| Ischemic stroke,a kind of cerebrovascular disease characterized by high morbidity,mortality and disability,poses a serious threat to human health,consequently,it is a problem in the field of neuroscience need to be solved immediately.At present,with the continuous improvement of diagnosis and treatment technology,but there is still lack of effective treatment for the disease.Therefore,it is urgent to clarify the pathogenesis of ischemic stroke and find effective therapeutic methods.The medical community has reached a consensus that the focus of focal cerebral ischemia was consist of ischemic core and penumbra previously.The manners of cell death in the ischemic core was mainly necrosis,which was irreversible injury,and in the penumbra,apoptosis was dominant as a form of reversible injury.Hence,inhibition of cell apoptosis in the penumbra was the key method to treat ischemic stroke.However,increasing studies have found that anti-apoptotic therapy using broad-spectrum Caspase inhibitors did not reduce the degree of brain damage caused by cerebral ischemia.Autophagy,as a kind of intracellular lysosomal-dependent degradation pathway,was definitely activated before apoptosis in the penumbra reported by a large number of literatures in recent years,and interacted with apoptosis as well as played an important role in cerebral ischemic injury,but its role is still unclear.Accordingly,it will be helpful for finding a new effective target for the prevention and treatment of ischemic stroke to further observe the activation and change rule of autophagy in the penumbra as well as confirm its role in cerebral ischemia injury.Domestic and overseas studies have reported that Caveolin-1(Cav1)is involved in the regulation of autophagy in lung epithelial cells,breast cancer cells,and so on.At the same time,some scholars find that Cav1 knockout aggregates cerebral ischemic injury.So,does Cav1 involve in the regulation of autophagy after cerebral ischemia? And what is the underlying mechanism? It is not yet clarified.Increasing researches indicates that the mammalian target of rapamycin(m TOR)/Unc-51-like kinase 1(ULK1)pathway is the crucial pathway that regulating autophagy after cerebral ischemia,so,does Cav1 play a neuroprotective role in cerebral ischemic injury through regulating the activity of m TOR/ULK1 pathway to influence the level of autophagy in the penumbra? Therefore,we intend to elucidate the role and possible mechanism of Cav1 in the regulation of autophagy in the penumbra after cerebral ischemia on the basis of permanent middle cerebral artery occlusion(p MCAO)model in Cav1 knockout(KO)and wild type(WT)mice.Buyang Huanwu Decoction(BHD),as a classical prescription,was first developed by Wang Qingren who was a doctor in Qing Dynasty,and was applied to treat hemiplegia after stroke.Nowadays,we usually use BHD to treat the ischemic stroke and have achieved good clinical efficacy.A large number of studies have demonstrated that BHD has the effect of anti cerebral ischemia injury,but its mechanism is not fully understood.Thus,it is of great practical significance to further confirm and clarify the neuroprotectective effect and the underlying mechanism of BHD.The present study is consisted of four parts.The first part: Establishment of identification and breeding methods of Cav1 gene knockout mice,and the expression and localization of Cav1 in the brain of normal C57BL/6 WT mice;The second part: To observe the activation of autophagy and change rule in the cerebral ischemic penumbra and confirm its role in cerebral ischemia injury;The third part: To elucidate the role of Cav1 in the regulation of autophagy and the possible mechanism in cerebral ischemia penumbra;The fourth part: On the possible mechanism of BHD protect cerebral ischemia injury from the perspective of autophagy mediated by Cav1/m TOR/ULK1 pathway through the method of drug pretreatment.Part 1 Identification and breeding methods of Cav1 gene knockout mice,and the expression and localization of Cav1 in the brain of wild type miceObjective: To investigate the identification and optimal breeding method of Cav1 gene knockout mice and observe the expression as well as localization of Cav1 in the brain of normal C57BL/6 WT mice,for providing ideal animal model to further study the role and mechanism of Cav1 in the cerebral ischemia injury.Methods: According to the primer sequences provided by the Jackson Laboratory of America,the polymerase chain reaction(PCR)was used to amplify mouse tail genomic DNA and agarose gel electrophoresis was used to detect the genotypes of Cav1 gene knockout mice;With the following four different ways of mating: Cav1 heterozygote intercrossing,Cav1 heterozygous and homozygous mateing(orthogonal and reverse cross)as well as homozygous intercrossing,the parental mice’s pregnancy rate and offspring mice’shomozygous rate were observed;IHC staining was used to detect the expression and distribution of Cav1 in the brain of normal C57BL/6 WT mice.Results: Agarose gel electrophoresis results suggested the size of the PCR products was about 200 bp and 661 bp that was in consistent with the expected target gene fragment,and PCR method identified Cav1 gene knockout mice of different genotypes successfully.The offspring mice’s genotype distribution of the four different mating patterns mentioned above were basically in agreement with Mendel rule,the homozygous rate was 23.52%,47.36%,52.54% and 100%,respectively;Both the female and male Cav1 homozygous mice had the ability of breeding,the female mice’s pregnancy rate of Cav1 heterozygote intercrossing,Cav1 heterozygous and homozygous mateing(orthogonal and reverse cross)as well as homozygous intercrossing was 100%,87.50% and 75%,respectively,moreover,there was significant difference in the pregnancy rate between the heterozygous intercrossing and the other three reproductive modes in female mice(all P < 0.05,n=8).The expression of Cav1 was mainly located in the cell membrane and cytoplasm in neurons,glial cells and endothelial cells,and which was extremely abundant in neurons;In addition,Cav1 positive cells were widely distributed in the cortex, striatum,hippocampus,subventricular zone and other areas.Conclusion: PCR can identify the genotypes of Cav1 knockout mice fastly and reliably;Combining the breeding methods of intercrossing of Cav1 heterozygous mice and intercrossing of Cav1 homozygous mice is probably a good way to obtain enough homozygous mice and homologous WT mice in the short term;Cav1 is expressed extensively in the brain of normal C57BL/6 WT mice.Part 2 Activation and role of autophagy in the penumbra after cerebral ischemiaObjective: To observe the activation and change rule of autophagy in the penumbra after focal cerebral ischemia,explore the role of autophagy in cerebral ischemia injury and find a novel effective target for the prevention and treatment of ischemic stroke.Methods: Focal cerebral ischemia model was established by the method of p MCAO.The C57BL/6 WT mice were randomly divided into Sham group and Model group,then the ratio of microtubule-associated protein 1 light chain 3 Ⅱ(LC3-Ⅱ)/LC3-Ⅰand expression of ubiquitin binding protein P62(sequestosome 1,SQSTM1)were detected by western blot(WB)in the penumbra cortex at 3h,6h,12 h and 24 h in mice subjected to cerebral ischemia,besides,the number of autophagome(AP)was also observed through transmission electron microscopy(TEM),for clarifying the activation and change rule of autophagy in the penumbra after cerebral ischemia.In order to further confirm the role of autophagy in the penumbra following cerebral ischemia injury,we utilized the method of bidirectional regulation of autophagy by the autophagy inhibitor 3-methyladenine(3-MA)and inducer Rapamycin.Then,the C57BL/6 WT mice were randomly divided into Sham,Model,3-MA,Rapamycin and 5% dimethyl sulfoxide(DMSO)group,which was intraperitoneally injected with 3-MA(15mg·kg-1·d-1),Rapamycin(6mg·kg-1·d-1)and 5% DMSO respectively for 7d before p MCAO.The neurological function was evaluated at 3h,6h,12 h and 24 h post cerebral ischemia,after that we evaluated the cerebral infarct volume,histopathological change,the expression of autophagy marker protein P62 and the ratio of LC3-Ⅱ/LC3-Ⅰas well as the number of AP in the penumbra cortex of mice at the time point of autophagy peak post ischemic stroke by 2,3,5-triphenyltetrazolium chloride(TTC)staining,hematoxylin and eosin(HE)staining,WB and TEM technology,respectivly.Results:(1)The activation of autophagy in the penumbra following cerebral ischemia In order to observe the activation of autophagy and change rule in the penumbra following cerebral ischemia,we detected the autophagy level of cerebral ischemic penumbra cortex in C67BL/6 WT mice at 3h,6h,12 h and 24 h post p MCAO with the WB and TEM technology.WB results showed that the LC3-Ⅱ/LC3-Ⅰratio in Model group at the four time points increased significantly(all P < 0.05,n=4),and the expression of P62 reduced significantly in comparison with Sham group(all P < 0.05,n=4);Compared with Model group(p MCAO 3h),the LC3-Ⅱ/LC3-Ⅰratio at 6h post p MCAO further increased significantly(P < 0.05,n=4),the expression of P62 further reduced significantly(P < 0.05,n=4);Compared with Model group(p MCAO 6h),the LC3-Ⅱ/LC3-Ⅰratio at 12 h post p MCAO reduced significantly(P < 0.05,n=4),while P62 were significantly upregulated(P < 0.05,n=4);The LC3-Ⅱ/LC3-Ⅰratio and expression of P62 at 24 h post p MCAO had no significant difference in comparison with Model group(p MCAO 12h)(all P > 0.05,n=4).TEM results showed that only a small amount of APs formed in Sham group;The number of AP in Model group was significantly increased in comparison with Sham group(P < 0.05,n=4);Compared with Model group(p MCAO 3h),the number of AP at 6h post p MCAO further increased significantly(P < 0.05,n=4);While the number of AP at 12 h post p MCAO reduced significantly when compared with Model group(p MCAO 6h)(P < 0.05,n=4);However,no significant difference in the number of AP at 24 h post p MCAO was found when compared with Model group(p MCAO 12h)(P > 0.05,n=4).The above results indicated that autophagy in the penumbra was activated after focal cerebral ischemia,the autophagy level increased at 3h,peaked at 6h,began to decline at 12 h and still maintain a high level at 24 h,which indicated an obvious dynamic change rule.(2)The role of penumbra autophagy in cerebral ischemia injury In order to clarify the role of penumbra autophagy in cerebral ischemic injury,we used the method of bidirectional regulation of autophagy via inhibiting and inducing autophagy,the neurological function was evaluated at 3h,6h,12 h and 24 h after cerebral ischemia in C57BL/6 WT mice,moreover,the cerebral infarct volume,histopathological change,the expression of autophagy marker protein P62 and the ratio of LC3-Ⅱ/LC3-Ⅰas well as the number of AP in the penumbra cortex of mice with ischemic stroke at 6h by TTC staining,HE staining,WB and TEM technology,respectively.The neurological deficit score results demonstrated that the scores of Model group at the four time points post p MCAO were all significantly increased in comparison with Sham group of the same time(all P < 0.05,n=4);Compared with Model group of the same time,the neurological deficit scores of 3-MA group at the four time points after p MCAO were all significantly decreased(all P < 0.05,n=4),while that of Rapamycin group was significantly increased(all P < 0.05,n=4),however,no significant difference of the neurological deficit scores was found in 5% DMSO group(all P > 0.05,n=4).TTC staining suggested that normal brain tissue was bright red,and infarct brain tissue was white;Compared with Sham group,the relative infarct volume of Model group increased significantly(P < 0.05,n=4);Compared with Model group,the relative infarct volume of 3-MA group decreased significantly(P < 0.05,n=4),and that of Rapamycin group increased significantly(P < 0.05,n=4),however,no significant difference of the relative infarct volume was found in 5% DMSO group(P > 0.05,n=4).HE staining results showed that the cortical tissue of Sham group was normal in morphology,the structure was clear and arranged neatly,the neuronal somas were round or fusiform,the nuclei were larger,the staining was light,the nucleoli was clear,the caryotheca was intact,the nuclei of the glial cells were smaller and the staining was darker than the neurons,the nerve fibers were in the consistent shape,the staining of mesenchyme was uniform.Compared with Sham group,the histomorphological changes of the penumbra cortical tissue of Model group showed obvious pathological changes after ischemia,the tissue structure was in disorder,the number of neurons decreased,the arrangement of the cells was irregularity and the cell gap was increased,partial shrinking cells showed eosinophilic change,karyopyknosis and darker-staining,the cell morphology became triangular or elongated,a small amount of glial cells gathered,the nerve fibers were in the disorder shape,the mesenchyme was edema,the staining was light,but there were still some normal neurons;Compared with Model group,the pathological change of 3-MA group was improved while Rapamycin group was worse,and no obvious difference was found in 5% DMSO group.WB results suggested that the ratio of LC3-Ⅱ/ LC3-Ⅰin Model group was significantly higher(P < 0.05,n = 4),while the expression of P62 was downregulated when compared with Sham group(P < 0.05,n=4);Compared with Model group,the ratio of LC3-Ⅱ/ LC3-Ⅰin 3-MA group was significantly lower(P < 0.05,n = 4),while the expression of P62 was upregulated(P < 0.05,n=4),and the results of Rapamycin group were contrary to3-MA group(all P < 0.05,n = 4),besides,no significant difference of LC3-Ⅱ/ LC3-Ⅰratio and P62 expression were found in 5% DMSO group(all P > 0.05,n = 4).TEM results suggested that only a small amount of APs were formed in Sham group;Compared with Sham group,the number of AP in Model group was significantly increased(P < 0.05,n = 4);Compared with Model group,the number of AP in the 3-MA group decreased significantly(P < 0.05,n = 4),while the results of Rapamycin group were contrary to 3-MA group(P < 0.05,n = 4),and no significant difference of the number of AP was found in 5% DMSO group(P > 0.05,n = 4).The above results showed that pretreatment of autophagy inhibitor 3-MA reduced the neurological deficit score,the relative infarct volume and improved the penumbra cortex pathological morphology,which could play a neuroprotective role in the cerebral ischemia injure in mice by downregulating of the auophagy level in the penumbra cortex,with inhibiting the transformation of LC3-Ⅰto LC3-Ⅱand P62 protein degradation as well as AP formation;However,the autophagy inducer Rapamycin pretreatment aggravated the brain damage.Conclusion: Autophagy is activated in the penumbra after focal cerebral ischemia,and it has obvious dynamic changes;The penumbra autophagy plays a role of damage in permanent cerebral ischemic injury.cerebral ischemia through the m TOR/ULK1 pathway Part 3 Cav1 regulates autophagy in the penumbra afterObjective: To clarify the role and possible molecular mechanisms of Cav1 in the regulation of autophagy in the penumbra after cerebral ischemia,further enrich the understanding of autophagy mechanism of cerebral ischemic injury and provide potential molecular targets for clinical prevention and treatment of ischemic stroke.Methods: The model of focal cerebral ischemia was established by p MCAO method.The Cav1 knockout(Cav1 KO)mice and homologous WT mice were randomly divided into Sham group and Model group.The ratio of LC3-Ⅱ/LC3-Ⅰand the expression of P62 in the penumbra were detected by WB,and the number of AP was observed by TEM at the autophagy peak 6h after cerebral ischemia,for clarifying the role of Cav1 in the regulation of penumbra autophagy after cerebral ischemia.To further elucidate the molecular mechanism of Cav1 in the regulation of autophagy in the penumbra after cerebral ischemia,the expression of m TOR,p-m TOR(Ser2448)and ULK1,p-ULK1(Ser757)protein in the cerebral ischemic penumbra were detected by WB,and the expression of p-m TOR(Ser2448)and p-ULK1(Ser757)were further detected by IHC,real time quantitative PCR(RT-q PCR)was used to detect the m RNA expression of m TOR and ULK1.Results:(1)The role of Cav1 in the regulation of penumbra autophagy after cerebral ischemia In order to clarify the role of Cav1 in the regulation of penumbra autophagy after cerebral ischemia,we detected the autophagy level of the penumbra cortical tissue at 6 h in WT and Cav1 KO mice with ischemic stroke by WB and TEM technology.WB results showed that no significant difference was found on the ratio of LC3-Ⅱ/ LC3-Ⅰand the expression of P62 protein in Cav1 KO Sham group when compared with WT Sham group(all P > 0.05,n = 4);The ratio of LC3-Ⅱ/ LC3-Ⅰin WT Model group was significantly higher than that in WT Sham group(P < 0.05,n = 4),and P62 protein expression was further significantly downregulated(P < 0.05,n = 4);Compared with WT Model group,the ratio of LC3-Ⅱ/ LC3-Ⅰin Cav1 KO Model group was further significantly upregulated(P < 0.05,n = 4),and the P62 protein expression was significantly downregulated(P < 0.05,n=4).TEM results showed that only a small amount of APs were detected in the cortical penumbra in WT Sham group;Compared with WT Model group,the number of AP in Cav1 KO Sham group was not significantly different from that in WT Model group(P > 0.05,n = 4);The number of AP in WT Model group was significantly increased than that in WT Sham group(P < 0.05,n=4);Compared with WT Model group,the number of AP in Cav1 KO Model group further increased significantly(P < 0.05,n = 4).The above results showed that Cav1 knockout did not affect the level of autophagy in the normal brain,but which could upregulate of the autophagy level via promoting the transformation of LC3-Ⅰto LC3-Ⅱand P62 protein degradation and increase the number of AP in the penumbra cortex after focal cerebral ischemia.(2)The molecular mechanism of Cav1 on regulating autophagy in the penumbra after cerebral ischemia In order to further elucidate the molecular mechanism of Cav1 on regulating autophagy in the penumbra after cerebral ischemia,we used WB,IHC and RT-q PCR to detect the protein expression of the m TOR,p-m TOR(Ser2448)and ULK1,p-ULK1(Ser757)and m RNA expression of m TOR,ULK1.The results of WB showed that there was no significant difference on the protein expression of m TOR,p-m TOR(Ser2448)and ULK1,p-ULK1(Ser757)in Cav1 KO Sham group when compared with WT Sham group(all P > 0.05,n = 4);The protein expression of p-m TOR(Ser2448)and p-ULK1(Ser757)were downregulated in WT Model group(all P < 0.05,n = 4),but there was no significant difference on the protein expression of m TOR and ULK1 between WT Sham group and Cav1 KO Sham group(all P > 0.05,n = 4);Compared with WT Model group,the protein expression of p-m TOR(Ser2448)and p-ULK1(Ser757)in Cav1 KO Model group was further downregulated(all P < 0.05,n = 4),while no significant difference was found on m TOR and ULK1 protein expression(all P > 0.05,n = 4).IHC results showed that a large number of p-m TOR(Ser2448)and p-ULK1(Ser757)positive cells were found in the cortical brain tissue of WT Sham group,both of which were mainly expressed in the cytoplasm;Compared with WT Sham group,the p-m TOR(Ser2448)and p-ULK1 (Ser757)positive cells and mean optical density(MOD)were not significantly different in Cav1 KO Sham group(all P > 0.05,n = 4),while the p-m TOR(Ser2448)and P-ULK1(Ser757)positive cells and MOD were significantly decreased in WT Model group(all P < 0.05,n = 4);Compared with WT Model group,the number of p-m TOR(Ser2448)and p-ULK1(Ser757)positive cells in Cav1 KO Model group were further significantly reduced(all P < 0.05,n = 4).The results of IHC were consistent with those of WB.RT-q PCR results showed that there was no significant difference on the expression of m TOR and ULK1 m RNA between Cav1 KO Sham group and WT Model group(all P > 0.05,n = 4);Compared with WT Model group,there was no significant difference on the expression of m TOR and ULK1 m RNA in Cav1 KO Model group(all P > 0.05,n = 4);The m RNA results were consistent with the protein expression.The above results suggested that Cav1 knockout did not affect the level of key sites phosphorylation and protein as well as m RNA expression of m TOR and ULK1 in normal brian,but which downregulated the expression of p-m TOR(p-m TOR)Ser2448)and p-ULK1(Ser757)protein while had no effect on m TOR and ULK1 protein and m RNA expression in the penumbra after cerebral ischemia.Conclusion: Cav1 has definite regulatory effect on autophagy in the penumbra after cerebral ischemia.It may be the molecular mechanism of Cav1 on regulating autophagy in the penumbra after cerebral ischemia to controll the phosphorylation of p-m TOR(Ser2448)and p-ULK1(Ser757)at a certain level and affect the activity of m TOR/ULK1 pathway.Part 4 The mechanism of Buyang Huanwu Decoction on regulating autophagy against cerebral ischemia via Cav1/ m TOR/ULK1 pathwayObjective: To investigate the mechanism of BHD on cerebral ischemic injury from the perspective of autophagy mediated by Cav1/m TOR/ULK1 pathway,provide experimental evidence for clinical application of this prescription in the prevention and treatment of ischemic stroke.Methods: With the method of drug pretreatment,we induced autophagy by Cav1 gene knockout and Rapamycin to double block Cav1/m TOR/ULK1 pathway,for elucidating the mechanism of BHD against cerebral ischemia injury.The C57BL/6 WT mice were randomly divided into Model group,BHD group and BHD + Rapamycin group;The Cav1 KO mice were randomly divided into BHD group and BHD + Rapamycin group.Then,Rapamycin(6 mg ·kg-1 · d-1)intraperitoneal injection and BHD(18.5g · kg-1 · d-1)intragastric administration were pretreated for 7 days,respectively.The model of focal cerebral ischemia was established by p MCAO method.The neurological deficit score was evaluated at 3h,6h,12 h and 24 h after cerebral ischemia.Then,the infarct volume was measured by TTC staining and the relative infarct volume was calculated,HE staining was used to observe the histopathological changes of the penumbra cortex,WB was used to detect the ratio of LC3-Ⅱ/ LC3-I and the expression of P62,and the number of AP was observed by TEM at the autophagy peak 6h.In order to further elucidate the molecular mechanism of BHD on autophagy in the penumbra after cerebral ischemia,we examined the expression of m TOR,p-m TOR(Ser2448)and ULK1,p-ULK1(Ser7548)by WB,at the same time,p-m TOR(ser2448)and p-ULK1(Ser757)protein expression was detected by IHC.And RT-q PCR was used to detect the m RNA expression of m TOR and ULK1.Results:(1)Autophagy mechanism of BHD against cerebral ischemic injury In order to clarify the relationship between the protective effect of BHD and the level of autophagy in the penumbra after cerebral ischemia,we examined the neurological function of mice at 3h,6h,12 h and 24 h after p MCAO.Then,the relative infarct volume,histopathology of penumbra cortex,the ratio of LC3-Ⅱ/ LC3-Ⅰand the expression of P62 as well as the number of AP were detected at the autophagy peak 6h.The results of neurological deficit score showed that the scores of WT BHD group were significantly lower at four time points when compared with WT Model group of the same time(all P < 0.05,n = 4).Compared with WT BHD group of the same time,the scores of WT BHD + Rapamycin group,Cav1 KO BHD group and Cav1 KO BHD + Rapamycin group were increased in varying degrees at four time points after p MCAO(all P < 0.05,n = 4),moreover,the scores of Cav1 KO BHD + Rapamycin group increased more significantly(P <0.05,n = 4).TTC staining showed that the normal brain tissue was bright red and the infarct brain tissue was white;Compared with WT Model group,the relative infarct volume of WT BHD group was significantly lower than that of WT BHD group(P < 0.05,n = 4);Compared with WT BHD group,the relative infarct volume of WT BHD + rapamycin group,Cav1 KO BHD group and Cav1 KO BHD + Rapamycin group were increased in varying degrees(all P < 0.05,n = 4),moreover,the relative infarct volume of Cav1 KO BHD + Rapamycin group increased more significantly(P <0.05,n = 4).The results of HE staining showed that obvious pathological changes in the penumbra cortex tissue were detected in WT Model group,the tissue structure was in disorder,the number of neurons decreased,the arrangement of the cells was irregularity and the cell gap was widened,partial shrinking cells showed eosinophilic change,karyopyknosis and darker-staining,the cell morphology became triangular or elongated,a small amount of glial cells gathered,the nerve fibers were in the disorder shape,the mesenchyme was edema,the staining was light,but there were still some normal neurons;Compared with Model group,the pathological change of WT BHD group was marked improved,the nucleus pyknosis cells in the cortical penumbra were significantly decreased,the cell morphology was improved,the cells were neatly arranged,the nucleus was clear and the cell gap was more uniform.Compared with WT BHD,the pathological morphology of WT BHD + Rapamycin group,Cav1 KO BHD group and Cav1 KO BHD + Rapamycin group showed different degrees of deterioration,and among which Cav1 KO BHD + Rapamycin group was more serious.The results of WB showed that the ratio of LC3-Ⅱ/ LC3-Ⅰin WT BHD group was significantly lower than that in WT Model group(P < 0.05,n = 4),the P62 expression was significantly upregulated(P < 0.05,n = 4);Compared with WT BHD group,the ratio of LC3-Ⅱ/ LC3-Ⅰin WT BHD + Rapamycin group,Cav1 KO BHD group and Cav1 KO BHD + Rapamycin group was significantly upregulated in different degrees(all P < 0.05,n = 4),and the expression of P62 was decreased in different degrees(all P <0.05,n = 4),and the expression of LC3-Ⅱ/ LC3-Ⅰand P62 protein in Cav1 KO BHD + Rapamycin group were more significant(P <0.05,n = 4).TEM results showed that a large number of APs were found in the cortical penumbra of WT Model group;Compared with WT Model group,the number of AP in WT BHD group was significantly decreased(P < 0.05,n = 4);Compared with WT BHD group,the number of AP in WT BHD + Rapamycin group,Cav1 KO BHD group and Cav1 KO BHD + Rapamycin group were all significantly increased in different degrees(all P < 0.05,n = 4),and the number of AP in Cav1 KO BHD + Rapamycin group increased more significantly(P <0.05,n = 4).The above results showed that pretreatment of BHD reduced the neurological deficit score and relative cerebral infarction volume as well as improve the pathological morphology of penumbra cortex tissue in mice subjected to p MCAO,played a protective role in cerebral ischemia jnjure through downregulating the autophagy level in the penumbra cortex,with inhibiting the transformation of LC3-Ⅰto LC3-Ⅱ,the degradation of P62 protein and reducing the formation of AP.However,the autophagy inducer Rapamycin and /or knockout Cav1 offset the effect in varying degrees.(2)The molecular mechanism of BHD on regulating autophagy in the penumbra after cerebral ischemia In order to further elucidate the molecular mechanism of BHD on regulating autophagy in cerebral ischemia penumbra,we used WB,IHC and RT-q PCR to detect the m TOR,P-m TOR(Ser2448)and ULK1,p-ULK1(Ser757)protein expression and m TOR,ULK1 m RNA expression of penumbra cortex cerebral ischemia in mice at 6h after p MCAO.WB results showed that the protein expression of p-m TOR(Ser2448)and p-ULK1(Ser757)in WT BHD group was significantly upregulated when compared with WT Model group(all P < 0.05,n = 4),while no significant difference was found on the expression of m TOR and ULK1 protein(all P > 0.05,n = 4);Compared with WT BHD group,the expression of p-m TOR(Ser2448)and p-ULK1(Ser757)protein in WT BHD + Rapamycin group and Cav1 KO BHD group were both significantly downregulated(all P < 0.05,n = 4),while the expression of which in Cav1 KO BHD + Rapamycin group was more significantly lower(all P < 0.05,n =4).The results of IHC showed that only a small amount of p-m TOR(Ser2448)and p-ULK1(Ser757)positive cells were found in WT Model group,both of which were mainly expressed in the cytoplasm;Compared with WT Model group,the p-m TOR(Ser2448)and p-ULK1(Ser757)positive cells and MOD in WT BHD group were significantly increased(all P < 0.05,n = 4);Compared with WT BHD group,the p-m TOR(Ser2448)and p-ULK1(Ser757)positive cells as well as MOD in WT BHD + Rapamycin group and Cav1 KO BHD group were significantly decreased(all P < 0.05,n = 4),while that in Cav1 KO BHD + Rapamycin group decreased more markly(all P < 0.05,n = 4).WB results are consistent with the results of IHC.RT-q PCR results showed that the expression of m TOR and ULK1 m RNA in WT BHD group were not significantly different from that in WT Model group(all P > 0.05,n = 4);Compared with WT BHD group,there was no significant difference on the m TOR and ULK1 m RNA expression in WT BHD + Rapamycin group,Cav1 KO BHD group and Cav1 KO BHD+Rapamycin group(all P > 0.05,n = 4);The m RNA expression were consistent with the protein expression.The above results indicated that the pretreatment of BHD could upregulate the expression of p-m TOR(Ser2448)and p-ULK1(Ser757)protein in the penumbra cortex after focal cerebral ischemia in mice,while with no effect on the protein expression of m TOR and ULK1 and m RNA expression.Conclusion: It may be one of the mechanisms of brain protective function of BHD to inhibit the level of autophagy in the penumbra after cerebral ischemia via increasing phosphorylation levels of m TOR(Ser2448)and ULK1(Ser757)protein and regulating the activity of Cav1/m TOR/ULK1 pathway. |
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