Study On The Mechanism Of Electroacupuncture Combined With Exercise Training In Improving Ischemic Stroke Injury Based On Wnt/β-catenin Pathway

Objective: To observe the effect of electroacupuncture combined with exercise training on the improvement of ischemic stroke injury,and to explore its possible mechanism.Methods: Adult male Sprague Dawley rats were subjected to the establishment of the left middle cerebral artery occlusion(MCAO)model.The Sham operation group had the same procedure,except the suture was not inserted.The rats who successfully modeled were randomly divided into a model(MCAO)group,an electroacupuncture(EA)group,an Exercise group,and an EA plus Exercise(EA+Exercise)group.24 hours after modeling,EA was applied to Baihui(GV 20),Dazhui(GV 14),Quchi(LI 11),and Neiguan(PC 6)on the hemiplegic side in the EA group,30 minutes each time for 9consecutive days.The Exercise group performed corresponding treadmill training for 30 minutes a day for 9 days.The EA+Exercise group received EA treatment followed by exercise training for 9 days.After 9 days of treatment,neurological severity scores(NSS),Foot-Fault test,and rotarod fatigue test were used to evaluate the improvement of rats’ neurological function,balance and coordination ability.Cerebral infarction volume was detected by 2,3,5-triphenyl tetrazolium chloride(TTC)staining.Immunofluorescence was used to observe the number of the double cortex(DCX)and neural nuclei(Neu N)cells in the ischemic area and its vicinity.Western blot was used to detect the expressions of DCX and Wnt/β-catenin pathway-related proteins in the cerebral ischemic cortex of rats.Results:1.The behavioral results showed that compared with the MCAO group,the EA,Exercise,and EA+Exercise groups had significantly lower NSS scores(P<0.001).Compared with the EA and Exercise groups,the EA+Exercise group had a slightly lower NSS score(P=0.286,P=0.590).Compared with the MCAO group,the scores of the Foot-Fault test were increased in EA,Exercise,and EA+Exercise groups(P<0.05).Compared with the EA and Exercise groups,the EA+Exercise group had a slight increase in the Foot-Fault test score(P=0.101,P=0.222).Compared with the MCAO group,the residence time of the rotational fatigue test was significantly increased in the EA,Exercise,and EA+Exercise groups(P<0.01).Compared with the EA and Exercise groups,the rotarod fatigue test time was increased in the EA+Exercise group(P=0.181,P=0.308).2.TTC staining results showed that the infarct volume in EA,Exercise,and EA+Exercise groups was significantly lower than that in the MCAO group(P<0.01).The EA+Exercise group had a slightly smaller infarct volume than the EA and Exercise groups,but the difference was not significant(P=0.936,P=0.875).3.Immunofluorescence results showed that compared with the MCAO group,the number of DCX,Neu N,and DCX/Neu N cells in and around the ischemic area was increased in the EA and Exercise groups(P>0.05),and the number of DCX,Neu N,and DCX/Neu N cells in the EA+Exercise group was significantly increased(P<0.05).Compared with the EA group and the Exercise group,the number of DCX cells in and around the ischemic area in the EA+Exercise group was slightly increased(P=0.070,P=0.616),the number of Neu N cells was slightly increased(P=0.120,P=0.513),and the number of DCX/Neu N cells was slightly increased(P=0.445,P=0.938).4.Western blot results showed that compared with the MCAO group,the EA,Exercise,and EA+Exercise groups significantly increased DCX(P<0.05),Wnt3a(P<0.001),P-GSK-3β(ser9)/GSK-3β(P<0.01),Cyclin D1(P<0.05)and Ngn1(P<0.05).Compared with the EA group,the expressions of Wnt3 a and Ngn1 proteins in the ischemic cortex were slightly increased in the EA+Exercise group(P=0.537,P=0.472).Compared with the Exercise group,the expression of Wnt3 a protein in the ischemic cortex was slightly increased in the EA+Exercise group,but the difference was not significant(P=0.269).Conclusion:1.EA,exercise training,and their combination can improve balance and coordination ability,enhance exercise endurance,reduce cerebral infarction volume,and reduce neurological deficits in rats with cerebral ischemia.And electroacupuncture combined with exercise training was slightly better than electroacupuncture or exercise training alone,but the difference was not significant.2.EA,exercise training,and their combination can promote the differentiation of neural stem cells(NSCs)into DCX,migration to the infarct area,and further differentiation into mature neurons in rats with cerebral ischemia,and electroacupuncture combined with exercise has a better effect,but the difference is not significant.3.EA,exercise training,and their combination may promote the differentiation of NSCs in rats with cerebral ischemia by activating the Wnt/β-catenin pathway and promoting the expression of related proteins in this pathway.Moreover,EA combined with exercise training can increase the expression of Wnt3 a ligand protein.The activation of the Wnt/β-catenin pathway was slightly stronger than that of EA or exercised alone,but there was no significant difference.In conclusion,EA combined with exercise training can reduce cerebral infarction volume,alleviate neurological impairment in rats with cerebral ischemia,promote the differentiation of NSCs in the ischemic area and its adjacent areas into DCX and further differentiate into mature neurons,and up-regulate the expression of related proteins in the Wnt/β-catenin pathway.In terms of behavior,infarct volume,NSCs differentiation,and Wnt3 a ligand expression,the combined treatment had better effects than EA or exercised alone,but the differences were not significant.