RNA sequencing reveals the potential mechanism of exercise preconditioning for cerebral ischemia reperfusion injury in rats.

INTRODUCTION: Cerebral ischemia reperfusion injury (CIRI) often leads to deleterious complications after stroke patients receive reperfusion therapy. Exercise preconditioning (EP) has been reported to facilitate brain function recovery. We aim to explore the specific mechanism of EP in CIRI. METHODS: Sprague-Dawley rats were randomized into Sham, middle cerebral artery occlusion (MCAO), and EP groups (n = 11). The rats in the EP group received adaptive training for 3 days (10 m/min, 20 min/day, with a 0° incline) and formal training for 3 weeks (6 days/week, 25 m/min, 30 min/day, with a 0° incline). Then, rats underwent MCAO surgery to establish CIRI models. After 48 h, neurological deficits and cerebral infarction of the rats were measured. Neuronal death and apoptosis in the cerebral cortices were detected. Furthermore, RNA sequencing was conducted to investigate the specific mechanism of EP on CIRI, and qPCR and Western blotting were further applied to confirm RNA sequencing results. RESULTS: EP improved neurological deficit scores and reduced cerebral infarction in MCAO rats. Additionally, pre-ischemic exercise also alleviated neuronal death and apoptosis of the cerebral cortices in MCAO rats. Importantly, 17 differentially expressed genes (DEGs) were identified through RNA sequencing, and these DEGs were mainly enriched in the HIF-1 pathway, cellular senescence, proteoglycans in cancer, and so on. qPCR and Western blotting further confirmed that EP could suppress TIMP1, SOCS3, ANGPTL4, CDO1, and SERPINE1 expressions in MCAO rats. CONCLUSION: EP can improve CIRI in vivo, the mechanism may relate to TIMP1 expression and HIF-1 pathway, which provided novel targets for CIRI treatment.

Promotion of mature angiogenesis in ischemic stroke by Taohong Siwu decoction through glycolysis activation.

Backgrounds: Mature angiogenesis plays a critical role in improving cerebral ischemia-reperfusion injury (CIRI). Glycolysis serves as the primary energy source for brain microvascular endothelial cells (BMECs), whereas other vascular cells rely on aerobic respiration. Therefore, intercellular variations in energy metabolism could influence mature angiogenesis. Taohong Siwu Decoction (THSWD) has demonstrated efficacy in treating ischemic stroke (IS), yet its potential to promote mature angiogenesis through glycolysis activation remains unclear. Methods: In this study, we established a middle cerebral artery occlusion/reperfusion (MCAO/R) model in vivo and an oxygen-glucose deprivation/reoxygenation (OGD/R) model in vitro. We assessed neuroprotective effects using neurobehavioral scoring, 2,3,5-triphenyltetrazolium chloride (TTC) staining, Hematoxylin-eosin (HE) staining, and Nissl staining in MCAO/R rats. Additionally, we evaluated mature angiogenesis and glycolysis levels through immunofluorescence, immunohistochemistry, and glycolysis assays. Finally, we investigated THSWD's mechanism in linking glycolysis to mature angiogenesis in OGD/R-induced BMECs. Results: In vivo experiments demonstrated that THSWD effectively mitigated cerebral damage and restored neurological function in MCAO/R rats. THSWD significantly enhanced CD31, Ang1, PDGFB, and PDGFR-ß expression levels, likely associated with improved glucose, pyruvate, and ATP levels, along with reduced lactate and lactate/pyruvate ratios. In vitro findings suggested that THSWD may boost the expression of mature angiogenesis factors (VEGFA, Ang1, and PDGFB) by activating glycolysis, increasing glucose uptake and augmenting lactate, pyruvate, and ATP content, thus accelerating mature angiogenesis. Conclusion: THSWD could alleviate CIRI by activating the glycolysis pathway to promote mature angiogenesis. Targeting the glycolysis-mediated mature angiogenesis alongside THSWD therapy holds promise for IS treatment.

Glycyrrhetinic acid triggers a protective autophagy by inhibiting the JAK2/STAT3 pathway in cerebral ischemia/reperfusion injury.

Cerebral ischemia/reperfusion injury (CIRI) is a common feature of ischemic stroke leading to a poor prognosis. Effective treatments targeting I/R injury are still insufficient. The study aimed to investigate the mechanisms, by which glycyrrhizic acid (18ß-GA) in ameliorates CIRI. Our results showed that 18ß-GA significantly decreased the infarct volume, neurological deficit scores, and pathological changes in the brain tissue of rats after middle cerebral artery occlusion. Western blotting showed that 18ß-GA inhibited the expression levels of phosphorylated JAK2 and phosphorylated STAT3. Meanwhile, 18ß-GA increased LC3-II protein levels in a reperfusion duration-dependent manner, which was accompanied by an increase in the Bcl-2/Bax ratio. Inhibition of 18ß-GA-induced autophagy by 3-methyladenine (3-MA) enhanced apoptotic cell death. In addition, 18ß-GA inhibited the JAK2/STAT3 pathway, which was largely activated in response to oxygen-glucose deprivation/reoxygenation. However, the JAK2/STAT3 activator colivelin TFA abolished the inhibitory effect of 18ß-GA, suppressed autophagy, and significantly decreased the Bcl-2/Bax ratio. Taken together, these findings suggested that 18ß-GA pretreatment ameliorated CIRI partly by triggering a protective autophagy via the JAK2/STAT3 pathway. Therefore might be a potential drug candidate for treating ischemic stroke.

[Exogenous leptin improves cerebral ischemia-reperfusion-induced glutamate excitotoxic injury in mice by up-regulating GLT-1 and GLAST expression in astrocytes].

OBJECTIVE: To investigate the protective effect of exogenous leptin against focal cerebral ischemia-reperfusion (I/R) injury in mice and explore the underlying mechanism. METHODS: A total of 100 C57BL/6 mice were randomly divided into 5 groups, including a sham-operated group, cerebral I/R model group, and 3 leptin treatment groups with intraperitoneal injections of 0.5, 1.0 or 2.0 leptin immediately after occlusion of the internal carotid artery. At 24 h after reperfusion, neurological function scores of the mice were assessed, and TTC staining was used to determine the area of cerebral infarction. The pathological changes in the cortical brain tissue of the mice were observed using HE staining, and degenerative damage of the cortical neurons were assessed with Fluoro-Jade C staining. The expression of glial fibrillary acidic protein in cortical brain tissues was detected using immunohistochemistry and Western blotting. In another 45 C57BL/6 mice with sham operation, I/R modeling, or leptin (1 mg/kg) treatment, glutamic acid in the cortical brain tissue was detected using glutamate assay, and cortical glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) protein expressions were detected using immunohistochemistry. RESULTS: Compared with the I/R model mice, the leptin-treated mice had significantly lower neurological deficit scores, smaller cerebral infarct area, milder pathologies in the cortical brain tissue, and lessened cortical neuronal damage with normal morphology and less excessive proliferation of the astrocytes. Leptin treatment significantly up-regulated the expressions of GLT-1 and GLAST and lowered the content of glutamic acid in the brain tissue of the I/R mice. CONCLUSION: Exogenous leptin has obvious neuroprotective effect against cerebral I/R injury in mice, mediated probably by controlling excessive astrocyte proliferation and up-regulating cortical GLT-1 and GLAST expressions to reduce glutamate-mediated excitotoxic injury of the astrocytes.

The Effects and Mechanisms of AM1241 in Alleviating Cerebral Ischemia-Reperfusion Injury.

OBJECTIVE: Research has shown that cerebral ischemia-reperfusion injury (CIRI) involves a series of physiological and pathological mechanisms, including inflammation, oxidative stress, and cell apoptosis. The cannabinoid receptor 2 agonist AM1241 has been found to have anti-inflammatory and anti-oxidative stress effects. However, it is unclear whether AM1241 has a protective effect against brain ischemia-reperfusion injury, and its underlying mechanisms are not yet known. METHODS: In this study, we investigated the anti-inflammatory, anti-oxidative stress, and anti-apoptotic effects of AM1241 and its mechanisms in BV2 cells stimulated with H2O2 and in a C57BL/6 mouse model of CIRI in vitro and in vivo, respectively. RESULTS: In vitro, AM1241 significantly inhibited the release of pro-inflammatory cytokines TNF-α and IL-6, reactive oxygen species (ROS), and the increase in Toll-like receptor 4/myeloid differentiation protein 2 (MD2/TLR4) complex induced by H2O2. Under H2O2 stimulation, MD2 overexpression resulted in increased levels of MD2/TLR4 complex, TNF-α, IL-6, NOX2, BAX, and Cleaved-Caspase3 (C-Caspase3), as well as the activation of the MAPK pathway and NF-κB, which were reversed by AM1241. In addition, molecular docking experiments showed that AM1241 directly interacted with MD2. Surface Plasmon Resonance (SPR) experiments further confirmed the binding of AM1241 to MD2. In vivo, AM1241 significantly attenuated neurofunctional impairment, brain edema, increased infarct volume, oxidative stress levels, and neuronal apoptosis in CIRI mice overexpressing MD2. CONCLUSION: Our study demonstrates for the first time that AM1241 alleviates mouse CIRI by inhibiting the MD2/TLR4 complex, exerting anti-inflammatory, anti-oxidative stress and anti-apoptotic effects.

Research progress of propofol in alleviating cerebral ischemia/reperfusion injury.

Ischemic stroke is a leading cause of adult disability and death worldwide. The primary treatment for cerebral ischemia patients is to restore blood supply to the ischemic region as quickly as possible. However, in most cases, more severe tissue damage occurs, which is known as cerebral ischemia/reperfusion (I/R) injury. The pathological mechanisms of brain I/R injury include mitochondrial dysfunction, oxidative stress, excitotoxicity, calcium overload, neuroinflammation, programmed cell death and others. Propofol (2,6-diisopropylphenol), a short-acting intravenous anesthetic, possesses not only sedative and hypnotic effects but also immunomodulatory and neuroprotective effects. Numerous studies have reported the protective properties of propofol during brain I/R injury. In this review, we summarize the potential protective mechanisms of propofol to provide insights for its better clinical application in alleviating cerebral I/R injury.

The neuroprotective effect of Chinese herbal medicine for cerebral ischemia reperfusion injury through regulating mitophagy.

The incidence of ischemic stroke has been increasing annually with an unfavorable prognosis. Cerebral ischemia reperfusion injury can exacerbate nerve damage. Effective mitochondrial quality control including mitochondrial fission, fusion and autophagy, is crucial for maintaining cellular homeostasis. Several studies have revealed the critical role of mitophagy in Cerebral ischemia reperfusion injury. Cerebral ischemia and hypoxia induce mitophagy, and mitophagy exhibits positive and negative effects in cerebral ischemia reperfusion injury. Studies have shown that Chinese herbal medicine can alleviate Cerebral ischemia reperfusion injury and serve as a neuroprotective agent by inhibiting or promoting mitophagy-mediated pathways. This review focuses on the mitochondrial dynamics and mitophagy-related pathways, as well as the role of mitophagy in ischemia reperfusion injury. Additionally, it discusses the therapeutic potential and benefits of Chinese herbal monomers and decoctions in the treatment of ischemic stroke.

Urine Proteomic Signatures of Mild Hypothermia Treatment in Cerebral Ischemia-Reperfusion Injury in Rats.

Mild hypothermia (MH) is an effective measure to alleviate cerebral ischemia-reperfusion (I/R) injury. However, the underlying biological mechanisms remain unclear. This study set out to investigate dynamic changes in urinary proteome due to MH in rats with cerebral I/R injury and explore the neuroprotective mechanisms of MH. A Pulsinelli's four-vessel occlusion (4-VO) rat model was used to mimic global cerebral I/R injury. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to profile the urinary proteome of rats with/without MH (32 °C) treatment after I/R injury. Representative differentially expressed proteins (DEPs) associated with MH were validated by western blotting in hippocampus. A total of 597 urinary proteins were identified, among which 119 demonstrated significant changes associated with MH. Gene Ontology (GO) annotation of the DEPs revealed that MH significantly enriched in endopeptidase activity, inflammatory response, aging, response to oxidative stress and reactive oxygen species, blood coagulation, and cell adhesion. Notably, changes in 12 DEPs were significantly reversed by MH treatment. Among them, 8 differential urinary proteins were previously reported to be closely associated with brain disease, including NP, FZD1, B2M, EPCR, ATRN, MB, CA1and VPS4A. Two representative proteins (FZD1, B2M) were further validated by western blotting in the hippocampus and the results were shown to be consistent with urinary proteomic analysis. Overall, this study strengthens the idea that urinary proteome can sensitively reflect pathophysiological changes in the brain, and appears to be the first study to explore the neuroprotective effects of MH by urinary proteomic analysis. FZD1 and B2M may be involved in the most fundamental molecular biological mechanisms of MH neuroprotection.

Crosstalk between ferroptosis and necroptosis in cerebral ischemia/reperfusion injury and Naotaifang formula exerts neuroprotective effect via HSP90-GCN2-ATF4 pathway.

BACKGROUND: Cerebral ischemia/reperfusion injury (CIRI) is a sequence of pathophysiological processes after blood recanalization in the patients with ischemic stroke, and has become the hinder for the rehabilitation. Naotaifang formula (NTF) has exhibited the clinical effectiveness for this disease. However, its action effects and molecular mechanisms against CIRI are not fully elucidated. PURPOSE: The research was to clarify the crosstalk between ferroptosis and necroptosis in CIRI, and uncover the mechanism underlying the neuroprotection of NTF. METHODS: This study established MCAO/R rat models with various reperfusion times. Western blot, transmission electron microscope, laser speckle imaging, immunofluorescence, immunohistochemistry and pathological staining were conducted to detect and analyze the obtained results. Subsequently, various NTF doses were used to intervene in MCAO/R rats, and biology experiments, such as western blot, Evans blue, immunofluorescence and immunohistochemistry, were used to analyze the efficacy of NTF doses. The effect of NTF was further clarified through in vitro experiments. Eventually, HT22 cells that suffered OGD/R were subjected to pre-treatment with plasmids overexpressing HSP90, MLKL, and GPX4 to indicate the interaction among ferroptosis and necroptosis. RESULTS: There was a gradual increase in the Zea Longa score and cerebral infarction volume following CIRI with prolonged reperfusion. Furthermore, the expression of factors associated with pro-ferroptosis and pro-necroptosis was upregulated in the cortex and hippocampus. NTF alleviated ferroptosis and necroptosis in a dose-dependent manner, downregulated HSP90 levels, reduced blood-brain barrier permeability, and thus protected nerve cells from CIRI. The results in vitro research aligned with those of the in vivo research. HSP90 and MLKL overexpression promoted necroptosis and ferroptosis while activating the GCN2-ATF4 pathway. GPX4 overexpression had no effect on necroptosis or the associated signaling pathway. The administration of NTF alone, as well as its combination with the overexpression of HSP90, MLKL, or GPX4 plasmids, decreased the expression levels of factors associated with pro-ferroptosis and pro-necroptosis and reduced the protein levels of the HSP90-GCN2-ATF4 pathway. Moreover, the regulatory effects of the NTF alone group on GSH, ferrous iron, and GCN2 were more significant compared with those of the HSP90 overexpression combination group. CONCLUSION: Ferroptosis and necroptosis were gradually aggravated following CIRI with prolonged reperfusion. MLKL overexpression may promote ferroptosis and necroptosis, while GPX4 overexpression may have little effect on necroptosis. HSP90 overexpression accelerated both forms of cell death via the HSP90-GCN2-ATF4 pathway. NTF alleviated ferroptosis and necroptosis to attenuate CIRI by regulating the HSP90-GCN2-ATF4 pathway. Our research provided evidence for the potential of drug development by targeting HSP90, MLKL, and GPX4 to protect against ischemic stroke.

Precisely targeted drug delivery by mesenchymal stem cells-based biomimetic liposomes to cerebral ischemia-reperfusion injured hemisphere.

The precise and targeted delivery of therapeutic agents to the lesion sites remains a major challenge in treating brain diseases represented by ischemic stroke. Herein, we modified liposomes with mesenchymal stem cells (MSC) membrane to construct biomimetic liposomes, termed MSCsome. MSCsome (115.99 ± 4.03 nm) exhibited concentrated accumulation in the cerebral infarcted hemisphere of mice with cerebral ischemia-reperfusion injury, while showing uniform distribution in the two cerebral hemispheres of normal mice. Moreover, MSCsome exhibited high colocalization with damaged nerve cells in the infarcted hemisphere, highlighting its advantageous precise targeting capabilities over liposomes at both the tissue and cellular levels. Leveraging its superior targeting properties, MSCsome effectively delivered Dl-3-n-butylphthalide (NBP) to the injured hemisphere, making a single-dose (15 mg/kg) intravenous injection of NBP-encapsulated MSCsome facilitate the recovery of motor functions in model mice by improving the damaged microenvironment and suppressing neuroinflammation. This study underscores that the modification of the MSC membrane notably enhances the capacity of liposomes for precisely targeting the injured hemisphere, which is particularly crucial in treating cerebral ischemia-reperfusion injury.

Neuroprotective effect of autologous mitochondrial transplantation against global ischemia/reperfusion injury in a rat model of cardiac arrest.

BACKGROUND: Mitochondria have emerged as a promising target for ischemic disease. A previous study reported the application of mitochondrial transplantation in focal cerebral ischemia/reperfusion injury, but it is unclear whether exogenous mitochondrial transplantation could be a therapeutic strategy for global ischemia/reperfusion injury induced by cardiac arrest. METHODS: We hypothesized that transplantation of autologous mitochondria would rescue hippocampal cells and alleviate neurological impairment after cardiac arrest. In this study, we employed a rat cardiac arrest-global cerebral ischemia injury model (CA-GCII) and transplanted isolated mitochondria intravenously. Behavior test was applied to assess neurological deficit. Apoptosis and mitochondria permeability transition pore opening in hippocampus was determined using immunoblotting and swelling assay, respectively. RESULTS: Transplanted mitochondria distributed throughout hippocampal cells and reduced oxidative stress. An improved neurological outcome was observed in rats receiving autologous mitochondria. In the hippocampus, mitophagy was enhanced while cell apoptosis was induced by ischemia/reperfusion insult was downregulated by mitochondrial transplantation. Mitochondrial permeability transition pore (MPTP) opening in surviving hippocampal cells was also suppressed. CONCLUSIONS: These results indicated that transplantation of autologous mitochondria rescued hippocampal cells from ischemia/reperfusion injury and ameliorated neurological impairment caused by cardiac arrest.

Targeting TNF-α: The therapeutic potential of certolizumab pegol in the early period of cerebral ischemia reperfusion injury in mice.

The neuroinflammatory response triggered by cerebral ischemia-reperfusion injury (CIRI) is characterized by the upsurge of pro-inflammatory cytokines, including TNF-α, IL-1ß, and IL-6, which promote leukocyte infiltration and subsequent accumulation in the ischemic zone. This accumulation further intensifies inflammation and aggravates ischemic damage. Certolizumab pegol (CZP), a monoclonal antibody targeting TNF-α, is widely used in treating various inflammatory diseases. This study explored the therapeutic potential of CZP in a mouse model of CIRI, induced by middle cerebral artery occlusion (MCAO), focusing on its influence on the microglial inflammatory response. In vitro analyses revealed that CZP markedly inhibits TNF-α-stimulated inflammation in primary microglia with an EC50 of 1.743 ng/mL. In vivo, MCAO mice treated with CZP (10 µg/mouse, i.p.) for 3 days showed reduced infarct volume, partially improved neurological function, and diminished blood-brain barrierdisruption. Additionally, CZP treatment curtailed microglial activation and the release of pro-inflammatory mediators in the early stages of stroke. It also favorably modulated microglial M1/M2 polarization, rebalanced Th17/Treg cells dynamics, and inhibited Caspase-8-mediated GSDMD cleavage, preventing microglial pyroptosis. Collectively, this study described that the treatment with CZP reversed damaging process caused by CIRI, offering a promising therapeutic strategy for the treatment of ischemic stroke.

Ferritinophagy and Ferroptosis in Cerebral Ischemia Reperfusion Injury.

Cerebral ischemia-reperfusion injury (CIRI) is the second leading cause of death worldwide, posing a huge risk to human life and health. Therefore, investigating the pathogenesis underlying CIRI and developing effective treatments are essential. Ferroptosis is an iron-dependent mode of cell death, which is caused by disorders in iron metabolism and lipid peroxidation. Previous studies demonstrated that ferroptosis is also a form of autophagic cell death, and nuclear receptor coactivator 4(NCOA4) mediated ferritinophagy was found to regulate ferroptosis by interfering with iron metabolism. Ferritinophagy and ferroptosis are important pathogenic mechanisms in CIRI. This review mainly summarizes the link and regulation between ferritinophagy and ferroptosis and further discusses their mechanisms in CIRI. In addition, the potential treatment methods targeting ferritinophagy and ferroptosis for CIRI are presented, providing new ideas for the prevention and treatment of clinical CIRI in the future.

SIRT3 facilitates mitochondrial structural repair and functional recovery in rats after ischemic stroke by promoting OPA1 expression and activity.

BACKGROUND: Optical atrophy 1 (OPA1), a protein accountable for mitochondrial fusion, facilitates the restoration of mitochondrial structure and function following cerebral ischemia/reperfusion (I/R) injury. The OPA1-conferred mitochondrial protection involves its expression and activity, which can be improved by SIRT3 in non-cerebral ischemia. Nevertheless, it remains obscure whether SIRT3 enhances the expression and activity of OPA1 after cerebral I/R injury. METHODS: Mature male Sprague Dawley rats were intracranially injected with adeno-associated viral-Sirtuin-3(AAV-SIRT3) and AAV-sh_OPA1, followed by a 90-min temporary blockage of the middle cerebral artery and subsequent restoration of blood flow. Cultured cortical neurons of rats were transfected with LV-SIRT3 or LV-sh_OPA1 before a 2-h oxygen-glucose deprivation and reoxygenation. The rats and neurons were subsequently treated with a selective OPA1 activity inhibitor (MYLS22). The interaction between SIRT3 and OPA1 was assessed by molecular dynamics simulation technology and co-immunoprecipitation. The expression, function, and specific protective mechanism of SIRT3 were examined by various analyses. RESULTS: SIRT3 interacted with OPA1 in the rat cerebral cortex before and after cerebral I/R. After cerebral I/R damage, SIRT3 upregulation increased the OPA1 expression, which enhanced deacetylation and OPA1 activity, thus alleviating cerebral infarct volume, neuronal apoptosis, oxidative pressure, and impairment in mitochondrial energy production; SIRT3 upregulation also improved neuromotor performance, repaired mitochondrial ultrastructure and membrane composition, and promoted the mitochondrial biogenesis. These neuroprotective effects were partly reversed by OPA1 expression interference and OPA1 activity inhibitor MYLS22. CONCLUSION: In rats, SIRT3 enhances the expression and activity of OPA1, facilitating the repair of mitochondrial structure and functional recovery following cerebral I/R injury. These findings highlight that regulating SIRT3 may be a promising therapeutic strategy for ischemic stroke.

Therapeutic effect and mechanisms of traditional Chinese medicine compound (Qilong capsule) in the treatment of ischemic stroke.

Background Qilong capsule (QLC) is a well-known traditional Chinese medicine compound extensively used in clinical practice. It has been approved by the China's FDA for the treatment of ischemic stroke (IS). In our clinical trial involving QLC (ClinicalTrials.gov identifier: NCT03174535), we observed the potential of QLC to improve neurological function in IS patients at the 24th week, while ensuring their safety. However, the effectiveness of QLC beyond the initial 12-week period remains uncertain, and the precise mechanisms underlying its action in IS have not been fully elucidated. Purpose In order to further explore the clinical efficacy of QLC in treating IS beyond the initial 12-week period and systematically elucidate its underlying mechanisms. Study Design This study employed an interdisciplinary integration strategy that combines post hoc analysis of clinical trials, transcriptome sequencing, integrated bioinformatics analysis, and animal experiments. Methods In this study, we conducted a post-hoc analysis with 2302 participants to evaluate the effectiveness of QLC at the 12th week. The primary outcome was the proportion of patients achieving functional independence at the 12th week, defined as a score of 0-2 on the modified Rankin Scale (mRS), which ranges from 0 (no symptoms) to 6 (death). Subsequently, we employed RNA sequencing (RNA-Seq) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) techniques in the QLC trial to investigate the potential molecular mechanisms underlying the therapeutic effect of QLC in IS. Simultaneously, we utilized integrated bioinformatics analyses driven by external multi-source data and algorithms to further supplement the exploration and validation of QLC's therapeutic mechanism in treating IS. This encompassed network pharmacology analysis and analyses at the mRNA, cellular, and pathway levels focusing on core targets. Additionally, we developed a disease risk prediction model using machine learning. By identifying differentially expressed core genes (DECGs) between the normal and IS groups, we quantitatively predicted IS occurrence. Furthermore, to assess its protective effects and determine the key regulated pathway, we conducted experiments using a middle cerebral artery occlusion and reperfusion (MACO/R) rat model. Results Our findings demonstrated that the combination of QLC and conventional treatment (CT) significantly improved the proportion of patients achieving functional independence (mRS score 0-2) at the 12th week compared to CT alone (n = 2,302, 88.65 % vs 87.33 %, p = 0.3337; n = 600, 91.33 % vs 84.67 %, p = 0.0165). Transcriptome data revealed that the potential underlying mechanism of QLC for IS is related to the regulation of the NF-κB inflammatory pathway. The RT-qPCR results demonstrated that the regulatory trends of key genes, such as MD-2, were consistent with those observed in the RNA-Seq analysis. Integrated bioinformatics analysis elucidated that QLC regulates the NF-κB signaling pathway by identifying core targets, and machine learning was utilized to forecast the risk of IS onset. The MACO/R rat model experiment confirmed that QLC exerts its anti-CIRI effects by inhibiting the MD-2/TLR-4/NF-κB signaling axis. Conclusion: Our interdisciplinary integration study has demonstrated that the combination of QLC with CT exhibits significant superiority over CT alone in improving functional independence in patients at the 12th week. The potential mechanism underlying QLC's therapeutic effect in IS involves the inhibition of the MD-2/TLR4/NF-κB inflammatory signaling pathway, thereby attenuating cerebral ischemia/reperfusion inflammatory injury and facilitating neurofunctional recovery. The novelty and innovative potential of this study primarily lie in the novel finding that QLC significantly enhances the proportion of patients achieving functional independence (mRS score 0-2) at the 12th week. Furthermore, employing a "multilevel-multimethod" integrated research approach, we elucidated the potential mechanism underlying QLC's therapeutic effect in IS.

TET1 mediated m5C modification of RelB aggravates cerebral ischemia/reperfusion-induced neuroinflammation through regulating microglia polarization.

Microglia mediated neuroinflammation is one of the major contributors to brain damage in cerebral ischemia reperfusion injury (CI/RI). Recently, RNA modification was found to contribute to the regulation of microglia polarization and the subsequent development of cerebral I/R neuroinflammation. Herein, we investigated the effect and mechanism of m5C RNA modification in the microglia induced CI/RI neuroinflammation. We found that the m5C RNA modification levels decreased in the primary microglia isolated from a mouse model of intraluminal middle cerebral artery occlusion/reperfusion (MCAO/R) and the BV2 microglial cells subjected to oxygen-glucose deprivation and reoxygenation (OGD/R), and this change was accompanied by an increase in the M1/M2 polarization ratio. Furthermore, the expression of m5C demethylase TET1 in microglia increased, which promoted M1 polarization but impeded M2 polarization. Mechanistically, the higher TET1 expression decreased the m5C modification level of RelB and enhanced its mRNA stability, which subsequently increased the M1/M2 polarization ratio. In conclusion, this study provides insight into the role of m5C RNA modification in the pathogenesis of cerebral I/R neuroinflammation and may deepen our understanding on clinical therapy targeting the TET1-RelB axis.

Astrocyte modulation in cerebral ischemia-reperfusion injury: A promising therapeutic strategy.

Cerebral ischemia-reperfusion injury (CIRI) poses significant challenges for drug development due to its complex pathogenesis. Astrocyte involvement in CIRI pathogenesis has led to the development of novel astrocyte-targeting drug strategies. To comprehensively review the current literature, we conducted a thorough analysis from January 2012 to December 2023, identifying 82 drugs aimed at preventing and treating CIRI. These drugs target astrocytes to exert potential benefits in CIRI, and their primary actions include modulation of relevant signaling pathways to inhibit neuroinflammation and oxidative stress, reduce cerebral edema, restore blood-brain barrier integrity, suppress excitotoxicity, and regulate autophagy. Notably, active components from traditional Chinese medicines (TCM) such as Salvia miltiorrhiza, Ginkgo, and Ginseng exhibit these important pharmacological properties and show promise in the treatment of CIRI. This review highlights the potential of astrocyte-targeted drugs to ameliorate CIRI and categorizes them based on their mechanisms of action, underscoring their therapeutic potential in targeting astrocytes.

JR14a: A novel antagonist of C3aR attenuates neuroinflammation in cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury (CIRI), a prevalent stroke-related complication, can lead to severe brain damage. Inflammation is a crucial factor in CIRI pathogenesis, and the complement component 3a receptor (C3aR) could be a key mediator in the post-CIRI inflammatory cascade. In this study, the role of C3aR in CIRI was investigated utilizing a middle cerebral artery occlusion (MCAO) model in C3aR knockout (KO) mice. Magnetic resonance imaging (MRI) and neurofunctional assessments revealed that C3aR KO mice exhibited significantly diminished cerebral infarction and improved neurological impairments. Consequently, the focus shifted to searching for a small molecule antagonist of C3aR. JR14a, a new potent thiophene antagonist of C3aR, was injected intraperitoneally into mice 1-h post-MCAO model implementation. The mass spectrometry (MS) results indicated the ability of JR14a to penetrate the blood-brain barrier. Subsequent TTC staining and neurofunctional assessments revealed the efficacy of JR14a in reducing cerebral infarct volume and neurological impairment following MCAO. In addition, immunofluorescence (IF) and immunohistochemistry (IHC) demonstrated attenuated microglial activation, neutrophil infiltration, and blood-brain barrier disruption by JR14a in the MCAO model. Furthermore, enzyme-linked immunosorbent assay (ELISA) and Western blotting supported the role of JR14a in downregulating the expression levels of C3aR, tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), as well as the phosphorylation of p65. In conclusion, the findings suggested that C3aR could be a potential therapeutic target for CIRI, and JR14a emerged as a promising treatment candidate.

MyD88 Inhibition Attenuates Cerebral Ischemia-reperfusion Injury by Regulating the Inflammatory Response and Reducing Blood-brain Barrier Damage.

Myeloid differentiation primary response gene 88 (MyD88), a downstream molecule directly linked to Toll-like receptor (TLRs) and IL1 receptor, has been implicated in ischemia-reperfusion injury across various organs. However, its role in cerebral ischemia-reperfusion injury (CIRI) remains unclear. Five transient middle cerebral artery occlusion (tMCAO) microarray datasets were obtained from the Gene Expression Omnibus (GEO) database. We screened these datasets for differentially expressed genes (DEGs) using the GSE35338 and GSE58720 datasets and performed weighted gene co-expression network analysis (WGCNA) using the GSE30655, GSE28731, and GSE32529 datasets to identify the core module related to tMCAO. A protein-protein interaction (PPI) network was constructed using the intersecting DEGs and genes in the core module. Finally, we identified Myd88 was the core gene. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) validated that TNFα, IL17, and MyD88 signaling pathways were significantly enriched in tMCAO. Subsequently, we investigated the mechanistic role of MyD88 in the tMCAO model using male C57BL/6 mice. MyD88 expression increased significantly 24 h after reperfusion. After intraperitoneal administration of TJ-M2010-5, a MyD88-specific inhibitor, during reperfusion, the infarction volumes in the mice were ameliorated. TJ-M2010-5 inhibits the activation of microglia and astrocytes. Moreover, it attenuates the upregulation of inflammatory cytokines TNFα, IL17, and MMP9 while preserving the expression level of ZO1 after tMCAO, thereby safeguarding against blood-brain barrier (BBB) disruption. Finally, our findings suggest that MyD88 regulates the IRAK4/IRF5 signaling pathway associated with microglial activation. MyD88 participates in CIRI by regulating the inflammatory response and BBB damage following tMCAO.

Maresin1 alleviates neuroinflammation by inhibiting caspase-3/ GSDME-mediated pyroptosis in mice cerebral ischemia-reperfusion model.

OBJECTIVE: To explore the mechanism of Maresin1 in reducing cerebral ischemia-reperfusion injury. MATERIALS AND METHODS: Male C57BL/6 mice were randomly divided (n = 5 in each group), and focal middle cerebral artery occlusion (MCAO) model was used to simulate cerebral ischemia/reperfusion injury. TTC and the Longa score were used to detect the degree of neurological deficits. Western blot was used to detect the expression levels of GSDME, GSDME-N, caspase-3 and cleaved caspase-3 in cerebral ischemic penumbra tissue, and immunofluorescence was used to detect the expression levels of GSDME-N. The mRNA expression levels of GSDME and pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) were detected by RT-PCR. RESULTS: Compared with sham group, GSDME mRNA levels in MCAO group were significantly increased at 12 h and 24 h after reperfusion, and GSDME and GSDME-N significantly increased at 6-48 h after reperfusion. Compared with sham group, the percentage of infarct size, the Longa score, the mRNA expression levels of IL-1ß, IL-6 and TNF-α, and GSDME, GSDME-N, caspase-3 and cleaved caspase-3 in MCAO group was significantly increased. Then, the percentage of infarct size and the Longa score significantly decreased after MaR1 administration, the mRNA expression levels of IL-1ß and IL-6 downregulated, and GSDME, GSDME-N, caspase-3 and cleaved caspase-3 were also reduced. After administration of Z-DEVD-FMK(ZDF), the expression of caspase-3, cleaved caspase-3 and GSDME-N was decreased, which in MCAO+MaR1+ZDF group was not statistically significant compared with MCAO+ ZDF group. CONCLUSION: Maresin1 alleviates cerebral ischemia/reperfusion injury by inhibiting pyroptosis mediated by caspase-3/GSDME pathway and alleviating neuroinflammation.