Experimental Study Of Acupuncture Regulating ERK1/2 Signaling To Extend Thrombolytic Time Window Of Cerebral Infarction

Objective:At present,ultra-early thrombolysis is the most effective treatment for cerebral infarction,but it must be implemented within 4.5h time window,otherwise it is easy to destroy the blood-brain barrier(BBB)and lead to complications such as cerebral edema and hemorrhagic transformation of intracranial thrombolysis,resulting in increased mortality rate.If a method of extending the time window can be found,more treatment opportunities will be won for patients.The therapeutic effect of acupuncture on cerebral infarction is generally recognized,but whether early acupuncture can improve thrombolytic safety and further extend thrombolytic time window for cerebral infarction is still unclear.Therefore,this study firstly clarifies the effect of acupuncture on extending the thrombolytic time window of cerebral infarction,and secondly discusses the mechanism of acupuncture on improving the thrombolytic safety of cerebral infarction and prolonging the thrombolytic time window from multiple levels.Methods:The acupuncture method of "Xingnao Kaiqiao" was used in this study.The improved model of cerebral ischemia caused by autologous thromboembolism was used as the research object,and rtPA was used as intravenous thrombolysis.Experiment 1:SD rats were randomly divided into sham operation group,model group,4.5h rtPA group,6h rtPA group,9h rtPA group,acupuncture+4.5h rtPA group,acupuncture+6h rtPA group,acupuncture+9h rtPA group.To observe the effect of acupuncture intervention on infarct volume,neurobehavioral score,BBB permeability,intracranial thrombolytic hemorrhagic transformation and cerebral edema of rats in each group,and to clarify the effect of acupuncture on extending thrombolytic time window of cerebral infarction.Experiment 2:SD rats were randomly divided into sham operation group,model group,6h rtPA group and acupuncture+6h rtPA group.Western blot and immunofluorescence(IF)were used to detect protein expression changes of ERK1/2 signal pathway indicators(MEK1/2,ERK1/2),transmembrane protein(Claudin5),cytoplasmic adhesion protein(ZO-1),cytoskeleton protein(MAP-2)and MMP9.Real time-PCR and Western blot were used to detect the mRNA and protein expression of NF-κB p65.The ultrastructural changes of BBB in each group were observed by transmission electron microscope.The content of inflammatory factors(TNF-α,IL-1β)in ischemic brain tissue was detected by ELISA Experiment 3:SD rats were randomly divided into sham operation group,6h rtPA group,6h rtPA group+ERK1/2 signaling pathway inhibitor(U0126)group.Western blot was used to detect protein expression changes of p-ERK 1/2、MMP9 and p-NF-κB p65.Through experiments 2 and 3,we clarified the mechanism of acupuncture regulating ERK1/2 signaling pathway to extend thrombolytic time window of cerebral infarction.Results:1.Comparison of neurobehavioral scores of rats in different groupsCompared with the model group,the neurobehavioral score of 4.5h rtPA group was decreased with statistically significant difference(P<0.01).Compared with the model group,the neurobehavioral scores of the thrombolytic group(6h rtPA group,9h rtPA group)beyond the time window had no significant difference(P>0.05).Compared with the model group and the 6h rtPA group,the neurobehavioral scores in the acupuncture+6h rtPA group were all decreased,and the difference was statistically significant(P<0.05).This indicated acupuncture improves neurobehavioral scores of ischemic rats with thrombolysis beyond the time window.2.Comparison of cerebral infarction volumes of rats in different groupsCompared with the model group,the cerebral infarction volumes in 4.5h rtPA group,acupuncture+4.5h rtPA group and acupuncture+6h rtPA group was all significantly reduced(P<0.01).The volumes of cerebral infarction in 6h rtPA group,9h rtPA group and acupuncture+9h rtPA group were all significantly larger than that in model group(p<0.01,P<0.05).Compared with 6h rtPA group,infarct volume of acupuncture+6h rtPA group was decreased significantly(P<0.01).This indicated acupuncture reduced cerebral infarction volume of ischemic rats with thrombolysis beyond the time window3.Comparison of cerebral hemorrhage transformation of rats in different groupsCompared with the model group,hemoglobin contents of 6h rtPA group,9h rtPA group and acupuncture+9h rtPA group were significantly increased(P<0.01),indicating that thrombolysis beyond the time window significantly increased the risk of hemorrhagic transformation.Compared with 6h rtPA group,the hemoglobin content of acupuncture+6h rtPA group was decreased(P<0.05).This indicated acupuncture reduced cerebral hemorrhage transformation of ischemic rats with thrombolysis beyond the time window.4.Comparison of BBB permeability of rats in different groupsCompared with the model group,EB content of 4.5h rtPA group was decreased,however,The EB content of 6h,9h rtPA group were all increased,and these differences were statistically significant(P<0.01).Compared with model group,EB content of acupuncture+4.5h rtPA group and acupuncture+6h rtPA group were all decreased,the EB content of acupuncture+9h rtPA group was increased,and these differences had statistically significant(P<0.01).Compared with 6h rtPA group,the EB content of acupuncture+6h rtPA group was decreased(P<0.05).This indicated acupuncture reduced BBB permeability of ischemic rats with thrombolysis beyond the time window.5.Comparison of brain water content of rats in different groupsCompared with the model group,the cerebral water content of 4.5h rtPA group,acupuncture+4.5h rtPA group and acupuncture+6h rtPA group were significantly reduced(P<0.01).The brain water content of 6h rtPA group and 9h rtPA group were significantly increased(P<0.01).Compared with 6h rtPA group,brain water content of acupuncture+6h rtPA group was decreased significantly(P<0.01).This indicated acupuncture reduced brain water content of ischemic rats with thrombolysis beyond the time window.6.Comparison of BBB ultrastructure of rats in different groupsTransmission electron microscopy showed that the endothelial cells and basement membrane in the model group were obviously swollen and irregular,and the tight junction was obviously destroyed.The swelling and deformation of BBB brain microvascular endothelial cells and basement membrane were further aggravated in the 6h rtPA group,and a large number of vacuoles and pinocytosis vesicles were formed,the tight junction was broken.In acupuncture+6h rtPA group,the destruction of BBB ultrastructure was reduced,endothelial cells and basement membrane were slightly swollen,and the destruction of tight junction was significantly reduced.This indicated acupuncture improved the destruction of BBB ultrastructure in ischemic rats with thrombolysis beyond the time window.7.Effect of acupuncture on ERK1/2 signal pathway in thrombolytic rats with cerebral infarctionWB and IF results showed that compared with sham operation,p-MEK1/2/t-MEK1/2 and p-ERK1/2/t-ERK1/2 in model group were increased significantly(P<0.01).Compared with 6h rtPA group and model group,p-MEK1/2/t-MEK1/2 and p-ERK 1/2/t-ERK1/2 in acupuncture+6h rtPA group were significantly decreased(P<0.01).This indicated acupuncture inhibited the activation of ERK1/2 signaling pathway in ischemic rats with thrombolysis beyond the time window.8.Comparison of MMP9 expression in each groupWB and IF results showed that compared with sham operation,the expression of MMP9 in model group was increased(P<0.01).Compared with the model group,MMP9 expression in 6h rtPA group was futher increased(P<0.05).The expression of MMP9 in acupuncture+6h rtPA group was significantly lower than that in model group and 6h rtPA group respectively(P<0.01).This indicated acupuncture attenuated MMP9 expression in ischemic rats with thrombolysis beyond the time window.9.Comparison of ZO-1,Claudin5 and MAP-2 expression in each groupWB and IF results showed that compared with sham operation,ZO-1,Claudin5 and MAP-2 expressions in model group were all decreased with statistically significant difference(P<0.01).The expression of ZO-1,Claudin5,and MAP-2 in 6h rtPA group was further decreased than that in model group(P<0.05).The expression of ZO-1,Claudin5 and MAP-2 protein in acupuncture+6h rtPA group was significantly higher than that in model group and 6h rtPA group respectively(P<0.05;P<0.01).This indicated acupuncture increased expression of ZO-1,Claudin5 and MAP-2 in ischemic rats with thrombolysis beyond the time window.10.Comparison of NF-κB p65 expression in each groupWB and RT-PCR results showed that compared with sham operation,the expression of NF-κB p65 mRNA and protein in model group was significantly increased(P<0.01).Compared with the model group,the expression level of NF-κB p65 mRNA and protein in 6h rtPA group was further increased(P<0.01).Compared with the model group and 6h rtPA group,the expression level of NF-κB p65 mRNA and protein in acupuncture+6h rtPA group were significantly decreased(P<0.01).This indicated acupuncture attenuated expression of NF-κB p65 in ischemic rats with thrombolysis beyond the time window.11.Comparison of TNF-α and IL-1β contents in each groupELISA results showed that compared with sham operation,TNF-α and IL-1β contents in brain tissue of rats of model group were increased significantly(P<0.01).Compared with the model group,TNF-α and IL-1β contents in the 6h rtPA group were further increased(P<0.01).The contents of TNF-α and IL-1βin acupuncture+6h rtPA group were significantly lower than those in model group and 6h rtPA group respectively(P<0.01).This indicated acupuncture decreased the contents of TNF-α and IL-1β in brain tissue of thrombolytic rats12.Effect of ERK1/2 signaling pathway on the expression of MMP9 and NF-κB p65WB results showed that,compared with sham operation,the expressions of p-ERK1/2,MMP9 and p-NF-κB p65 in 6h rtPA group were increased with statistically significant difference(P<0.01).Compared with 6h rtPA group,the expressions of p-ERK1/2,MMP9 and p-NF-κB p65 in 6h rtPA+U0126 group were significantly decreased(P<0.01).This indicated ERK1/2 signaling pathway was located in the upstream of MMP9 and NF-κB p65 and it can regulate the protein expression level.Conclusion:1.The timely intervention of acupuncture reduced the complications of thrombolysis,improved the safety of thrombolysis,and exert the effect of extending the thrombolytic time window of cerebral infarction.This experimental study shows that acupuncture can extend the safety time window to 6h.2.Acupuncture can inhibit the activation of ERK1/2 signaling pathway,and then down-regulate the expression of MMP9 and NF-κB p65,up-regulate the expression of blood-brain barrier structure-related proteins(Claudin5,ZO-1,MAP-2)in rats,and reduce the content of inflammatory factors(TNF-α,IL-1β)in the brain tissue,improve the destruction of the blood-brain barrier.This indicated that acupuncture can extend the thrombolytic time window effect of cerebral infarction by regulating the ERK 1/2 signal pathway.