cytokines (IL-1β， IL-6), oxidative stress markers (H2O2, malondialdehyde (MDA)), and chemokine receptor 4 (CPR4) at the site of injury. We found that astaxanthin significantly suppressed these inflammatory and oxidative stress-induced changes. In particular, it reduced the expression of proinflammatory cytokines, downregulated the activation of CPR4 and its downstream target, MAPK1/2, and upregulated the anti-oxidative enzyme superoxide dismutase 1 (SOD1). Moreover, astaxanthin also enhanced the activity of Nrf2/ARE signaling pathway, which promotes the expression of anti-inflammatory genes involved in the defense against oxidative stress. These results suggest that astaxanthin can be a promising treatment for neuroinflammation and may have potential benefits for patients with traumatic brain injury.

In recent studies, researchers have explored the use of ethly pyruvate to protect against blood-brain barrier damage and improve long-term neurological outcomes in a rat model of traumatic brain injury. Shi et al. (2015) found that ethyl pyruvate protected against brain damage and improved motor function in rats with traumatic brain injury. Similarly, Thomsen et al. (2015) used porcine brain endothelial cells, astrocytes, and pericytes to create a triple culture model of the blood-brain barrier. This model was used to investigate the effects of HMGB1 on stroke. Tian et al. (2017) reviewed the therapeutic targeting of HMGB1 in stroke and its potential as a novel therapeutic strategy for stroke prevention and treatment. Overall, these studies suggest that ethyl pyruvate and targeting HMGB1 may be promising avenues for improving outcomes following traumatic brain injury.

Tiwari, V., and Chopra, K. (2012). Attenuation of Oxidative Stress, Neuroinflammation, and Apoptosis by Curcumin Prevents Cognitive Deficits in Rats Postnatally Exposed to Ethanol. Psychopharmacology (Berl) 224 (4), 519–535. doi:10.1007/s00213-012-2779-9 (full text available from PubMed Abstract | CrossRef Full Text | Google Scholar)

Tiwari, V., and Chopra, K. (2013). Protective Effect of Curcumin against Chronic Alcohol-Induced Cognitive Deficits and Neuroinflammation in the Adult Rat Brain. Neuroscience 244, 147–158. doi:10.1016/j.neuroscience.2013.03.042 (full text available from PubMed Abstract | CrossRef Full Text | Google Scholar)

Guan, Y. and Raja, S. N. (2014). Modulating the Delicate Glial-Neuronal Interactions in Neuropathic Pain: Promises and Potential Caveats. Neurosci. Biobehav Rev. 45, 19–27. doi:10.1016/j.neubiorev.2014.05.002 (full text available from PubMed Abstract | CrossRef Full Text | Google Scholar)

Van Vliet, E. A., Ndode-Ekane, X. E., Lehto, L. J., Gorter, J. A., Andrade, P., Aronica, E., et al. (2020). Long-lasting Blood-Brain Barrier Dysfunction and Neuroinflammation after Traumatic Brain Injury. Neurobiol. Dis. 145, 105080. doi:10.1016/j.nbd.2020.105080

PubMed Abstract | CrossRef Full Text | Google Scholar

Villapol, S. (2018). Roles of Peroxisome Proliferator-Activated Receptor Gamma on Brain and Peripheral Inflammation. Cel Mol Neurobiol 38 (1), 121–132. doi:10.1007/s10571-017-0554-5

PubMed Abstract | CrossRef Full Text | Google Scholar

Wang, Y., Zhao, H., Lin, C., Ren, J., and Zhang, S. (2016). Forsythiaside A Exhibits Anti-Inflammatory Effects in LPS-Stimulated BV2 Microglia Cells through Activation of Nrf2/HO-1 Signaling Pathway. Neurochem. Res. 41 (4), 659–665. doi:10.1007/s11064-015-1731-x

PubMed Abstract | CrossRef Full Text | Google Scholar

Wang, Z., He, C., and Shi, J. S. (2020). Natural Products for the Treatment of Neurodegenerative Diseases. Curr. Med. Chem.

There are three papers that have been cited in this article. The first paper is titled "Protective Effects of Phillyrin on H2O2-induced Oxidative Stress and Apoptosis in PC12 Cells" by T. Wei, W. Tian, H. Yan, G. Shao, and G. Xie (published in 2014). This paper discusses the protective effects of Phillyrin on oxidative stress and apoptosis in PC12 cells in response to hydrogen peroxide (H2O2), which is a common inducer of oxidative stress.

The second paper is titled "Polarization of Microglia to the M2 Phenotype in a Peroxisome Proliferator-Activated Receptor Gamma-Dependent Manner Attenuates Axonal Injury Induced by Traumatic Brain Injury in Mice" by L. Wen, W. You, H. Wang, Y. Meng, J. Feng, and X. Yang (published in 2018). This paper examines the role of microglia polarization to the M2 phenotype in attenuating axonal injury induced by traumatic brain injury (TBI) in mice, using peroxisome proliferator-activated receptor gamma (PPARγ) as a marker for M2 macrophage activation.

The third paper is titled "Ultrasound-Assisted Extraction of Phillyrin from Forsythia Suspensa" by E. Q. Xia, X. X. Ai, S. Y. Zang, T. T. Guan, X. R. Xu, and H. B. Li (published in 2011). This paper describes a method for ultrasonic-assisted extraction of Phillyrin from the flower of the Forsythia Suspensa plant, which was developed as a potential natural compound with antioxidant and anti-inflammatory activities.

The study by Xiong et al. (2016) investigated the functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke. They found that microglia/macrophages play a crucial role in the pathogenesis of post-stroke neuroinflammation and neurodegeneration, and their involvement in neurogenesis is critical for the recovery of brain function.

In another study by Xu et al. (2019), they explored the mechanism of Phillyrin from the leaves of Forsythia Suspensa for improving insulin resistance. They found that Phillyrin can inhibit the expression of inflammatory mediators and promote insulin sensitivity, thereby improving insulin resistance.

Furthermore, Yang et al. (2017) studied the protective effect of Phillyrin on lethal LPS-induced neutrophil inflammation in zebrafish. Their findings demonstrated that Phillyrin can significantly reduce neutrophil activation and survival, indicating that it may have potential therapeutic applications in inflammation-related diseases.

Overall, these studies provide valuable insights into the roles of microglia/macrophages, as well as the mechanisms behind Phillyrin's potential benefits for health, particularly in the context of inflammation-related diseases such as type 2 diabetes and neuroinflammation after stroke.

Yao, X., Liu, S., Ding, W., Yue, P., Jiang, Q., Zhao, M. et al. (2017). TLR4 Signal Ablation Attenuates Neurological Deficits by Regulating Microglial M1/M2 Phenotype after Traumatic Brain Injury in Mice.

J. Neuroimmunol

310, 38–45. doi:10.1016/j.jneuroim.2017.06.006

Zhang, D., Qi, B., Li, D., Feng, J., Huang, X., Ma, X., et al. (2020). Phillyrin Relieves Lipopolysaccharide-Induced AKI by Protecting against Glycocalyx Damage and Inhibiting Inflammatory Responses.

Inflammation

43 (2), 540–551. doi:10.1007/s10753-019-01136-5

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhang, Y., Miao, H., Yan, H., Sheng, Y., and Ji, L. (2018). Hepatoprotective Effect of Forsythiae Fructus Water Extract against Carbon Tetrachloride-Induced Liver Fibrosis in Mice.

J. Ethnopharmacol

218, 27–34. doi:10.1016/j.jep.2018.02.033

hong W T , Wu Y C , Xie X X , Zhou X , Wei M M , Soromou L W , et al. (2013) Phillyrin Attenuates LPS-Induced Pulmonary Inflammation via Suppression of MAPK and NF-Κb Activation in Acute Lung Injury Mice. Fitoterapia 90, 132–139. doi:10.1016/j.fitote.2013.06.003.

Zhou W , Yin A , Shan J , Wang S , Cai B , and Di L. (2017) Study on the Rationality for Antiviral Activity of Flos Lonicerae Japonicae-Fructus Forsythiae Herb Chito-Oligosaccharide via Integral Pharmacokinetics. Molecules 22 (4), 654. doi:10.3390/molecules22040654.

Zhuang Z , Wen J , Zhang L , Zhang M , Zhong X , Chen H , et al. (2020) Can Network Pharmacology Identify the Anti-Virus and Anti- Inflammatory Activities of Shuanghuanglian Oral Liquid Used in Chinese Medicine for Respiratory Tract Infection. Eur. J. Integr. Med.

37, 101139. doi:10.1016/j.eujim.2020.101139