| Objective: To study the effects of Huoxue Huayu Jiedu Prescription on the macroscopic symptoms,neurological injury score,pathological morphology of cerebral cortex on the ischemic side,expression of related factors of autophagy and PI3K/Akt/mTOR signal pathway in rats with cerebral ischemia-reperfusion injury,and to explore the mechanism of Huoxue Huayu Jiedu Prescription on the pathogenesis of "Blood Stasis Toxin interaction" of cerebral ischemia-reperfusion injury by regulating PI3K/Akt/ mTOR signal pathway mediated autophagy.Methods: Sixty SPF male SD rats were randomly divided into sham operation group,model group,edaravone group,Huoxue Huayu Jiedu prescription group,rapamycin group and Huoxue Huayu Jiedu prescription+rapamycin group.The focal occlusion cerebral ischemia model of right middle cerebral artery in rats was prepared by modified Zea longa suture method,and reperfusion was performed 2 hours after ischemia.After 7 days of treatment,the rats were killed.After modeling,the rats were scored for nerve defect on days 1,3,5 and 7.The macroscopic symptoms during the experiment were observed.The serum contents of tumor necrosis factor alpha(TNF-α),malondialdehyde(MDA)and glutamic acid(GLU)were detected by enzyme-linked immunosorbent assay(ELISA),hematoxylin eosin(HE)staining was used to observe the pathological morphology,western blot(WB)detect the expression of related proteins on autophagy and PI3K/Ak T/mTOR pathway.Fluorescence real-time quantitative polymerase chain reaction(RT-PCR)to detect the expression of Beclin1 m RNA.The expression of LC3 in neurons,astrocytes and cerebrovascular endothelial cells after cerebral ischemia-reperfusion injury was observed by double immunofluorescence labeling.Results:1.Macroscopic symptoms:The rats in the sham operation group has good spirit,moderate body shape,weight between 260 g and 330 g,bright red lip and tongue color,no abnormal shape of tongue body,soft and moist hair,bright red color of auricles,limbs and tail,no abnormality,mild temperament,soft stool,normal urine color,normal drinking,eating and action.The rats in the model group have poor spirit,thin body,weight between 180 g and 230 g,dark red lip and tongue color,skewed tongue,sever convulsions,unclear consciousness,dry hair,dark red ulcer on the auricle,irritable temperament,loose stool,less urine,yellow urine,poor appetite,crawling to the affected side and chasing the tail to the affected side.The rats in edaravone group and Huoxue Huayu Jiedu prescription group have better mental state than those in the model group,moderate body shape,weight between 220 g and260g,dark red lip and tongue color,better deflection of tongue body,significantly improved spirit,moist hair,better rotation of tongue body,significantly improved spirit,moist hair,better transformation of auricle ulcer,slightly irritable temperament,loose stool,yellow urine,general content,crawling slightly towards the affected side,and relatively convenient action.The rats in rapamycin group and Huoxue Huayu Jiedu prescription + rapamycin group have poor spirit,lean body,weight between 200 g and 240 g,dark red lip and tongue color,skewed tongue,dry and dull hair,dark red ulcer on auricle,irritable temperament,loose stool,less urine,yellow urine and less sodium.They tend to crawl to the affected side and chase the tail to the affected side.2.ELISA:Compared with the sham operation group,the serum contents of TNF-α,MDA and Glu in model group increased significantly(P<0.01,P<0.05);Compared with the model group,the serum contents of TNF-α,MDA and Glu in edaravone group and Huoxue Huayu Jiedu prescription decreased significantly(P<0.01,P<0.05);Compared with Huoxue Huayu Jiedu prescription group,the serum contents of TNF-α,MDA and Glu in Huoxue Huayu Jiedu prescription and rapamycin increased significantly(P<0.01,P<0.05);3.m-NSS score:1d: Compared with the sham operation group,the m-NSS scores of other groups increased significantly(P<0.01);3d: Compared with the sham operation group,the m-NSS scores of the model group were significantly increased(P<0.01);Compared with the model group,the scores of edaravone group and Huoxue Huayu Jiedu prescription group were significantly lower(all P<0.01);Compared with Huoxue Huayu Jiedu prescription group,the scores of Huoxue Huayu Jiedu prescription and rapamycin group were significantly higher(P<0.05);5d: Compared with the sham operation group,the m-NSS scores of the model group were significantly increased(P<0.01);Compared with the model group,the scores of edaravone group and Huoxue Huayu Jiedu prescription group were significantly lower(P<0.01,P<0.05);Compared with rapamycin group,the scores of Huoxue Huayu Jiedu prescription and rapamycin group were significantly higher(P<0.01);7d: Compared with the sham operation group,the m-NSS scores of the model group were significantly increased(P<0.01);Compared with the model group,the scores of edaravone group and Huoxue Huayu Jiedu prescription group were significantly lower(all P<0.01);Compared with Huoxue Huayu Jiedu prescription group,the score of Huoxue Huayu Jiedu prescription and rapamycin group was significantly higher(P<0.01).4.HE staining:In sham operation group,the morphology of nerve cells in cortical area was normal,the boundary was clear,the structure was complete,the pyramidal cells were arranged orderly,and the nucleolus and nuclear membrane were clearly visible;The cortical tissue structure of the model group was abnormal,the cell gap was widened,there were many vacuoles around,most of them showed punctate necrosis,the cells were arranged in disorder,and the nucleus was obviously broken,pyknotic and deeply stained.In edaravone group and Huoxue Huayu Jiedu prescription group,the number of intact nerve cells in cortical tissue decreased,the cell gap was slightly wider,the arrangement was slightly disordered,and the phenomena of cell shrinkage and pyknosis and dense staining of nucleus were still visible,but the degree was significantly reduced compared with the model group.In rapamycin group and Huoxue Huayu Jiedu prescription+ rapamycin group,the cortical tissue structure was abnormal,the number of pyramidal cell layers decreased,the cell gap was wide,the arrangement of nerve cells was disordered,and the nucleus was obviously broken,pyknosis and deep staining,but the damage was more serious than that in Huoxue Huayu Jiedu prescription group.5.RT-PCR:Compared with the sham operation group,the expression level of Beclin1 m RNA in the model group was significantly higher(P<0.01);Compared with the model group,the expression level of Beclin1 m RNA in edaravone group and Huoxue Huayu Jiedu presrciption group decreased significantly(P<0.01);Compared with Huoxue Huayu Jiedu prescription group,the expression level of Beclin1 m RNA in Huoxue Huayu Jiedu prescription and rapamycin group increased significantly(P<0.05).6.Double immunofluorescence staining:LC3 was expressed in neurons,astrocytes and brain microvascular endothelial cells.LC3 was more expressed in the neurons of the model group,and the expression of LC3 decreased after treatment in the drug group.7.Western blot:Compared with the sham operation group,the expression levels of PDK1,p-PI3 K,p-AKT and p-mTOR protein in the model group were significantly decreased(P<0.01);Compared with the model group,the expression levels of PDK1,p-PI3 K,p-AKT and p-mTOR protein in edaravone group and Huoxue Huayu Jiedu prescription group were significantly up-regulated(P<0.01);Compared with Huoxue Huayu Jiedu prescription group,the expression of PDK1,p-PI3 K,p-AKT and p-mTOR protein in Huoxue Huayu Jiedu prescription and rapamycin group was significantly down-regulated(P<0.01).Conclusion:1.Huoxue Huayu Jiedu prescription can improve the symptoms of neurological deficit in rats with cerebral ischemia-reperfusion injury,improve pathomorphological structure and reduce the contents of TNF-α,MDA and Glu have a certain protective effect on the brain tissue of cerebral ischemia-reperfusion injury model rats.2.Huoxue Huayu Jiedu prescription can down regulate the expression of Beclin1 m RNA,reduce the expression of LC3 in nerve cells,and up regulate the protein expression of PDK1,p-pi3 k,p-Akt and p-mTOR in cerebral cortex of ischemic side.3.Huoxue Huayu Jiedu prescription can activate PI3K/Akt/mTOR signal pathway,inhibit autophagy and protect brain. |
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