Buyang Huanwu Decoction Alleviates Cerebral Ischemia-Reperfusion Injury Via Sirtuin1/Autophagy Pathway

ObjectiveIschemic stroke due to vascular occlusion and insufficient blood perfusion accounts for approximately 87%of total stroke cases.After cerebral ischemia,it leads to tissue damage and even infarction,causing a series of cascade reactions such as abnormal neurological function and hypoactive consciousness.The body damage caused by ischemic stroke caused cell metabolism disorder and stagnation of some organelle renewal,which made the long-term dynamic balance of intracellular autophagy activity broken.The literature survey found that autophagy-mediated neuronal survival is related to the regulation of the Sirtuins family(the main regulators of metabolism and longevity),and Sirtuin 1(SIRT1)is one of the most characteristic members.Buyang huanwu decoction(BHD),as a commonly used clinical prescription for Yiqi Huoxue,is mostly used to treat ischemic stroke and its sequelae.There is evidence that the serum containing Buyang Huanwu Decoction can up-regulate SIRT1 expression.The curative effect of this compound has been widely recognized clinically,and related experimental studies have not yet elucidated its mechanism of action in the process of nerve repair.Therefore,we propose a hypothesis:the effective dose of BHD provides the possibility of behavioral recovery and neural recovery of the body.At the same time,SIRT1/autophagy pathway may be involved in the process of ischemic stroke injury and treatment.In this experiment,an experimental animal model of ischemic stroke is used to explore and verify the mechanism from the overall level and the molecular level.Methods1.Effect of Buyang Huanwu Decoction on cerebral ischemia-reperfusion injury in rats.Middle cerebral artery ischemia-reperfusion(MCAO-R)was used as the experimental model;male SD rats were randomly divided into five groups:sham operation group(SHAM),middle cerebral artery occlusion and reperfusion group(MCAO-R),yang Huanwutang low-dose group(BHD5 g/kg),Buyang Huanwutang medium-dose group(BHD10 g/kg),Buyang Huanwutang high-dose group(BHD20 g/kg);by NSS scale,TTC staining,HE staining and Nissl staining of frozen sections of the brain were used to detect neurological deficit score,cerebral infarct volume,changes in brain tissue cell morphology,and the survival of nerve cells Nissl body.2.Promoting nerve regeneration of Buyang Huanwu Decoction in treating ischemic stroke and the mechanism of autophagy.MCAO-R as an experimental model;male SD rats were randomly divided into four groups:sham operation group(SHAM),middle cerebral artery occlusion and reperfusion group(MCAO-R),Buyang Huanwu Decoction group(BHD),Lei Rapamycin group(Rapa);detection of protein expression levels of SIRT1,autophagy-related proteins LC3,P62,Beclin-1 by Western blot;detection of brain-derived neurotrophic factor BDNF by single immunofluorescence labeling and double immunofluorescence labeling,Immature neuronal marker DCX,neural stem cell marker Nestin and autophagy marker LC3 protein expression.Results1.Effect of Buyang Huanwu Decoction on cerebral ischemia-reperfusion injury in rats.The NSS score showed that the NSS score(0.00±0.00)of the SHAM group was very small,and the behavior and perception evaluation were normal in all aspects.The MCAO-R group score(8.33±1.40)was significantly higher than that of the SHAM group(P<0.01);the postoperative low-dose and middle-dose BHD5 g/kg,BHD 10 g/kg group rats score(7.42 ± 1.24,4.63±0.91)compared with MCAO-R group rats slightly decreased(P<0.05,P<0.01);the postoperative high-dose BHD20 g/kg group’s score(2.42±0.79)was lower than that of the MCAO-R group(P<0.01).TTC staining results showed that the whole brain tissue(0.00±0.00)of the SHAM group was completely stained red,while the brain tissue of the MCAO-R group rats(29.68±0.61)was partially white during surgical ischemia,indicating cortical infarction,or even infarction Has been extended to the entire hemibrain;the middle and high doses of Buyang Huanwu Decoction,that is,the infarct.volume of rats in the BHD 10 g/kg and BHD20 g/kg groups(13.29±0.57,6.92±0.45)compared with the MCAO-R group Significantly reduced(P<0.01,P<0.01).HE staining results showed that a large number of small vacuoles appeared in the cells of the MCAO-R group,and the nuclei were difficult to stain,suggesting that the brain tissue cells were damaged.In the BHD group at 20 g/kg after operation,vacuoles appeared in the cells,and the nuclei were blue.The results of Nissl staining showed that the Nissl bodies of nerve cells in the SHAM group were large and dense,and were massive.In the MCAO-R group,the cytoplasm disappeared,the swollen body was swollen,and the nucleus coagulated.The morphology and coloring of Nissl body in BHD group at 20 g/kg after operation were significantly better than those in MCAO-R group.2.Promoting nerve regeneration of Buyang Huanwu Decoction in treating ischemic stroke and the mechanism of autophagy.WB results showed that compared with SHAM group,MCAO-R group brain tissue autophagy index LC3-Ⅱ,Beclin-1 expression was increased(P<0.05),P62 expression was down-regulated(P<0.01),which prompted this experiment At 5 days after ischemia-reperfusion injury in rats,the autophagy of the brain tissue cells was activated.Compared with the MCAO-R group,the autophagy status was significantly increased after BHD treatment.The autophagy agonist Rapa treatment group showed a consistent trend with the BHD group.Compared with SHAM group,SIRT1 expression in MCAO-R group was increased at 5 days after ischemia-reperfusion injury in rats(P<0.05);compared with MCAO-R group,SIRT1 expression in BHD group and autophagy agonist Rapa group Significantly increased(P<0.01),suggesting that BHD can promote the expression of SIRT1 in cerebral ischemic injury,and this promotion may be related to the activation of autophagy.The immunostaining experiments of brain slices focused on the central nervous system effects of Buyang Huanwu Decoction on ischemia-reperfusion injury.Neural stem cells can differentiate into neurons.The fluorescent labeling results of BDNF and DCX confirmed that the administration of Buyang Huanwu Decoction after cerebral ischemia-reperfusion injury in rats played a role in promoting the protection and regeneration of neurons.The number of Nestin-positive cells increased after administration of Buyang Huanwu Decoction,indicating that the role of neural stem cells was activated,thereby further growth and differentiation,and promote nerve regeneration.Conclusion1.Cells are damaged or even died after ischemia-reperfusion injury,especially nerve cells.This process will initiate a self-rescue procedure,that is,cell autophagy;2.Buyang Huanwu Decoction may promote nerve regeneration and exert protective effects after cerebral ischemia-reperfusion injury in rats;3.After cerebral ischemia-reperfusion injury,the role of Buyang Huanwu Decoction may be related to the participation of SIRT1/autophagy pathway.In summary,in this experiment,the intervention of Buyang Huanwu Decoction after ischemia-reperfusion injury may be effective through the SIRT1/autophagy pathway,which is a process;it may be achieved by promoting nerve regeneration to recover after injury,which is the result.