Study Of Orientin On Autophagy And JNK/ERK Signal Pathagy In Rats Following Cerebral Ischemia-Reperfusion Injury

Ischemic stroke is one of the leading cause of death in our country,with high morbidity,disability and mortality rate,which severely threaten people's health.The main treatment after cerebral ischemia in clinic is to recover blood supply,however,the recovery of blood flow could cause reperfusion injury at the same time.Which receives widely concern in medical field.There are a number of mechanisms involved in cerebral ischemia-reperfusion(I/R)injury,such as oxidative stress,inflammatory reaction,cell apoptosis and mitochondria damage,etc.These factors interact with each other and cause pathological cascade reaction,leading to brain damage.In addition,there is still lack of effective treatments.With the study on autophagy,the effects and mechanisms of drugs on cerebral I/R injury has become a hotspot in modern medical research.Autophagy is a special catabolism and metabolic pathway,which plays an important role in maintaining cell survival and homeostasis.In normal physiological status,autophagy maintains at a low level and was activated when in the stimulus of ischemia or hypoxia.Studies have shown that autophagy plays a dual role in cells,while proper activation can protect cells and excessive autophagy could cause damage to cells.Currently,the research of autophagy remains immature and the specific molecular mechanisms and signaling pathways is still not completely clear.Orientin is a monomer of flavonoid C-glycosides and the main active components in Chinese traditional medicines Trollius chinensis Bunge and Polygonum orientale L.Which can be used for treatment of coronary heart disease,angina pectoris,blood stasis resistance,and so on.The previous research in our team found that orientin could protect cerebral I/R injury and the mechanisms may be related to anti-inflammatory,antioxidant effect and the decrease on neurotoxicity of excitatory amino acids.However,whether the effect of autophagy was mediated by orientin on cerebral I/R injury has not been discussed.Consequently,from the point of autophagy,we investigated the protective effects of orientin on cerebral I/R injury and participated JNK/ERK signal pathway,which was expected to provide new target for the clinical application of orientin.In the present study,middle cerebral artery occlusion(MCAO)model was established in rats according to Longa's suture method.All rats were anesthetized and then were subjected to ischemia,followed by reperfusion after ischemia for 2 h at the right side of the brain.Neurological deficit scores were evaluated when rats were revived.The results demonstrated that MCAO rats can't fully extended their forepaw,circle or fall into the contra of the surgery,and the scores of model rats were 1~3,showing the successful model.1 The dynamic expression of Beclin 1 and LC3 in ischemic areas of I/R ratsRats were randomly divided into Sham group and I/R group.Then the latter is divided into different subgroups including 1 h,3 h,6 h,12 h,24 h,48 h and 72 h of reperfusion.In sham rats,common carotid artery was separated and no suture was inserted.All experimental rats of reperfusion were decaptured at different time points.Immunohistochemistry(IHC),real-time fluorescent quantitative PCR and western blot methods were applied to determine the positive cells,protein and mRNA levels of Beclin1 and LC3 in all groups to select the appropriate time for the following experiment.The results of IHC showed that compared to Sham group,the number of positive cells of Beclin1 and LC3 in I/R rats were significantly increased(P<0.05,P<0.01).The increase was slow from 1 h to 6 h of reperfusion,and the peak appeared at 12 h and maintained to 24 h,after which the positive cells were declined.Real-time fluorescent quantitative PCR results showed that mRNA levels of Beclin1 and LC3 were significantly increased compared with Sham group(P<0.05,P<0.01),and the peak appeared at 24 h of reperfusion and then declined after 24 h.In the western blot,protein expression of Beclin1 and LC3 in the model rats showed an significant increase compared to control group(P<0.05,P<0.01),and the increase continued until 24 h of reperfusion.LC3 expression displayed a slight decline and Beclin1 declined after 24 h of reperfusion.2 Study of orientin on autophagy in rats after cerebral I/R injuryExperimental rats were randomly divided into Sham group,I/R group,I/R+orientin group(Ori),I/R+orientin+rapamycin group(Ori+RAP),I/R+orientin+3-methyladenine group(Ori+3-MA).The administration was intraperitoneal injection.Rats were executed after 24 h of reperfusion.TTC staining was used to detect cerebral infarction volume of rats,and western blot method was applied to determine protein levels of Beclin1,LC3 and phosphorylated Akt and mTOR.TTC results showed that compared with control group,cerebral infarction volume were significantly increased in I/R group(P<0.01).While orientin significantly reduced infarction volume(P<0.05,P<0.01).Compared with orientin group,RAP group elevated cerebral infarction volume(P<0.05),while 3-MA group showed a significant decrease(P<0.05).The results of western blot showed that proteins expression of Beclin1,LC3,p-Akt and p-mTOR increased significantly in comparison to sham group(P<0.01).Orientin significantly reduced these proteins levels(P<0.05,P<0.01).Compared with orientin group,RAP group significantly increased proteins expression of Beclin 1,LC3 and p-Akt(P<0.05),while p-mTOR showed no significant increase.3-MA group reduced protein levels of Beclin1,p-Akt and p-mTOR(P<0.05),while LC3 showed no significant decline.3 Study of orientin on JNK/ERK signal pathways in rats after cerebral I/R injuryRats were randomly divided into Sham group,I/R group,I/R+orientin group(Ori),I/R+orientin+JNK inhibitor(Ori+JNK),I/R+orientin+ERK inhibitor(Ori+ERK).The protein levels of Beclin1,LC3 and p-JNK and p-ERK were detected by using western blot method.The results displayed that I/R significantly increased the protein expression of Beclin1 and LC3,as well as p-ERK and p-JNK compared to Sham group(P<0.01).After treatment with orientin,there was a significant decline in these proteins expression(P<0.05,P<0.01).Compared with orientin group,JNK inhibitor greatly reduced the proteins expression of Beclin 1 and LC3(P<0.05),however,ERK inhibitor decreased the expression of p-ERK and Beclin 1(P<0.05),while LC3 expression has no significant difference.In summary,from the perspective of autophagy,we explored the protective effects of orientin on rats after cerebral I/R injury.The results demonstrated that inhibition of autophagy may be one of the protective mechanism of orientin on rats following cerebral I/R injury.Moreover,JNK and ERK signaling pathway may be involved in the possible mechanism by orientin.