| Objective:To observe the effect of "Xingnao Kaiqiao" electroacupuncture on neurovascular units and Wnt/ β-catenin signal pathway in(Ischemic Cerebral Stroke,ICS)rats with ischemic stroke,and to explore the mechanism of electroacupuncture in the treatment of ischemic stroke.Methods:(1)Animal grouping and model preparation.Forty-eight healthy male SD rats were randomly divided into control group(n = 12),model group(n = 12),electroacupuncture group(n = 12),electroacupuncture group(n =12)and blocker group(n = 12).The rat model of middle cerebral artery occlusion(MCAO)was made according to Zea Longa thread occlusion method and modified(MCAO),.According to Zea Longa neural function score,the model reached 1-3 points in accordance with the inclusion criteria.(2)Intervention methods.1.Electroacupuncture intervention refers to the acupuncture method of "Xingnao Kaiqiao" initiated by Academician Shi Xuemin of Tianjin University of traditional Chinese Medicine.The electroacupuncture group was given acupuncture at "Baihui","Shuigou",bilateral "Sanyinjiao" and bilateral "Neiguan".The ipsilateral "Neiguan" and "Sanyinjiao" were connected to the same electrode,and dense wave,2 Hz/100 Hz,2～4 V,and needle retention for 30 min were applied in the electroacupuncture group.The first electroacupuncture was performed within 2 hours after operation,and then once every morning with the same method for 7 days.2.Before the establishment of the model,the rats in the xav939 blocker group were injected in lateral ventricle with xav-939 0.1mg/kg,a blocker of Wnt/ β-catenin pathway.(3)Evaluation of neurological function.1.Neurological function score of rats in each group after 7 days of intervention,according to Zealonga score,0: no neurological defect;1: right forelimb flexion,without other abnormalities;2: turning on the right side when crawling;3: difficulty in walking,dumping to the right;4: unable to walk spontaneously,loss of consciousness.two。.Falling latency test after 7 days of intervention,roller exercise test was used to observe the falling latency of rats in each group.(4)Evaluation of cerebral infarction volume.The changes of cerebral infarction volume of rats in each group were detected under the in vivo mechanism of 9.4T MRI.(5)Detection by molecular biological methods.1.Immunohistochemical method was used to detect the expression of VEGF,GFAP,Neu N,Wnt3 a,β-catenin,GSK-3 β and Axin2 positive cells in ischemic brain tissue of rats.The protein expressions of VEGF,GFAP,Neu N,Wnt3 a,β-catenin,GSK-3 β and Axin2 in ischemic brain tissue of rats were detected by 2.Western blot.The relative expressions of VEGF m RNA,GFAP m RNA,Neu N m RNA,Wnt3 a m RNA,β-catenin m RNA,GSK-3 β m RNA and Axin2 m RNA in ischemic brain tissue were detected by RT-q PCR.Results:(1)Evaluation of neurological function.1.Zea Longa score.There was no neurological deficit in the control group,compared with the control group,the neurological function of the model group was significantly impaired(P < 0.01),the neurological function score of the blocker group was significantly lower than that of the model group(P < 0.01),and the neurological function score of the electroacupuncture group was significantly lower than that of the blocker group(P < 0.01).two。2.Fall latency test.The rats in the control group could maintain balance and motor rhythm continuously in the inclined rotating drum,and the falling latency of the model group was significantly lower than that of the control group(P < 0.01).Compared with the model group,the falling latency of rats in the blocker group and electroacupuncture group was increased(P < 0.01),and the falling latency in the electroacupuncture group was significantly higher than that in the blocker group(P < 0.01).(2)the volume of cerebral infarction in vivo(9.4T MRI).There was no cerebral infarction in the control group.Compared with the control group,cerebral infarction occurred in model group,blocker group and electroacupuncture group.Compared with the model group,the cerebral infarction volume in the blocker group was significantly lower than that in the blocker group(P < 0.01),and that in the electroacupuncture group was significantly lower than that in the blocker group(P <0.01).(3)Immunohistochemical method was used to detect the expression of VEGF,GFAP,Neu N,Wnt3 a,β-catenin,GSK-3 β and Axin2 positive cells in ischemic brain tissue of rats.After 7 days of intervention,compared with the control group,the positive expression of VEGF and GFAP in the model group was significantly higher than that in the model group,the positive expression of VEGF and GFAP in the blocker group was significantly higher than that in the model group(P > 0.05,P < 0.01),and the positive expression of VEGF and GFAP in the electroacupuncture group was significantly higher than that in the blocker group(P < 0.01,P < 0.01).Compared with the control group,the positive expression of Neu N in the model group decreased significantly,compared with the model group,the positive expression of Neu N in the blocker group increased significantly,and the positive expression of Neu N in the electroacupuncture group increased significantly compared with the blocker group.Compared with the control group,the positive expression of Wnt3 and β-catenin in the model group increased(P <0.01,P < 0.01),the positive expression of Wnt3 and β-catenin in the blocker group increased compared with the model group(P > 0.05,P < 0.01),and the positive expression of Wnt3 and β-catenin in the electroacupuncture group increased compared with the blocker group(P < 0.01,P < 0.01).Compared with the control group,the positive expression of GSK-3 β and Axin2 in the model group was significantly lower than that in the model group(P < 0.01,P < 0.01),and the positive expression of GSK-3β and Axin2 in the blocker group was significantly lower than that in the model group(P < 0.01,P < 0.01).(4)Western blot was used to detect the protein expression of NVU related components(VEGF,GFAP,Neu N),Wnt/ β-catenin pathway related components(Wnt3a,β-catenin,GSK-3 β,Axin2)in ischemic brain tissue of rats.After 7 days of intervention,compared with the control group,the expression of VEGF and GFAP in the model group was significantly higher than that in the model group,the expression of VEGF and GFAP in the blocker group was significantly higher than that in the model group(P < 0.05,P < 0.05),and the expression of VEGF and GFA in the electroacupuncture group was significantly higher than that in the blocker group(P <0.05,P < 0.01).Compared with the model group,the relative expression of Neu N in the model group was significantly decreased,the positive expression of Neu N in the blocker group was increased,and the positive expression of Neu N in the electroacupuncture group was significantly higher than that in the blocker group.Compared with the control group,the relative expression of Wnt3 and β-catenin in the model group was significantly increased(P < 0.01,P < 0.01),the relative expression of Wnt3 and β-catenin in the blocker group was significantly higher than that in the model group(P < 0.01,P < 0.01),and the relative expression of Wnt3 and β-catenin in the electroacupuncture group was significantly higher than that in the blocker group(P <0.01,P < 0.01).Compared with the control group,the relative expression of GSK-3 βand Axin2 in the model group was significantly lower than that in the model group,the relative expression of GSK-3 β and Axin2 in the blocker group was significantly lower than that in the model group(P < 0.01,P < 0.01),and the relative expression of GSK-3β and Axin2 in the electroacupuncture group was significantly lower than that in the blocker group(P < 0.01,P < 0.01).(5)RT-q PCR method was used to determine the relative expression of NVU related components(VEGF,GFAP,Neu N)m RNA related to m RNA,Wnt/ β-catenin pathway(Wnt3a,β-catenin,GSK-3 β,Axin2)in ischemic brain tissue of rats in each group.After 7 days of intervention,compared with the control group,the relative expression of VEGF m RNA and GFAP m RNA in the model group was significantly higher than that in the model group,the relative expression of VEGF m RNA and GFAP m RNA in the blocker group was significantly higher than that in the model group(P < 0.05,P < 0.01),and the relative expression of VEGF m RNA and GFAP m RNA in the electroacupuncture group was significantly higher than that in the blocker group(P <0.01,P < 0.01).Compared with the control group,the relative expression of Neu N m RNA in the model group decreased significantly,compared with the model group,the relative expression of Neu N m RNA in the blocker group increased,and the relative expression of Neu N m RNA in the electroacupuncture group increased significantly compared with the blocker group.Compared with the control group,the relative expression of Wnt3 a m RNA and β-catenin m RNA in the model group was significantly increased(P < 0.01,P < 0.01),the relative expression of Wnt3 a m RNA and β-catenin m RNA in the blocker group was increased compared with the model group(P < 0.05,P< 0.05),and the relative expression of Wnt3 a m RNA and β-catenin m RNA in the electroacupuncture group was increased compared with the blocker group(P < 0.01,P <0.01).Compared with the control group,the relative expression of GSK-3 β m RNA and Axin2 m RNA in the model group was significantly lower than that in the model group,the relative expression of GSK-3 β m RNA and Axin2 m RNA in the blocker group was significantly lower than that in the blocker group(P < 0.05,P < 0.05),and the relative expression of GSK-3 β m RNA and Axin2 m RNA in the electroacupuncture group was significantly lower than that in the blocker group(P < 0.01,P < 0.01).Conclusion:1.The electroacupuncture method of "Xingnao Kaiqiao" can effectively improve the neurological function of MCAO rats,reduce the volume of cerebral infarction,and promote the reconstruction of the structure and function of neurovascular units.2."Xingnao Kaiqiao" electroacupuncture can promote the activation of Wnt/ β-catenin signal pathway in MCAO rats,and the regulation of electroacupuncture on neurovascular units is partly realized by Wnt/ β-catenin signal pathway. |
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