| Objective:To clarify the effect of acupuncture early intervention on prolonging the thrombolytic time window of cerebral infarction,and to explore the mechanism of acupuncture prolonging the thrombolytic time window of cerebral infarction from the interaction of autophagy and apoptosis.Methods:SPF grade SD rats were used,the modified autologous thromboembolic model was used as the research object of cerebral infarction,rtPA was used as the intravenous thrombolytic drug,and the "Xing Nao Kai Qiao" acupuncture method was used as the intervention method.It is divided into two parts:experiment 1 and experiment 2.Experiment 1 80 SD rats were randomly divided into sham operation group,model group,thrombolysis group 4.5h group,6h thrombolysis group,thrombolysis 7.5h group,acupuncture+4.5h thrombolysis group,acupuncture+6h Thrombolysis group,acupuncture+ 7.5h thrombolysis group.The effects of early acupuncture intervention on cerebral infarction volume,neurobehavioral scores,intracranial thrombolytic hemorrhagic transformation and cerebral edema in each group of rats were observed to determine the effect of acupuncture on prolonging the time window of thrombolysis for cerebral infarction.Experiment 2 After clarifying the effect of acupuncture on prolonging the time window of thrombolysis in cerebral infarction,50 SD rats were randomly divided into sham operation group,model group,thrombolysis group 6h group,acupuncture+6h thrombolysis group;Western blot was used,Real time-PCR to detect the expression of autophagy-lysosomal marker proteins LC3,Beclinl and p62 among different groups.The group remains unchanged.Western blot was used to observe the expression of the anti-apoptotic protein Bcl2 and the pro-apoptotic protein Bax in different groups,and observe the autophagy and apoptosisResult:(1)The neurobehavioral score of rats in each group:24h after treatment,compared with the model group,the neurobehavior score of the 4.5h thrombolysis group was lower(P<0.05),while the thrombolysis group outside the time window,the 6h thrombolysis group.There was no statistically significant difference in behavioral scores between the 7.5h thrombolysis group(P>0.05).Compared with the model group and the 6h thrombolysis group,the neurobehavioral scores of the acupuncture+6h thrombolysis group were reduced(P<0.05).However,compared with the model group and 7.5h thrombolysis group,the neurobehavioral scores of the acupuncture+7.5h thrombolysis group were not statistically different(P>0.05). It shows that acupuncture can improve the neurobehavioral score of thrombolytic rats outside the thrombolytic time window(6h).(2)The percentage of cerebral infarction volume of rats in each group:Compared with the model group,the volume of cerebral infarction in the 4.5h thrombolysis group,acupuncture+4.5h thrombolysis group,and acupuncture+6h thrombolysis group decreased(P<0.05).Compared with acupuncture+6h thrombolysis group and 6h thrombolysis group,the infarct volume was reduced(P<0.05).It shows that acupuncture can reduce the cerebral infarct volume of thrombolytic rats outside the time window(6h).(3)Cerebral hemorrhagic transformation of rats in each group:Compared with the model group,the hemoglobin content of the 6h thrombolysis group,7.5h thrombolysis group,and 7.5h acupuncture thrombolysis group were all increased(P<0.05),indicating over time Thrombolysis outside the window increases the risk of hemorrhagic transformation.Compared with acupuncture+6h thrombolysis and 6h thrombolysis,the hemoglobin content decreased(P<0.05).It shows that acupuncture can reduce the cerebral hemorrhagic transformation of thrombolytic rats outside the time window(6h).(4)Brain water content of rats in each group:Compared with the model group,the brain water content of the 4.5h thrombolysis group,acupuncture+4.5h thrombolysis group,and acupuncture+6h thrombolysis group all decreased(P<0.05);The brain water content of the thrombolysis group and 7.5 thrombolysis group increased at 6 hours outside the time window(P<0.05).Acupuncture+6h thrombolysis group compared with 6h thrombolysis group,the brain water content was lower(P<0.05).It shows that acupuncture can reduce the brain water content of thrombolytic rats outside the time window(6h).(5)Expression of autophagy level of cortical nerve cells in the IP area of rats in each group:①The WB results showed that,Compared with the sham operation group,the protein expression levels of Beclinl and LC3 in each group were increased(P<0.05),while the protein expression levels of P62 groups were significantly decreased(P<0.05).Compared with the model group,the expressions of Beclinl,LC3 and P62 in the 6h thrombolysis group showed no statistical differences(P>0.05),while the expressions of Beclinl and LC3 in the acupuncture+6h thrombolysis group increased,while the expression of P62 decreased(P<0.05).Compared with the 6h thrombolytic group,the expressions of Beclinl and LC3 in the acupuncture+6h thrombolytic group were increased,while the expression of P62 was decreased(P<0.05).②The RT-PCR results showed that,Compared with the sham operation group,the mRNA expression levels of Beclinl and LC3 in each group were increased,while the mRNA expression of P62 was decreased(P<0.05).Compared with the model group,the mRNA expressions of Beclinl and LC3 in the 6h thrombolytic group and the acupuncture+6h thrombolytic group were increased,while the mRNA expression of P62 in the acupuncture+6h thrombolytic group was decreased(P<0.05).Compared with the 6h thrombolytic group,the mRNA expressions of Beclinl and LC3 in the acupuncture+6h thrombolytic group were increased,while the expression of P62 was decreased(P<0.05).These results indicated that acupuncture could up-regulate Beclinl and LC3,and down-regulate the protein and mRNA expression levels of lysosomal marker p62 in rats with thrombecysis outside the window of time,and then moderately activate autophagy.(6)Expression of neuronal apoptosis in the cerebral cortex of rats in each group:WB results show that compared with the sham operation group,the expression of Bcl2 and Bax in the model group,6h thrombolysis group,and acupuncture+6h thrombolysis group increased(p<0.05).Compared with the model group,the expression of Bcl2 and Bax in the 6h thrombolysis group was not statistically different(p>0.05),but the expression of Bcl2 in the acupuncture+6h thrombolysis group increased,and the expression of Bax decreased(p<0.05).Compared with the 6h thrombolysis group,the Bcl2 expression in the acupuncture+6h thrombolysis group increased,and the Bax expression decreased(p<0.05),indicating that acupuncture can up-regulate the expression of anti-apoptotic Bcl2 protein in thrombolytic rats outside the time window,and down-regulate and promote apoptosis.Decrease the expression level of Bax protein,thereby inhibiting apoptosis.(7)The ratio of Bcl2/Beclinl and Bcl2/Bax in the cerebral cortex nerve cells of rats in each group:compared with the sham operation group,the ratio of Bcl2/Beclinl in the model group was up-regulated,the ratio of Bcl2/Bax was down-regulated,and the 6h thrombolytic group,Acupuncture+6h thrombolysis group Bcl2/Beclinl ratio decreased,Bcl2/Bax ratio increased(p<0.05);compared with the model group,6h thrombolysis group,acupuncture+6h thrombolysis group Bcl2/Beclinl ratio decreased.The ratio of Bcl2/Bax increased(p<0.05).Compared with the 6h thrombolysis group,the Bcl2/Beclin1 ratio was down-regulated and the Bcl2/Bax ratio was up-regulated in the acupuncture+6h thrombolysis group(p<0.05).Conclusion:(1)Timely intervention of acupuncture can reduce the complications of cerebral infarction thrombolysis and extend the safe thrombolysis time window to 6h;(2)The effect of acupuncture on prolonging the time window of thrombolysis in cerebral infarction is achieved by moderately activating neuronal autophagy after cerebral infarction and inhibiting apoptosis. |
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