B-Caryophyllene Alleviates Cerebral Ischemia/Reperfusion Injury Via Regulating Of Mitophagy

Objective:Mitophagy is a self-scavenging process of damaged mitochondria,it is an important link in maintaining the dynamic balance of mitochondrial quality and quantity in cells.It occurs in a variety of neurodegenerative diseases,including ischemic stroke.It has been found that in the model of cerebral ischemia-reperfusion injury,there is a neuroprotective effect through the regulation of mitophagy.β-caryophyllene（BCP）,a bicyclic sesquiterpene natural products.It can exert neuroprotective effect on cerebral ischemia-reperfusion injury through anti-apoptosis and antioxidant stress.However,whether it can play a protective role on cerebral ischemia-reperfusion injury by regulating mitophagy and its mechanism is still unclear.This topic will investigate whether BCP can improve the damage caused by cerebral ischemia-reperfusion by regulating mitophagy and the related mechanisms involved.Methods:1.In vivo experiments:C57BL/6J♂mice were randomly divided into five groups:normal group（Sham group）,model group（I/R group）,BCP administration group（BCP group）,BCP+Mitophagy inhibitor group（BCP+Mdivi-1group）and Inhibitor group（Mdivi-1 group）.Transient Middle Cerebral Artery Occlusion（MCAO）model was established by using the thread plug method.The mice in each group were given intragastrically for 5 days(BCP,72 mg.kg^-1)before operation,and Mdivi-1(50 mg.kg^-1)was intraperitoneal injected at the instant of reperfusion.24 h after operation,the neurobehavioral score was used to observe the neurological impairment,and the pathological changes of hippocampal neurons were observed by HE staining.Immunofluorescence staining and western blot were used to detect the expression of autophagy-related proteins LC3,P62,mitochondrial membrane proteins TOM20 and ion channel protein VDAC1 in the damaged site.Transmission electron microscopy was used to observe the occurrence of mitophagy in the damaged site,the mitochondrial ATP content and mitochondrial membrane potential（MMP）were detected to evaluate the mitochondrial function in each group.2.In vitro experiment:HT22 cell lines were cultured in vitro,and the cells were randomly divided into 5 groups:the normal control group without any treatment（Control group）,the OGD/R group（OGD/R group）,the BCP treatment group（BCP group）,the BCP+mitophagy inhibitor group（BCP+Mdivi-1 group）,and the mitophagy inhibitor group（Mdivi-1 group）.The OGD/R model was established after cell culture to about 80%.BCP（10μM）was pretreated for 12 h before model establishment,and mdivi-1（25μM）was added during reoxygenation.After 2 h of glucose deprivation and 24 h of reperfusion,samples were collected for correlation detection.We have adopted the cell activity determined by MTT method and flow cytometry technology measure cell death rate,Western blot used to detect the expression of mitophagy-related proteins LC3,P62,PINK1,PARK2 and mitochondrial membrane protein TOM20,co-localization of GFP-LC3 and TOM20 to detect mitophagy dynamic flow,immunofluorescence dual-label method to detect the overlap rate of PARK2/VDAC1 in hippocampal neurons,the content of mitochondrial DNA was detected by rel-timePCR（mt-Atp6/Rpl13）.The mitophagy of HT22 cell was observed by transmission electron microscopy transmission electron microscope.ATP content and MMP were measured to evaluate mitochondrial function.Result:1.WB results showed that compared with the I/R group,the expression of LC3B in the BCP group increased significantly,while the expression of P62 and TOM20 significantly decreased.Immunofluorescence showed that the coincidence rate of LC3B and VDAC1 was significantly increased,PCR results showed that mitochondrial DNA content（mt-Atp6/Rpl13）was significantly reduced,Transmission electron microscopy showed that more autophagosomes were observed.These results indicating that BCP up-regulated mitophagy induced by ischemia-reperfusion in vivo and in vitro.2.Neurobehavioral scores,cerebral infarct volume and hippocampal neuronal cell death in the BCP-treated mice were reduced.The results of flow cytometry showd that BCP treatment can significantly reduce the mortality of HT22cells and the inhibitor of mitophagy can reverse these phenomenon.These results suggest that BCP can reduce neuronal cell damage and death by up-regulating mitophagy after ischemia-reperfusion.3.Both the ATP content and MMP in the BCP group increased,indicating that BCP can improve mitochondrial function.4.WB and immunofluorescence results showed that,compared with the I/R group,the expression of PARK2 and PINK1 increased in the BCP group,and the coincidence rate of PARK2 and VDAC1 increased,suggesting that the pathway of BCP activating mitophagy was PINK1/PARK2.Conclusion:these results showd that BCP can reduce the death of neurons induced by cerebral ischemia-reperfusion injury by up-regulating mitophagy,which provides a new target and idea for the treatment of acute brain injury.