Thymol improves ischemic brain injury by inhibiting microglia-mediated neuroinflammation.

Microglia-mediated inflammation is a critical factor in the progression of ischemic stroke. Consequently, mitigating excessive microglial activation represents a potential therapeutic strategy for ischemic injury. Thymol, a monophenol derived from plant essential oils, exhibits diverse beneficial biological activities, including anti-inflammatory and antioxidant properties, with demonstrated protective effects in various disease models. However, its specific effects on ischemic stroke and microglial inflammation remain unexplored. Rodent transient middle cerebral artery occlusion (tMCAO) model was established to simulate ischemic stroke. TTC staining, modified neurological function score (mNSS), and behavioral tests were used to assess the severity of neurological damage. Then immunofluorescence staining and cytoskeleton analysis were used to determine activation of microglia. Lipopolysaccharide (LPS) was utilized to induce the inflammatory response of primary microglia in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were performed to exam the expression of inflammatory cytokines. And western blot was used to investigate the mechanism of the anti-inflammatory effect of thymol. In this study, we found that thymol treatment could ameliorate post-stroke neurological impairment and reduce infarct volume by mitigating microglial activation and pro-inflammatory response (IL-1β, IL-6, and TNF-α). Mechanically, thymol could inhibit the phosphorylation of phosphatidylinositol-3-kinase (PI3K), sink serine/threonine kinase (Akt), and mammalian target of rapamycin (mTOR), thereby suppressing the activation of nuclear factor-κB (NF-κB). Our study demonstrated that thymol could reduce the microglial inflammation by targeting PI3K/Akt/mTOR/NF-κB signaling pathway, ultimately alleviating ischemic brain injury. These findings suggest that thymol is a promising candidate as a neuroprotective agent against ischemic stroke.

Zhao C ，Sun L ，Zhang Y ，Shu X ，Hu Y ，Chen D ，Zhang Z ，Xia S ，Yang H ，Bao X ，Li J ，Xu Y ... -  《-》

Ethanol extracts of Cinnamomum migao H.W. Li attenuates neuroinflammation in cerebral ischemia-reperfusion injury via regulating TLR4-PI3K-Akt-NF-κB pathways.

Cinnamomum migao H.W. Li, commonly known as migao (MG), is used in the Miao region of China for treating cardiovascular and cerebrovascular diseases, attributed to its detoxifying (Jiedu in Chinese), activating blood circulation (Huoxue in Chinese), and promoting Qi circulation (Tongqi in Chinese) properties. However, its therapeutic potential for ischemic stroke (IS) remains unexplored. Therefore, this study was to explore the protective effect of MG against cerebral ischemia-reperfusion injury caused by IS. The aim of this study was to investigate whether ethanol extract of MG (EEMG) attenuates cerebral ischemia-reperfusion injury, and explored the underlying mechanisms. Middle cerebral artery occlusion and reperfusion (MCAO/R) was established, and the efficacy of EEMG was evaluated using triphenyltetrazolium chloride (TTC), immunofluorescence, hematoxylin-eosin (HE) staining, and real-time quantitative PCR (qRT-PCR). Qualitative analysis of EEMG was analyzed for chemical composition by liquid chromatography-mass spectrometry (LC-MS). The molecular mechanism of EEMG was explored by metabolomics, network pharmacology, immunoblotting, immunofluorescence staining, gene knockdown, and agonist treatment. The results showed that EEMG alleviates ischemic injury in MCAO/R-operated rats and reduces neuronal damage of OGD/R-treated SH-SY5Y cells. Specifically, EEMG inhibited the release of inflammatory factors and reversed serum metabolic profile disorders of MCAO/R rats. Network pharmacology analysis showed that the PI3K-Akt and NF-κB signaling pathways play a role in the neuroprotective effects of EEMG against ischemic injury and in mitigating the inflammatory response. Consistent with our expectations, EEMG activated PI3K-AKT and suppressed NF-kB signaling pathways both in MCAO/R-operated rats and OGD/R-treated BV2 cells. The results showed that knockdown of TLR4 abolished the EEMG-mediated inhibition on neuroinflammation in OGD/R-treated BV2 cells. After treating BV2 cells with the TLR4 agonist neoseptin 3, EEMG showed a trend toward inhibiting neuroinflammation, though the effect was not statistically significant. Additionally, EEMG was found to improve liver injury caused by cerebral ischemia-reperfusion, which is associated with NF-κB signaling pathway in this study. Collectively, this study demonstrated that EEMG attenuates neuroinflammation in cerebral ischemia-reperfusion injury via regulating TLR4-PI3K-Akt-NF-κB pathways.

Wu W ，Xu L ，Mu D ，Wang D ，Tan S ，Liu L ，Li Y ，Chai H ，Hou Y ... -  《-》

Triolein alleviates ischemic stroke brain injury by regulating autophagy and inflammation through the AKT/mTOR signaling pathway.

Triolein, a symmetric triglyceride exhibiting anti-inflammatory and antioxidant properties, has demonstrated potential in mitigating cellular damage. However, its therapeutic efficacy in ischemic stroke (IS) and underlying molecular mechanisms remain elusive. Given the critical roles of inflammation and autophagy in IS pathogenesis, this study aimed to elucidate the effects of triolein in IS and investigate its mechanism of action. We evaluated the impact of triolein using both in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) and in vivo middle cerebral artery occlusion (MCAO/R) models. Neurological function and cerebral infarct volume were assessed 72 h post-reperfusion. Autophagy was quantified through monodansyl cadaverine (MDC) labeling of autophagic vesicles and Western blot analysis of autophagy-related proteins. Microglial activation was visualized via immunofluorescence, while inflammatory cytokine expression was quantified using RT-qPCR. The cytoprotective effect of triolein on OGD/R-induced HT22 cells was evaluated using Cell Counting Kit-8 and lactate dehydrogenase release assays. The involvement of the Protein kinase B/Mechanistic target of rapamycin kinase (AKT/mTOR) pathway was assessed through Western blot analysis. Triolein administration significantly reduced infarct volume, enhanced neurological recovery, and attenuated M1 microglial activation and inflammation in MCAO/R-induced mice. Western blot analysis and MDC labeling revealed that triolein exerted an inhibitory effect on post-IS autophagy. Notably, in the BV2-induced OGD/R model, triolein demonstrated an autophagy-dependent suppression of the inflammatory response. Furthermore, triolein inhibited the activation of the AKT/mTOR signaling pathway, consequently attenuating autophagy and mitigating the post-IS inflammatory response. This study provides novel evidence that triolein exerts neuroprotective effects by inhibiting post-stroke inflammation through an autophagy-dependent mechanism. Moreover, the modulation of the AKT/mTOR signaling pathway appears to be integral to the neuroprotective efficacy of triolein. These findings elucidate potential therapeutic strategies for IS management and warrant further investigation.

Wang C ，Li Y ，Zhang Y ，Smerin D ，Gu L ，Jiang S ，Xiong X ... -  《-》

Silybin attenuates microglia-mediated neuroinflammation via inhibition of STING in experimental subarachnoid hemorrhage.

The primary cause of subarachnoid hemorrhage (SAH) is the rupture of intracranial aneurysms. Over-activation of microglia following SAH is a primary driving force in early brain injury (EBI), which is a leading cause of poor outcomes. Silybin is a flavonoid compound extracted from Silybum marianum, a plant belonging to the Asteraceae family. Its anti-inflammatory and antioxidant properties could provide neuroprotective effects. The mechanism of silybin on EBI after SAH is unclear. To determine the therapeutic effect of silybin on SAH and its underlying mechanisms. We used a prechiasmatic autologous arterial blood injection in vivo and hemoglobin in vitro to establish experimental SAH model. Dexamethasone was used as a positive control drug. We evaluated the neuroprotective effect of silybin on the in vivo SAH model by neurological function scores, rotarod test, and open field test, and explored the protective effect of silybin on neuroinflammation and apoptosis after SAH by quantitative polymerase chain reaction (qPCR), western blot (WB), Immunofluorescence (IF) and TUNEL staining. IF staining of CD86 and CD206 was used to assess microglial phenotype polarization. Then we used WB and IF labeling of STING to explore the effect of silybin on the STING pathway after SAH, and used a combination of transcriptomics and non-targeted metabolomics to study the potential mechanism of silybin in detail, and verified the essential genes by qPCR. We also extracted cerebrospinal fluid from SAH patients and detected the expression level of STING in cerebrospinal fluid by enzyme-linked immunosorbent assay (ELISA) to clarify the association between STING and neural function. Results showed that silybin ameliorated neuronal damage and improved short-term neurological function, and reduced inflammatory damage and neuronal apoptosis in SAH mice. Silybin inhibited the expression levels of TNF-α, IL-1β and NLRP3, and promoted the expression levels of CD206, Arg1 and IL-10. Notably, Silybin promoted M2 microglia polarization. Further studies found that silybin reduced the mRNA and protein levels of the stimulator of interferon genes (STING) in microglia. And the use of a specific activator of STING (CMA) disrupted the protective effect of silybin. A total of 358 differential expression genes were identified using transcriptomics, and 150 different metabolites abundance were identified using metabolomic screening. Analysis of the effects of STING on transcriptomics and metabolomics revealed that STING might impact metabolic pathways, including linoleic acid metabolism. The qPCR results confirmed the decreased expression of essential proteins involved in the pathway. Finally, we found that increased STING expression in the cerebrospinal fluid of SAH patients was associated with decreased neurological function scores and poor prognosis. Silybin had a therapeutic effect on SAH. The underlying mechanism involves linoleic acid metabolism, which is associated with the differential genes and metabolites detected in the study. This study presented a pharmacological rationale for using silybin to treat SAH.

Cui Y ，Zhi SM ，Ding PF ，Zhu T ，Chen XX ，Liu XZ ，Sheng B ，Li XJ ，Wang J ，Zhang JT ，Xu MX ，Jiang YX ，Hang CH ，Li W ... -  《-》

Danhong injection modulates microglial polarization and neuroinflammation via the JUNB/NF-κB pathway in ischemic stroke.

Ischemic stroke (IS) is a leading cause of death and disability in China. Danhong Injection (DHI) is a traditional Chinese medicine preparation made from Salvia miltiorrhiza var. miltiorrhiza and Carthamus tinctoriusL. It is used for treating stroke in China with proven safety and efficacy. Microglia M1/M2 polarization is a key factor in IS inflammatory response. However, the key transcription factors that regulate microglia polarization are unknown. It is also not clear how DHI exerts its mechanism in the treatment of IS. This research aimed to investigate the effect of DHI on microglial polarization and neuroinflammation associated with IS and to elucidate the underlying mechanisms, with an emphasis on the JUNB/NF-κB signaling pathway. An oxygen-glucose deprivation (OGD) damage cell model and a permanent middle cerebral artery occlusion (pMCAO) model in C57BL/6 mice were employed. Neurological deficits, cerebral infarct volume, and microglial activation were assessed. Non-targeted metabolomics analysis with UHPLC-QE-MS and molecular biology methods, including RT-qPCR and Western blot, were applied to investigate the mechanisms. In vivo, DHI decreased inflammation, reduced brain damage, and enhanced neurological function. DHI also ameliorated microglial activation and OGD-induced apoptosis in vitro. Metabolomics analysis identified significant metabolic changes, particularly in amino acid metabolism. Additionally, DHI treatment decreased the upregulated mRNA levels of ASS1 and ASL after OGD, indicating an influence on the arginine biosynthesis pathway, which is crucial for microglial function. DHI modulated the M1 to M2 phenotypes of microglial polarization and regulated microglial polarization through the JUNB/NF-κB signaling pathway. This was confirmed by JUNB silencing experiments. DHI exhibits neuroprotective effects via suppressing ASS1 through the JUNB/NF-κB pathway, promoting the M2 state of microglia, and lowering the expression of inflammatory cytokines. This research unveils the potential therapeutic target of JUNB for IS treatment and sheds light on the novel intervention mechanism of DHI in microglial cells.

Xie M ，Huang H ，Lu Y ，Chen L ，Wang S ，Xian M ... -  《-》