The Research Of Zhilong Capsule's Pretreatment Effects Of The Two Way Regulation Of Autophagy On Focal Cerebral Ischemia Reperfusion Injury In Rat

Objective: After the early intervention of Zhilong Capsule(ZLC)on the focal cerebral ischemia reperfusion injury rats,we give the rats autophagy inhibitor 3-MA or autophagy resultant rapamycin,and compare with the rats which only use autophagy inhibitor 3-MA or autophagy resultant rapamycin—observing the neurological score,the percent of Cerebral infarction volume ratio,the autophagy level of ischemic area and the expression changes of autophagy protein LC3–? and Beclin1 at different points.To study the impact of Zhilong Capsule for the ischemia reperfusion injury in rats in the two way regulation of autophagy.Methods : 1.Group technology:200 healthy male SD rats which weigh between 280 to 350 grams and are 6 to 8 weeks,are divided into a group,b group,c group and d group randomly—a group: blank group(Blank);b group:transient focal cerebral ischemia reperfusion injury model group(MCAO group);c group:Group c divided into c1 and c2group—c1:the intervention group modeling by ZLC and rapamycin(ZLC+Rap+ MCAO group),c2: the intervention group modeling by ZLC and 3-MA(ZLC+3-MA + MCAO group);d group: Group d divided into d1 and d2group—d1: the intervention group modeling by rapamycin(Rap + MCAO group),d2: the intervention group modeling by 3-MA(3-MA + MCAO group);2.Dosing method: ZLC+Rap + MCAO group and ZLC+3-MA + MCAO groupfeed the ZLC a half month;The ZLC+Rap + MCAO group injects the rapamycin through lateral ventricle 1 hour before the operation;The ZLC+3-MA + MCAO group injects the 3-MA through lateral ventricle 12 hour after the operation;Blank group,Model group,Rap + MCAO group and 3-MA+ MCAO group are given with the same volume distilled water as the ZLC+Rap + MCAO group and ZLC+3-MA + MCAO group a half month.The Rap + MCAO group injects the rapamycin through lateral ventricle 1 hour before the operation;The 3-MA + MCAO group injects the 3-MA through lateral ventricle 12 hour after the operation.Detection index: 1)Each group are scored by the neurological standard on focal cerebral ischemia after reperfusion0 hour,2 hour,6 hour,12 hour and 24 hour.2)After reperfusion 24 hour use the fresh brain tissue to TTC staining,observing the cerebral ischemia infarction area after 24 hours and calculating cerebral infarction volume ratio.3)Each group is observed with electron microscope for the change of autophagy body at every observation point;4)In each observation point collect the fresh brain tissue,then ues western blot method to detect autophagy related protein expression of LC3-?,Beclin-1 at every observation point.Results: 1.Use Garcia score Standard to evaluate each group's neurological score.To compare with Blank group,the neurological score of model group,ZLC+Rap + MCAO group,Rap + MCAO group,ZLC+3-MA + MCAO group,3-MA + MCAO group is decreased significantly in every experimental observation point,the difference is statistical significance(P < 0.01).To compare with model group,the neurological score of ZLC+Rap + MCAO group,Rap+ MCAO group,ZLC+3-MA + MCAO group is increased at all points,the difference is statistical significance(P < 0.01 or P < 0.05);the neurological score of 3-MA +MCAO group has no obvious difference at ischemia reperfusion 0 h,2 hour and6 hour,no statistical significance(P > 0.05);at ischemia reperfusion 12 h and24 h the neurological scores are higher,differences are statistical significance(P < 0.05).ZLC+Rap+ MCAO group compared with Rap+ MCAO group,the neurological score of ZLC+Rap+ MCAO group is significantly higher in the experiment at all the observation time points,the difference is statistical significance(P < 0.05);ZLC+3-MA + MCAO group compared with 3-MA +MCAO group,the neurological score of ZLC+3-MA + MCAO group is significantly higher in the experiment at all the observation time points,the difference is statistical significance(P < 0.05).2.Stained using TTC to calculate the infarct volume ratio.To compare with Blank group,the cerebral infarction volume ratio of model group,ZLC+Rap+ MCAO group,Rap+ MCAO group,ZLC+3-MA + MCAO group,3-MA + MCAO group are higher,the difference is statistical significance(P < 0.01).Compared with model group,the cerebral infarction volume ratio of ZLC+ Rap+ MCAO group,Rap+ MCAO group,ZLC+3-MA + MCAO group are decreased,and the difference is statistical significance(P < 0.01).ZLC+Rap+ MCAO group compared with Rap+ MCAO group,the cerebral infarction area is decreased;the difference is statistical significance(P < 0.01).ZLC+3-MA + MCAO group compared with 3-MA +MCAO group,the cerebral infarction area is decreased,and the difference is statistical significance(P < 0.01).3.Use electron microscope to observe the change of autophagic bodies.In the blank group,neurons of morphological structure is normal,there is normal nucleus,mitochondria,many endoplasmic reticulum and a few lysosome structure in these neurons.In the model group,there are free bubbles,markedly swollen mitochondria with free bubble formation and autophagy,and over time,the quantity of autophagy body increase gradually,about 12 h reperfusion the number of autophagy reaches its peak.The autophagy body number of ZLC+Rap+ MCAO group reaches its peak in ischemia reperfusion 6 hour.The autophagy body number of Rap+MCAO group is similar in ischemia reperfusion 6 hour and 12 hour,so we can conclude that the autophagy body number of Rap+ MCAO group's peak should be between reperfusion 6 h and 12 hour.ZLC+Rap+ MCAO group compared with Rap+ MCAO group,the autophagy body significantly increases In every time point and degrades faster,so this can illustrate that using ZLC with autophagy inducer can enhance the level of autophagy.ZLC+3-MA + MCAO group compared with 3-MA + MCAO group,the expression of autophagy body and lysosome increases at reperfusion 0 h,2 h and 6h;and the expression of autophagy body and lysosome reduces at reperfusion 12 h and 24h;so this can illustrate that using ZLC with autophagy inhibitor can inhibit autophagy process.4.Western blot method to detect the expression of autophagy related protein LC3-?,Beclin-1.Compared with the blank group,the expression ofprotein LC3-?,Beclin-1 in model group,ZLC+ Rap+ MCAO group,ZLC+3-MA + MCAO group,Rap+ MCAO group and 3-MA + MCAO group are enhanced obviously at each point,the differences are statistical significance(P < 0.01).Compared with the model group,the expression of protein LC3-?,Beclin-1 in ZLC+ Rap+ MCAO group and Rap+ MCAO are enhanced obviously at each point,the differences are statistical significance(P < 0.01);the expression of protein LC3-?,Beclin-1 in ZLC+3-MA + MCAO group is enhanced at reperfusion 0h,2h and 6 h,the difference is statistical significance(P < 0.05);the expression of protein LC3-?,Beclin-1 in ZLC+3-MA +MCAO group is weakened at reperfusion 12 h and 24 h,the difference is statistical significance(P < 0.05).the expression of protein LC3-?,Beclin-1 in3-MA + MCAO group is similar with model group at reperfusion 0h,2h and 6h,the difference has no significant(P > 0.05);the expression of protein LC3-?,Beclin-1 in 3-MA + MCAO group is weakened at reperfusion 12 h and 24 h,the difference is statistical significance(P < 0.05).ZLC+Rap+ MCAO group compared with Rap+ MCAO group,the expression of protein LC3-?,Beclin-1 are enhanced obviously at each point,the differences are statistical significance(P < 0.01 or P < 0.05);ZLC+3-MA+ MCAO group compared with3-MA+ MCAO group,the expression of protein LC3-?,Beclin-1 in ZLC+3-MA+ MCAO group is enhanced at reperfusion 0h,2h and 6 h(the difference is statistical significance(P<0.05))and reduced at reperfusion 12 h and 24 h(the difference is statistical significance(P<0.01orP<0.05)).Conclusion: 1?Through the detection of autophagosome and autophagy related proteins can instruct that there is abnormal autophagy in cerebral ischemia reperfusion injury.2?Use autophagy revulsant in the early cerebral ischemia reperfusion injury can enhance the level of autophagy,while use autophagy inhibitors in later can inhibit autophagy level.3?Zhilong Capsule can further improve the neurological score,reduced cerebral infarction volume of the focal transient ischemic reperfusion rats.It also can further induce the expression of autophagy related protein LC3-? and Beclin-1 in the early ischemia reperfusion process,while further inhibit its expression in the later process.4?The mechanism maybe that Zhilong Capsule has a two-way regulation for the autophagy in cerebral ischemia reperfusion injury,can enhance the effect of autophagy inducer and inhibitor.