Mechanism of Astragalus mongholicus Bunge ameliorating cerebral ischemia-reperfusion injury: Based on network pharmacology analysis and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus mongholicus Bunge (AMB) is a herb with wide application in traditional Chinese medicine, exerting a wealth of pharmacological effects. AMB has been proven to have an evident therapeutic effect on ischemic cerebrovascular diseases, including cerebral ischemia-reperfusion injury (CIRI). However, the specific mechanism underlying AMB in CIRI remains unclear. AIM OF THE STUDY: This study aimed to investigate the potential role of AMB in CIRI through a comprehensive approach of network pharmacology and in vivo experimental research. METHODS: The intersection genes of drugs and diseases were obtained through analysis of the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and Gene Expression Omnibus (GEO) database. The protein-protein interaction (PPI) network was created through the string website. Meanwhile, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was carried out using R studio, and thereafter the key genes were screened. Then, the molecular docking prediction was made between the main active ingredients and target genes, and hub genes with high binding energy were obtained. In addition, molecular dynamic (MD) simulation was used to validate the result of molecular docking. Based on the results of network pharmacology, we used animal experiments to verify the predicted hub genes. First, the rat middle cerebral artery occlusion and reperfusion (MACO/R) model was established and the effective dose of AMB in CIRI was determined by behavioral detection and 2,3,5-Triphenyltetrazolium chloride (TTC) staining. Then the target proteins corresponding to the hub genes were measured by Western blot. Moreover, the level of neuronal death was measured using hematoxylin and eosin (HE) and Nissl staining. RESULTS: Based on the analysis of the TCMSP database and GEO database, a total of 62 intersection target genes of diseases and drugs were obtained. The KEGG enrichment analysis showed that the therapeutic effect of AMB on CIRI might be realized through the advanced glycation endproduct-the receptor of advanced glycation endproduct (AGE-RAGE) signaling pathway in diabetic complications, nuclear factor kappa-B (NF-κB) signaling pathway and other pathways. Molecular docking results showed that the active ingredients of AMB had good binding potential with hub genes that included Prkcb, Ikbkb, Gsk3b, Fos and Rela. Animal experiments showed that AWE (60 g/kg) could alleviate CIRI by regulating the phosphorylation of PKCß, IKKß, GSK3ß, c-Fos and NF-κB p65 proteins. CONCLUSION: AMB exerts multi-target and multi-pathway effects against CIRI, and the underlying mechanism may be related to anti-apoptosis, anti-inflammation, anti-oxidative stress and inhibiting calcium overload.

Multiomics profiling of the therapeutic effect of Dan-deng-tong-nao capsule on cerebral ischemia-reperfusion injury.

BACKGROUND: Stroke is a complex physiological process associated with intestinal flora dysbiosis and metabolic disorders. Dan-deng-tong-nao capsule (DDTN) is a traditional Chinese medicine used clinically to treat cerebral ischemia-reperfusion injury (CIRI) for many years. However, little is known about the effects of DDTN in the treatment of CIRI from the perspective of gut microbiota and metabolites. PURPOSE: This study aimed to investigate the regulatory roles of DDTN in endogenous metabolism and gut microbiota in CIRI rats, thus providing a basis for clinical rational drug use and discovering natural products with potential physiological activities in DDTN for the treatment of CIRI. METHODS: The chemical composition of DDTN in vitro and in vivo was investigated using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLCHRMS), followed by target prediction using reverse molecular docking. Secondly, a biological evaluation of DDTN ameliorating neural damage in CIRI was performed at the whole animal level. Then, an integrated omics approach based on UHPLCHRMS and 16S rRNA sequencing was proposed to reveal the anti-CIRI effect and possible mechanism of DDTN. Finally, exploring the intrinsic link between changes in metabolite profiles, changes in the intestinal flora, and targets of components to reveal DDTN for the treatment of CIRI. RESULTS: A total of 112 chemical components of DDTN were identified in vitro and 10 absorbed constituents in vivo. The efficacy of DDTN in the treatment of CIRI was confirmed by alleviating cerebral infarction and neurological deficits. After the DDTN intervention, 21 and 26 metabolites were significantly altered in plasma and fecal, respectively. Based on the fecal microbiome, a total of 36 genera were enriched among the different groups. Finally, the results of the network integration analysis showed that the 10 potential active ingredients of DDTN could mediate the differential expression of 24 metabolites and 6 gut microbes by targeting 25 target proteins. CONCLUSION: This study was the first to outline the landscapes of metabolites as well as gut microbiota regulated by DDTN in CIRI rats using multi-omics data, and comprehensively revealed the systematic relationships among ingredients, targets, metabolites, and gut microbiota, thus providing new perspectives on the mechanism of DDTN in the treatment of CIRI.

Novel insight into the therapeutical potential of flavonoids from traditional Chinese medicine against cerebral ischemia/reperfusion injury.

Cerebral ischemia/reperfusion injury (CIRI) is a major contributor to poor prognosis of ischemic stroke. Flavonoids are a broad family of plant polyphenols which are abundant in traditional Chinese medicine (TCM) and have beneficial effects on several diseases including ischemic stroke. Accumulating studies have indicated that flavonoids derived from herbal TCM are effective in alleviating CIRI after ischemic stroke in vitro or in vivo, and exhibit favourable therapeutical potential. Herein, we systematically review the classification, metabolic absorption, neuroprotective efficacy, and mechanisms of TCM flavonoids against CIRI. The literature suggest that flavonoids exert potential medicinal functions including suppressing excitotoxicity, Ca2+ overloading, oxidative stress, inflammation, thrombin's cellular toxicity, different types of programmed cell deaths, and protecting the blood-brain barrier, as well as promoting neurogenesis in the recovery stage following ischemic stroke. Furthermore, we identified certain matters that should be taken into account in future research, as well as proposed difficulties and opportunities in transforming TCM-derived flavonoids into medications or functional foods for the treatment or prevention of CIRI. Overall, in this review we aim to provide novel ideas for the identification of new prospective medication candidates for the therapeutic strategy against ischemic stroke.

Moxibustion preconditioning reduces inflammatory response in rats with cerebral ischemia-reperfusion injury by regulating PI3K / AKT / mTOR signaling pathway. / 艾灸预处理通过调控PI3K/AKT/mTORä¿¡å·é€šè·¯å‡è½»è„‘ç¼ºè¡€å†çŒæ³¨æŸä¼¤å¤§é¼ ç‚Žæ€§ååº”.

OBJECTIVES: To observe the effect of moxibustion preconditioning on inflammatory response in rats with cerebral ischemia reperfusion injury (CIRI), so as to explore its mechanisms underlying improving CIRI. METHODS: Seventy-five male SD rats were randomly divided into sham operation, model, moxibustion preconditioning 3 days (Moxi 1), moxibustion preconditioning 5 days (Moxi 2) and moxibustion preconditioning 7 days (Moxi 3) groups, with 15 rats in each group. Moxibustion was applied at "Baihui"(GV20), "Dazhui"(GV14) and "Zusanli"(ST36) for 20 min once a day, totally for 3, 5 or 7 days. Thirty minutes after the last moxibustion treatment, the CIRI model was established by occlusion of the middle cerebral artery. The neurological deficit score was assessed by using Longa's method. The infarct size of the brain assessed after staining with 2% triphenyltetrazolium chloride (TTC). The morphological changes of cortical neurons were observed by HE staining. The contents of inflammatory factors interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), S-100ß protein (S-100ß) and neuron-specific enolase (NSE) were detected by ELISA. The expression of phosphatidylinositol-3-kinase (PI3K), p-PI3K, protein kinase B (AKT) and mammalian target of rapamycin (mTOR) proteins in the ischemic cortex tissues were detected by immunohistochemistry and Western blot. RESULTS: Compared with the sham operation group, the neurological function score and the percentage of cerebral ischemic volume were increased (P<0.01). The contents of serum IL-1ß, TNF-α, S-100ß and NSE were significantly increased (P<0.01), while the protein expressions of PI3K, p-PI3K, AKT and mTOR in the cerebral cortex were significantly decreased (P<0.01) in the model group. Compared with the model group, the neurological function score and the percentage of cerebral ischemic volume were significantly decreased (P<0.01). The contents of serum IL-1ß, TNF-α, S-100ß and NSE were significantly decreased (P<0.01), and the expressions of PI3K, p-PI3K, AKT and mTOR proteins in the cerebral cortex were significantly increased (P<0.01) in three moxibustion groups. Compared with the Moxi 1 and Moxi 2 groups, the above indicators were significantly improved in rats of the Moxi 3 group (P<0.01, P<0.05). CONCLUSIONS: Moxibustion preconditioning can significantly improve the neurological function of rats after ischemia-reperfusion, inhibit serum inflammatory factors IL-1 ß and TNF-α, inhibit brain tissue injury markers S-100ß and NSE, which may be related to the activation of PI3K/AKT/mTOR signaling pathway. The protective effect of moxibustion preconditioning for 7 days on CIRI was better than that of 5 days and 3 days.

Progress of researches on mechanisms of acupuncture underlying improvement of cerebral ischemia-reperfusion injury by regulating calcium overload. / é’ˆåˆºè°ƒæŽ§è„‘ç¼ºè¡€å†çŒæ³¨æŸä¼¤åŽé’™è¶ è½½æœºåˆ¶ç ”ç©¶è¿›å±•.

Ischemic stroke is currently the most common type of stroke, and the key pathological link is cerebral ischemia-reperfusion injury (CIRI), while the key factor leading to apoptosis and necrosis of ischemic nerve cells is calcium overload. Current studies have confirmed that acupuncture therapy has a good modulating effect on calcium homeostasis and can reduce cerebral ischemia-reperfusion induced damage of neuronal cells by inhibiting calcium overload. After reviewing the relevant literature published in the past 15 years, we find that acupuncture plays a role in regulating the pathological mechanism of calcium overload after CIRI by inhibiting the opening of connexin 43 hemichannels, regulating the intracellular free calcium ion concentration, suppressing the expression of calmodulin, and blocking the function of L-type voltage-gated calcium channels, thereby inhibiting calcium overload, regulating calcium homeostasis and antagonizing neuronal damage resulted from cerebral ischemia-reperfusion, which may provide ideas for future research.

Effect of electroacupuncture on global cerebral ischemia-reperfusion injury in rats: A urine proteome analysis.

BACKGROUND: This study aimed to investigate dynamic urinary proteome changes of electroacupuncture (EP) on cerebral ischemia-reperfusion (CI/R) injured rats and to explore the therapeutic biological mechanisms of EP. METHODS: First, changed urinary proteins were found in EP stimulation in healthy rats. Then, we used a CI/R injury rat model induced by Pulsinelli's four-vessel occlusion (4-VO) method to explore the function of EP on urinary proteome in CI/R injury. Urine samples were collected for proteome analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis. RESULTS: In total, 384 proteins were identified, among which 47 proteins (23 upregulated, 24 downregulated) were differentially expressed with 0.6-log FC and p < .05. Gene ontology analysis revealed that the cell redox homeostasis, acute-phase response, response to lipopolysaccharide, and cellular response to glucocorticoid stimulus were significantly enriched. The partially biologically connected differential proteins were found by the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis in the EP group. With the CI/R rat model, 80 proteins (27 upregulated, 53 downregulated) were significantly changed in the CI/R rats compared to the controls. Among these differentially expressed proteins (DEPs), 23 proteins (17 upregulated, six downregulated) showed significant changes after EP treatment (0.6-log FC change, p < .05). The main related biological processes were aging, immune response, acute-phase response, liver regeneration, protein catabolic process, and response to oxidative stress. Many metabolic pathways were enriched by KEGG analysis. CONCLUSION: Our results indicate that the EP could alleviate cerebral damage induced by ischemia-reperfusion through an anti-inflammatory and metabolism regulation mechanism. The urinary proteome might reflect the pathophysiological changes in EP pretreatment in the treatment and prevention of CI/R injury.

Effect of electroacupuncture on neuronal programmed necrosis by regulating RIP1/RIP3/MLKL pathway in rats with cerebral ischemia reperfusion injury. / 电针调节RIP1/RIP3/MLKLé€šè·¯å¯¹è„‘ç¼ºè¡€å†çŒæ³¨æŸä¼¤å¤§é¼ ç¥žç»å ƒç¨‹åºæ€§åæ­»çš„å½±å“.

OBJECTIVES: To investigate the neuroprotective effect of electroacupuncture (EA) at "Quchi"(LI11) and "Zusanli"(ST36) in the rats with cerebral ischemia reperfusion injury and its influence on programmed necrosis of cerebral cortical neurons. METHODS: Sixty male SD rats were randomly divided into sham-operation group, model group, EA group and inhibitor group, with 15 rats in each group. Left middle cerebral artery occlusion model was established using the modified thread embolism method. In the sham-operation group, the carotid artery was exposed and dissociated in each rat. EA was applied to "Quchi"(LI11) and "Zusanli"(ST36) on the right side for 30 min each time, once daily for 7 days in the rats of the EA group. The rats in the inhibitor group were intraperitoneally injected with norstatin-1 (0.6 mg/kg) for consecutive 7 days. The neurological deficit score of rats in each group was observed. HE staining was adopted to detect the degree of pathological damage of the cerebral cortex in the infarction area. Using TUNEL staining, the apoptosis of cortical neurons in the infarction area was determined；the contents of tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-6 were detected by ELISA；the mRNA and protein expression of the receptor interacting protein-1 (RIP1), the receptor interacting protein-3 (RIP3) and the substrate mixed lineage kinase like protein (MLKL) were detected by fluorescence quantitative PCR and Western blot, respectively. RESULTS: In comparison with the sham-operation group, the neurological deficit score in the model group was higher(P<0.01)；HE staining showed that there was the pathological damage in the infarction area；the neuron apoptosis rate, the contents of TNF-α, IL-1ß and IL-6, and the mRNA and protein expressions of RIP1, RIP3 and MLKL increased(P<0.01) in the model group. In the EA group, the neurological deficit score was reduced(P<0.01)；HE staining showed that the pathological damage was ameliorated in the infarction area；the neuron apoptosis rate, the contents of TNF-α, IL-1ß and IL-6, and the mRNA and protein expressions of RIP1, RIP3, MLKL decreased(P<0.05, P<0.01) when compared with those in the model group. CONCLUSIONS: EA can attenuate cerebral ischemia reperfusion injury and display its neuroprotective effect probably through inhibiting programmed necrosis of cerebral cortical neurons in the rats.

Xingnaojing injection alleviates cerebral ischemia/reperfusion injury through regulating endoplasmic reticulum stress in Vivo and in Vitro.

Background: Xingnaojing (XNJ) injection, an extract derived from traditional Chinese medicine, is commonly used to treat ischemic stroke (IS). Previous studies have shown that XNJ has the ability to alleviate apoptosis in cerebral ischemia-reperfusion injury. However, the potential mechanisms have not been clarified. Objective: To identify the neuroprotective effect of XNJ and explore whether XNJ inhibits cell apoptosis associated with endoplasmic reticulum stress (ERS) after IS. Methods: In this study, cultured hippocampal neurons from mouse embryos and Sprague-Dawley rats were assigned randomly to four groups: sham, model, XNJ, and edaravone. The treatment groups were administered 2 h after modelling. Neurological deficit scores and motor performance tests were performed after 24 h of modelling. Additionally, pathomorphology, cell apoptosis and calcium content were evaluated. To ascertain the expression of ERS proteins, western blotting and polymerase chain reaction were employed. Results: The results indicated that XNJ treatment resulted in a notable decrease in infarct volume, apoptosis and missteps compared with the model group. XNJ also exhibited improvements in neurological function, grip strength and motor time. The calcium content significantly reduced in XNJ group. The XNJ administration resulted in a reduction in the levels of proteins associated with ERS including CHOP, GRP78, Bax, caspase-12, caspase-9, and cleaved-caspase-3, but an increase of the Bcl-2/Bax ratio. Furthermore, the downregulation of mRNA expression of CHOP, GRP78, caspase-12, caspase-9, and caspase-3 was confirmed in both cultured neurons and rat model. Conclusion: These findings suggest that XNJ may alleviate apoptosis by modulating the ERS-induced apoptosis pathway, making it a potential novel therapeutic approach for ischemic stroke.

Salvianolic acid A provides neuroprotective effects on cerebral ischemia-reperfusion injury in rats via PKA/CREB/c-Fos signaling pathway.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) is a phenomenon that pathological injury of ischemic brain tissue is further aggravated after the restoration of blood supply. The complex pathological mechanism of CIRI has led to the failure of multiple neuroprotective agents in clinical studies. Salvianolic acid A (SAA) is a neuroprotective extract from Salvia miltiorrhiza Bge., with significant pharmacological activities in the treatment of brain injury. However, the neuroprotective mechanisms of SAA remain unclear. PURPOSE: To explore the potential protective effect of SAA on CIRI and its mechanism, and to provide experimental basis for the research of new drugs for CIRI. STUDY DESIGN: A model of transient middle cerebral artery occlusion (tMCAO) in rats was used to simulate clinical CIRI, and the neuroprotective effect of SAA on tMCAO rats was investigated within 14 days after reperfusion. The improvement effects of SAA on cognitive impairment of tMCAO rats were investigated by behavioral tests from days 7-14. Finally, the neuroprotective mechanism of SAA was investigated on day 14. METHODS: The neuroprotective effects and mechanism of SAA were investigated by behavioral tests, HE and TUNEL staining, RNA sequence (RNA-seq) analysis and Western blot in tMCAO rats. RESULTS: The brain protective effects of SAA were achieved by alleviating cerebral infarction, cerebral edema, cerebral atrophy and nerve injury in tMCAO rats. Meanwhile, SAA could effectively improve the cognitive impairment and pathological damage of hippocampal tissue, and inhibit cell apoptosis in tMCAO rats. Besides, SAA could provide neuroprotective effects by up-regulating the expression of Bcl-2, inhibiting the activation of Caspase 3, and regulating PKA/CREB/c-Fos signaling pathway. CONCLUSION: SAA can significantly improve brain injury and cognitive impairment in CIRI rats, and this neuroprotective effect may be achieved through the anti-apoptotic effect and the regulation of PKA/CREB/c-Fos signaling pathway.

Nuciferine reduces inflammation induced by cerebral ischemia-reperfusion injury through the PI3K/Akt/NF-κB pathway.

BACKGROUND: Cerebral ischemia has the characteristics of high incidence, mortality, and disability, which seriously damages people's health. Cerebral ischemia-reperfusion injury is the key pathological injury of this disease. However, there is a lack of drugs that can reduce cerebral ischemia-reperfusion injury in clinical practice. At present, a few studies have provided some evidence that nuciferine can reduce cerebral ischemia-reperfusion injury, but its specific mechanism of action is still unclear, and further research is still needed. OBJECTIVE: In this study, PC12 cells and SD rats were used to construct OGD/R and MCAO/R models, respectively. Combined with bioinformatics methods and experimental verification methods, the purpose of this study was to conduct a systematic and comprehensive study on the effect and mechanism of nuciferine on reducing inflammation induced by cerebral ischemia-reperfusion injury. RESULTS: Nuciferine can improve the cell viability of PC12 cells induced by OGD/R, reduce apoptosis, and reduce the expression of inflammation-related proteins; it can also improve the cognitive and motor dysfunction of MCAO/R-induced rats by behavioral tests, reduce the area of cerebral infarction, reduce the release of inflammatory factors TNF-α and IL-6 in serum and the expression of inflammation-related proteins in brain tissue. CONCLUSION: Nuciferine can reduce the inflammatory level of cerebral ischemia-reperfusion injury in vivo and in vitro models by acting on the PI3K/Akt/NF-κB signaling pathway, and has the potential to be developed as a drug for the treatment of cerebral ischemia-reperfusion injury.

Electroacupuncture Ameliorates Neuronal Injury by NLRP3/ASC/Caspase-1 Mediated Pyroptosis in Cerebral Ischemia-Reperfusion.

NLRP3/ASC/Caspase-1 mediated pyroptosis is one of the important causes of cerebral ischemia-reperfusion (I/R) injury. Electroacupuncture (EA) is widely used in clinical treatment of ischemic stroke. However, mechanism of EA on ischemic stroke remains unclear. Therefore, on basis of a previous work, this study used middle cerebral artery occlusion (MCAO) 2 h and then reperfusion 7 days in rats to simulate brain I/R process. EA with Bahui (GV20) and Zusanli (ST36) and VX-765 (a specific inhibitor of Caspase-1) was performed. In this study, we found that EA improved cerebral infarct size and neuronal damage, including ultrastructural injury, and ameliorated nitro/oxidative stress in cerebral I/R. Additionally, EA treatment significantly decreased ASC, Caspase-1, GSDMD, and IL-1ß expression and VX-765 treatment significantly decreased NLRP3, Caspase-1, and IL-1ß expression. This proved that EA can regulate NLRP3/ASC/Caspase-1 mediated pyroptosis, improve neuronal injury during cerebral I/R, and provide basic experimental data for clinical treatment.

[Acacetin protects rats from cerebral ischemia-reperfusion injury by regulating TLR4/NLRP3 signaling pathway].

This study aims to investigate the mechanism of acacetin in protecting rats from cerebral ischemia-reperfusion injury via the Toll-like receptor 4(TLR4)/NOD-like receptor protein 3(NLRP3) signaling pathway. Wistar rats were randomized into sham, model, low-and high-dose acacetin, and nimodipine groups, with 10 rats in each group. The rat model of middle cerebral artery occlusion(MCAO) was established with the improved suture method in other groups except the sham group. The neurological deficit score and cerebral infarction volume of each group were evaluated 24 h after modeling. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1ß(IL-1ß), IL-6, tumor necrosis factor-α(TNF-α), malondialdehyde(MDA), supe-roxide dismutase(SOD), and glutathione(GSH). Western blot was employed to determine the expression levels of B-cell lymphonoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and TLR4/NLRP3 signaling pathway-related proteins(TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß) in the rat brain tissue. Hematoxylin-eosin(HE) staining was employed to reveal the histopathological changes in the ischemic area. Compared with the sham group, the modeling of MCAO increased the neurological deficit score and cerebral infarction volume, elevated the IL-1ß, IL-6, TNF-α, and MDA levels and lowered the SOD and GSH levels in the brain tissue(P<0.05). Compared with the MCAO model group, low-and high-dose acacetin and nimodipine decreased the neurological deficit score and cerebral infarction volume, lowered the IL-1ß, IL-6, TNF-α, and MDA levels and elevated the SOD and GSH levels in the brain tissue(P<0.05). Compared with the sham group, the model group showed up-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß and down-regulated protein level of Bcl-2 in the brain tissue(P<0.05). Compared with the MCAO model group, the acacetin and nimodipine groups showed down-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß and up-regulated protein level of Bcl-2 in the brain tissue(P<0.05). In conclusion, acacetin regulates the TLR4/NLRP3 signaling pathway to inhibit neuroinflammatory response and oxidative stress, thus exerting the protective effect on cerebral ischemia-reperfusion injury in rats.

A preliminary investigation of the regulatory effect of acupuncture on hippocampal circHDAC2/miR-3065/SLC30A3 axis in CIRI rats. / å¤§é¼ æµ·é©¬ç»„ç»‡circHDAC2/miR-3065/SLC30A3è½´å‚ä¸Žé’ˆåˆºè°ƒèŠ‚è„‘ç¼ºè¡€å†çŒæ³¨æŸä¼¤çš„åˆæ­¥ç ”ç©¶.

OBJECTIVES: To explore the mechanisms of acupuncture against cerebral ischemia/reperfusion injury (CIRI) through observing the expression of circular RNA HDAC2 (circHDAC2) in the hippocampus of rats. METHODS: SD rats were randomly divided into sham-operation, model and acupuncture groups, with 13 rats in each group. The rat model of CIRI was established by middle cerebral artery occlusion. In the acupuncture group, acupuncture was delivvered at "Dazhui" (GV14), "Shuigou" (GV26) and "Baihui" (GV20), and the needles were retained for 30 min each time and acupuncture was conducted once every 12 h for a total of 7 sessions. Before and after intervention, using modified Garcia scale, the neurological function of the rats were evaluated, and TTC staining was employed to determine the cerebral infarct area. Gene chip technology was used to screen the circRNAs with differential expressions in the ischemic hippocampus, and the circRNAs with co-differential expression (co-DE circRNAs) in the model group/sham-operation group, and the acupuncture group/model group separately. Among those circRNAs, the core circRNAs were screened according to P value, fold change (FC) and gene ontology (GO) analysis；and their expressions in the ischemic hippocampus were determined using quantitative real-time PCR (qPCR). Based on the verification results, a competing endogenous RNA (ceRNA) prediction network was constructed. The expression levels of microRNA (miRNA) and mRNA with high node centrality in the prediction network were validated by qPCR. RESULTS: Before intervention, compared with the sham-operation group, the modified Garcia score of each modeling group decreased (P<0.01). After intervention, the modified Garcia score was reduced and the cerebral infarct area ratio increased (P<0.01) in the model group when compared with the sham-operation group. In the acupuncture group, the modified Garcia score was higher and the cerebral infarct area ratio lower (P<0.01) than those of the model group. The microarray results of gene chip showed that 16 co-DE circRNAs were down-regulated in the model group and up-regulated in the acupuncture group, and 7 co-DE circRNAs up-regulated in the model group and down-regulated in the acupuncture group. The core circHDAC2 and circNTRK2 were screened according to P value, FC and the enrichment number of GO entries. QPCR results showed that, compared with the sham-operation group, the expression of circHDAC2 and circNTRK2 of the ischemic hippocampal tissue was down-regulated in the model group (P<0.01)；and that of circHDAC2 and circNTRK2 up-regulated in the acupuncture group when compared with the model group (P<0.01). The relevant ceRNA regulatory network was constructed for circHDAC2 and the prediction results showed that the regulatory networks contained 12 miRNAs and 31 mRNAs. Results of verifying miRNA with high network node centrality and mRNA relevant with nerve regulation showed that, when compared with the sham-operation group, the expression levels of miR-29a, miR-29b and the solute carrier family 30 member 3 (SLC30A3) mRNA in the ischemic hippocampus were down-regulated (P<0.01)；and those of miR-3065 and mercaptopyruvate sulfurtransferase (MPST) up-regulated (P<0.01) in the model group. Compared with the model group, the expressions of miR-29a, miR-29b and SLC30A3 mRNA in the ischemic hippocampus were up-regulated (P<0.01, P<0.05), while that of miR-3065 down-regulated (P<0.05) in the acupuncture group. CONCLUSIONS: Acupuncture significantly improves the neurological function and reduces the cerebral infarct area in CIRI rats, which may be related to the regulation of hippocampal circHDAC2/miR-3065/SLC30A3 axis.

Electroacupuncture alleviates neurological function via activating the Yap-OPA1 axis and mitochondrial fusion in rats with cerebral ischemia-reperfusion injury. / 电针通过Yesç›¸å ³è›‹ç™½-视神经萎缩蛋白1ä¿¡å·è½´æ¿€æ´»çº¿ç²’ä½“èžåˆåŠŸèƒ½å‡è½»å¤§é¼ è„‘ç¼ºè¡€å†çŒæ³¨æŸä¼¤çš„æœºåˆ¶ç ”ç©¶.

OBJECTIVES: To investigate the mechanism of electroacupuncture (EA) in alleviating cerebral ische-mia injury by activating the Yap-OPA1 signaling axis. METHODS: A total of 48 male SD rats were used in the present study. The focal CIRI model was established by occlusion of the middle cerebral artery and reperfusion (MCAO/R), followed by dividing the CIRI rats into model group, EA group and EA+Ver (Verteporfin, Yap antagonist) group (n=12 in each group). And another 12 normal rats were used as the sham operation group. For rats of the EA group, EA (4 Hz/20 Hz, 0.5 mA) was applied to "Baihui"(GV20) and "Shenting"(GV24) for 20 min, once daily for 7 days. The neurological deficit score (0 to 4 points) was given according to Longa's method. The infarct volume of rats in each group was assessed by TTC method, and the expression levels of Yes associated protein (Yap), Optic atrophy protein 1 (OPA1), mitofusin 1 (Mfn1), mitofusin 2 (Mfn2) proteins and mRNAs in cerebral cortex of infarcted side, as well as Bax (proapoptotic factor) and Bcl-1 (anti-apoptotic protein) proteins were detected by Westernblot, and real-time PCR, and the immunoactivity of Yap and OPA1 was detected by immunofluorescent staining. RESULTS: After modeling, the infarct volume, neurological deficit score and the expression of Bax were significantly increased (P<0.01), while the mRNA and protein expressions of Yap, OPA1, Mfn2, Mfn1, and Bcl-2 were significantly down-regulated in the model group relevant to the sham operation group (P<0.01, P<0.05). Compared with the model group, the neurological deficit score, infarct volume and the expression of Bax were significantly decreased (P<0.01), while the expression levels of Yap, OPA1, Mfn2, Mfn1 proteins and mRNAs and Bcl-2 protein, Yap and OPA1 immunofluorescence intensify were considerably up-regulated in the EA group (P<0.01, P<0.05). Following administration of Ver, the effects of EA in down-regulating the neurological score, infarct volume, and Bax expression and up-regulating the expressions of Yap, OPA1, Mfn1, Mfn2 proteins and mRNAs and Yap and OPA1 immunofluorescence intensify were eliminated. CONCLUSIONS: EA of GV20 and GV24 can improve the neurological function in rats with CIRI, which may be associated with its functions in activating mitochondrial fusion function and up-regulating Yap-OPA1 signaling axis.

Hyperbaric oxygen improves cerebral ischemia-reperfusion injury in rats via inhibition of ferroptosis.

BACKGROUND: Our previous study found that hyperbaric oxygen (HBO) attenuated cognitive impairment in mice induced by cerebral ischemia-reperfusion injury (CIRI). However, its mechanism of action is not fully understood. In this study, we aimed to establish a rat model of cerebral ischemia-reperfusion, explore the possible role of ferroptosis in the pathogenesis of CIRI, and observe the effect of HBO on ferroptosis-mediated CIRI. METHODS: Sprague Dawley (SD) rats were randomly divided into control, model, Ferrostatin-1 (Fer-1), HBO and Fer-1+ HBO groups. Morris water maze, myelin basic protein (MBP) and ß-tubulin immunoreactivity were assessed to evaluate the neuroprotective effects of HBO on cerebral ischemia reperfusion injury. Ferroptosis were examined to investigate the mechanism underlying the effects of HBO. RESULTS: Our result showed that Fer-1 and HBO improved learning and memory ability in the navigation trail and probe trail of the Morris water maze and increased MBP and ß-tubulin immunoreactivity of the cortex in the model rats. The levels of ferritin, malondialdehyde (MDA) and glutathione (GSH) in the serum were also reversed by Fer-1 and HBO treatment. Mitochondrial cristae dissolution and vacuolization were observed in the model group by transmission electron microscopy and these conditions were improved in the Fer-1 and HBO groups. Furthermore, Fer-1 and HBO treatment reversed Prostaglandin-Endoperoxide Synthase 2 (PTGS2), Iron Responsive Element Binding Protein 2 (IREB2), acyl-CoA synthetase long chain family member 4 (ACSL4) and Solute Carrier Family 7 Member 11 (SLC7A11) mRNA levels and Transferrin Receptor 1 (TFR1), ferritin light chain (FTL), ferritin heavy chain 1 (FTH1), glutathione peroxidase 4 (GPX4), Nuclear factor E2-related factor 2 (Nrf2), lysophosphatidylcholine acyltransferase 3 (LPCAT3), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (P-JNK) phosphorylated Extracellular signal-regulated protein kinase (P-ERK) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) protein levels. The above changes were more pronounced in Fer-1+ HBOGroup. DISCUSSION: The results of the present study indicated that HBO improves cerebral ischemia-reperfusion injury in rats, which may be related to inhibition of ferroptosis. This also means that ferroptosis may become a new target of HBO against CIRI.

Role of traditional Chinese medicine monomers in cerebral ischemia/reperfusion injury:a review of the mechanism.

Ischemia/reperfusion (I/R) injury is a pathological process wherein reperfusion of an ischemic organ or tissue exacerbates the injury, posing a significant health threat and economic burden to patients and their families. I/R triggers a multitude of physiological and pathological events, such as inflammatory responses, oxidative stress, neuronal cell death, and disruption of the blood-brain barrier (BBB). Hence, the development of effective therapeutic strategies targeting the pathological processes resulting from I/R is crucial for the rehabilitation and long-term enhancement of the quality of life in patients with cerebral ischemia/reperfusion injury (CIRI). Traditional Chinese medicine (TCM) monomers refer to bioactive compounds extracted from Chinese herbal medicine, possessing anti-inflammatory and antioxidative effects, and the ability to modulate programmed cell death (PCD). TCM monomers have emerged as promising candidates for the treatment of CIRI and its subsequent complications. Preclinical studies have demonstrated that TCM monomers can enhance the recovery of neurological function following CIRI by mitigating oxidative stress, suppressing inflammatory responses, reducing neuronal cell death and functional impairment, as well as minimizing cerebral infarction volume. The neuroprotective effects of TCM monomers on CIRI have been extensively investigated, and a comprehensive understanding of their mechanisms can pave the way for novel approaches to I/R treatment. This review aims to update and summarize evidence of the protective effects of TCMs in CIRI, with a focus on their role in modulating oxidative stress, inflammation, PCD, glutamate excitotoxicity, Ca2+ overload, as well as promoting blood-brain barrier repairment and angiogenesis. The main objective is to underscore the significant contribution of TCM monomers in alleviating CIRI.

[Electroacupuncture improves learning and memory impairment and enhances hippocampal synaptic plasticity through BDNF/TRKB/CREB signaling pathway in cerebral ischemia-reperfusion injury rats].

OBJECTIVE: To observe the effect of electroacupuncture on brain-derived neurotrophin factor (BDNF) / tyrosine kinase receptor B (TRKB) / cyclic adenosine monophosphate response element binding protein (CREB) pathway, synaptic plasticity marker protein and synaptic ultrastructure in the hippocampus of rats with learning and memory impairment induced by cerebral ischemia reperfusion (IR), so as to explore its mechanisms underlying improvement of cognitive impairment after stroke. METHODS: SD rats were randomly divided into blank, sham operation, model, and EA groups, with 12 rats in each group. The model of IR was established by occlusion of the middle cerebral artery. EA (2 Hz/10 Hz, 1-3 mA) was applied to "Shenting" (GV24) and "Baihui" (GV20) for 30 min, once daily for 14 days. The neurological function was evaluated according to the Zea Longa's score criteria. Morris water maze test was used to detect the learning and memory function of the rats. Nissl staining was used to observe the pathological morphology of the hippocampus. Transmission electron microscopy was used to observe the ultrastructure of the syna-pse in the hippocampus, the synaptic gap width and postsynaptic dense substance (PSD) thickness were measured. Immunofluorescence staining was used to observe the positive expression levels of BDNF, PSD-95 and synaptophysin (SYN) in hippocampal CA1 region. The protein expression levels of BDNF, TRKB, CREB, PSD-95, and SYN in hippocampal tissue were detected by Western blot. RESULTS: Compared with the sham operation group, the neurological function score and escape latency (EL) were significantly increased (P<0.01), the times of crossing the original platform were decreased (P<0.01), the number of neurons in the CA1 area of the hippocampus was reduced, with incomplete morphology, widened synaptic gaps and significantly decreased PSD thickness (P<0.01), the positive expressions of BDNF, PSD-95, SYN and the protein expression levels of BDNF, TRKB, CREB, PSD-95, SYN were significantly decreased (P<0.01) in the model group. Compared with the model group, the neurological function scores and EL on the 12th and 13th day were decreased (P<0.01, P<0.05), the times of crossing the original platform were increased (P<0.01), the morphology of hippocampal CA1 neurons improved, the synaptic gaps was decreased (P<0.01), the PSD thickness was significantly increased (P<0.01), the positive expressions of BDNF, PSD-95, SYN, and the protein expression levels of BDNF, TRKB, CREB, PSD-95, SYN were increased (P<0.05, P<0.01) in the EA group. CONCLUSION: EA can alleviate cognitive impairment in IR rats, which may be related to its function in up-regulating the proteins of BDNF/TRKB/CREB pathway, promoting the expressions of synaptic plasticity marker proteins PSD-95 and SYN, thus improving the synaptic plasticity.

[Effect of acupuncture on HIF-1α/NLRP3 inflammatory signaling pathway in the rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effects of Xingnao Kaiqiao (regaining consciousness and opening orifices) acupuncture therapy on the expression of hypoxia-inducible factor 1α (HIF-1α) and Nod-like receptor protein 3 (NLRP3) in cerebral ischemia-reperfusion rats, and to explore the mechanism of acupuncture against cerebral ischemia-reperfusion injury. METHODS: Seventy-two male SD rats were randomly divided into a sham-operation group, a model group, an acupuncture group and a non-point acupuncture group, with 18 rats in each one. Using modified Longa thread embolization method, the rat model of acute focal cerebral ischemia was prepared; and after 2 h ischemia, the reperfusion was performed to prepared the model of cerebral ischemia-reperfusion. Immediately after reperfusion, Xingnao Kaiqiao acupuncture method was applied to bilateral "Neiguan" (PC 6) and "Shuigou" (GV 26) in the acupuncture group, while in the non-point acupuncture group, acupuncture was delivered at non-points and all of the needles were retained for 30 min in these two groups. The samples were collected 24 h after reperfusion in the rats of each group. Zea-Longa neurological deficit score was used to evaluate the degree of cerebral neurological impairment, TTC staining was adopted to observe the volume percentage of cerebral infarction, HE staining was provided to observe the morphological changes of brain, and Western blot was applied for detecting the expression of HIF-1α and NLRP3 proteins in the cerebral cortex on the right side. RESULTS: Compared with the sham-operation group, neurological deficit score and volume percentage of cerebral infarction were increased in the model group (P<0.01), and HIF-1α and NLRP3 protein expression was elevated (P<0.01). Compared with the model group, neurological deficit score and volume percentage of cerebral infarction were decreased (P<0.01), and HIF-1α and NLRP3 protein expression was lower (P<0.01) in the acupuncture group. There was no significant difference in above indexes in the non-point acupuncture group compared with the model group (P>0.05). Compared with the sham-operation group, the brain tissue of the rats in the model group and the non-point acupuncture group was loose and edema, and the nuclei were shriveled. The brain tissue morphology in the acupuncture group was similar to that of the sham-operation group. CONCLUSION: Acupuncture can alleviate cerebral ischemia-reperfusion injury, and its mechanism may be related to the regulation of HIF-1α/NLRP3 signaling pathway to attenuate inflammatory response.

Electroacupuncture Pretreatment Alleviates Cerebral Ischemia-reperfusion Injury by Down-regulating Mir-155-5p.

BACKGROUND: Increasing evidence shows that electroacupuncture pretreatment (EP) plays a crucial role in cerebral ischemia-reperfusion (I/R) injury, and cerebral I/R injury is the most serious complication of ischemic stroke treatment. The role of miR-155-5p in cerebral I/R injury has been studied, but the regulation of EP on miR-155-5p has not been reported. METHODS: The middle cerebral artery occlusion (MCAO) mice were used to investigate the role of EP in cerebral I/R injury. Longa and modified neurological severity scores (mNSS) were used to evaluate neurological impairment. HE staining and TUNEL staining were used to evaluate brain injury. The expressions of miR-155-5p, Yin Yang 1 (YY1) and p53 were detected by qRT-PCR. The expressions of related proteins were detected by western blot. The binding of YY1 to miR- 155-5p was verified by dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. Mice brain microvascular endothelial cells (BMECs) were isolated and cultured for in vitro experiments. Oxygen-glucose deprivation/reoxygenation (OGD/R) was used to verify the role of YY1, p53 and miR-155-5p in cerebral I/R injury in vitro. RESULTS: MCAO modeling induced brain injury, apoptosis, and increased levels of miR-155-5p, YY1, and p53. EP markedly alleviated brain injury and reduced levels of miR-155-5p, p53, and YY1. miR-155 agomir markedly increased the expression of miR-155-5p and p53. miR-155 antagomir decreased the levels of miR-155-5p and p53. Dual-luciferase reporter and ChIP assay verified that YY1 regulated miR-155-5p expression. YY1 shNRA greatly decreased miR-155-5p and p53. Inhibition of p53 decreased miR-155-5p expression. Both miR-155-5p inhibitor and YY1 shRNA promoted proliferation, inhibited apoptosis, and decreased levels of ICAM-1 and Eselectin of OGD/R-treated BMECs. Inhibition of p53 strengthened the effect of miR-155-5p inhibitor and YY1 shNRA on BMECs. CONCLUSION: Electroacupuncture pretreatment alleviates cerebral ischemia-reperfusion injury by regulating the YY1/p53/miR-155-5p axis.

[Effect of electroacupuncture pretreatment on ferroptosis in neurons of rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture（EA）preconditioning on ferroptosis in rats with cerebral ischemia-reperfusion injury （CIRI）, so as to explore the neuroprotective mechanism of EA preconditioning. METHODS: Male SD rats were randomly divided into sham operation, model, EA, inhibitor and inducer groups with 20 rats in each group. The CIRI model was established by modified Zea Longa occlusion of the middle cerebral artery. Before modeling, EA treatment （2 Hz/15 Hz, 1-2 mA） was applied to "Baihui"（GV20）, "Fengfu"（GV16） and "Dazhui"（GV14） for rats of the EA group, 20 min a day for 7 consecutive days. Rats of the inhibitor group were intraperitoneally injected with ferristatin-1（25 mg/kg）at a slow and uniform rate. Rats of the inducer group were intraperitoneally injected with Erastin（100 mg/kg） after 7 days of EA preconditioning, once every 2 h for a total of 4 times. The CIRI models were prepared 2 d later after the above interventions finished by thread-occlusion. The degree of neurological impairment was evaluated by modified Zea Longa score. The percentage of infarct size was calculated by TTC staining. The ultrastructure of neurons in hippocampus was observed by transmission electron microscope. The contents of ferrous ion （Fe2+）, malondialdehyde （MDA） and glutathione （GSH） in cerebral tissue and reactive oxygen species （ROS） in serum were determined by biochemical method. The changes of mitochondrial membrane potential in rats brain tissues were detected by flow cytometry. The mRNA and protein expression levels of glutathione peroxidase 4 （GPX4）, acyl-CoA synthetase long-chain family member 4 （ACSL4）, transferrin receptor （TFRC）, 15-lipoxygenase （15-LOX） and cyclooxygenase-2 （COX-2） in the ischemic hippocampal region of CIRI rats were detected by real-time quantitative PCR and Western blot, respectively. RESULTS: Compared with the sham operation group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression levels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01）, and mitochondria in brain tissue were significantly damaged （P<0.01） in the model group. Compared with the model group, the above indexes were all reversed （P<0.05, P<0.01） in the EA group and inhibitor group. Compared with the EA group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression le-vels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.05, P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01, P<0.05）, and mitochondria in brain tissue were significantly damaged （P<0.05） in the inducer group. CONCLUSION: EA preconditioning has neuroprotective effect on CIRI rats, which may be related to inhibiting ACSL4/TFRC/15-LOX/COX-2 expression and increasing GSH/GPX4 expression.