| ObjectiveIn this study,we used the animal models of traumatic brain injury(TBI)of rats as the research object,and to reveal the mechanism of electroacupuncture treatment of craniocerebral injury through PI3K/AKT signal transduction pathway that study the behavior,brain histopathological,and brain neurons apoptosis and edema changes in TBI rats,in order to find the best the time window of interventional treatment of electro-acupuncture,and to provide scientific experimental basis for clinical acupuncture treatment of TBI.MethodA total of 170 healthy SD rats were randomly divided into blank group(n=10),sham operation group(n=10),model group(n=60,each sampling time point 10 rats)electroacupuncture treatment group1(n=60,each sampling time point 10 rats)and electroacupuncture treatment group2(n=30,each sampling time point 10 rats).The electric Cortical Contusion Injury(eCCI)apparatus was used(Speed: 5.30 m/s,depth: 4.5 mm,residence time: 400 ms)on the left side of the rat brain(3 mm from the coronal suture and 2.5 mm in the sagittal seam)to Prepare TBI model(blank group and sham operation group without blow molding).take the rat acupoints “Baihui,Guan yuan”,“Quchi,Hegu”of the right forelimb,“Zusanli,yongquan” of right hindlimb,EA group 1 after modeling 4 hours to accept electroacupuncture treatment,EA group2 on the seventh day received electroacupuncture treatment,treatment of the two groups ended up at fourteenth day.the therapeutic effect of electroacupuncture treatment is evaluated by the following aspects:(1)At 24 h,7d,14 d and 21 d after modeling,the neurological function,the balance function,the walking function and the retraction force of the forelimb and hindlimb that affected were evaluated.(2)At 24 h,72h,7d,10 d,14d after modeling:(1)Pathological changes of brain tissue were observed by HE staining under light microscope;(2)Detection of apoptosis in brain cells by in situ terminal labeling(TUNEL);(3)The concentration changes of TNF-α and TIMP-1 in brain tissue and NSE in serum of TBI rats were detected by ELISA.(4)The expression of AKT,TNF-α,Caspase-3,ERK,AQP-4,TIMP-1and MMP-9 in brain tissue were detected by immunohistochemistry;(5)The expression of AKT,TNF-α,Caspase-3,ERK,AQP-4 and TIMP-1 in brain tissue were detected by immunofluorescence;(6)Western blot was used to detect the expression of AKT,TNF-α,Caspase-3,MMP-9 changes;(7)The mRNA expression of AKT mRNA,TNF-α mRNA,Caspase-3 mRNA,ERK mRNA,AQP-4 mRNA,MMP-9 mRNA and A20 mRNA in brain tissue were detected by fluorescence quantitative PCR;(8)The expression of A20 mRNA in brain tissue was detected by in situ hybridization.Result(1)The effect of electroacupuncture treatment on neurological function,balance function,walking function and retraction force of the forelimb and hindlimb that affected in TBI rats with the corresponding groups before modeling,in 24 h were significant difference(P<0.01).The therapeutic effect of electroacupuncture treatment in group1 was better than that of model group at 24 h,7d,14 d and 21 d after operation(P<0.01),and was better than that in electroacupuncture treatment group2(P <0.05)at 14 days and 21 days.At 21 days,the therapeutic effect of electroacupuncture treatment in the group1 was close to that of the before modeling,and there was no statistical significance(P>0.05).The therapeutic effect of electroacupuncture treatment in group2 was better than that of model group at 14 d and 21d(P<0.05).(2)The histopathological changes of the injured brain tissue showed that the structure of brain tissue was complete in the blank group and the sham operation group,and the morphology of the brain nerve cells was normal.The changes of brain tissue inflammation and edema in electroacupuncture treatment group 1 were better than those in model group at 24 h,72h,7d,14 d after operation.The pathology of brain tissue inflammation and edema in electroacupuncture treatment group 2 were better than those in model group,and poor than those in electroacupuncture treatment group 1 that observed at 10 d and 14 d after operation.(3)Changes of serum NSE levels: the serum NSE concentration in the model group,electroacupuncture group 1 was significantly increased after modeling 24 h,reached the highest at 72 h,and decreased at the 7th day and the 10 th day.At 14 d,the electroacupuncture group 1 compared with the blank group and sham operation group there was no significant difference(P>0.05).The concentration of NSE in serum of electroacupuncture group 1 was lower than that of electroacupuncture group 2(P <0.01)at 10 d and 14 d,lower than that of model group(P<0.01)at 24 h,72h,7d,10 d and 14d(P <0.01).The concentration of NSE in serum of electroacupuncture group 2 was lower than that in model group(P<0.01)at 10 d and 14 d.(4)The changes of apoptosis of the brain nerve cells: the model group,electroacupuncture treatment group 1,electroacupuncture treatment group 2 were all higher than those in the sham operation group and the blank group(P<0.01)at 24 h,72h,7d,10 d.The model group was higher than that of electroacupuncture treatment group 1(P<0.01),the apoptosis of brain nerve cells was significantly increased in the two groups after modeling 24 h,and reached the maximum at 72 h,decreased at 7d and 10 d,respectively.The apoptotic rate of electroacupuncture treatment group 2 at 10 day and 14 day was significantly higher than that of electroacupuncture treatment group 1(P<0.01),but lower than that of model group(P<0.01).There was no significant difference compared the electroacupuncture treatment group 1 with sham operation group and the blank group(P>0.05),but there was significant difference compared with the model group and electroacupuncture treatment group 2(P<0.05)at 14 day.(5)The Changes of TNF-α,AKT,Caspase-3,ERK and its mRNA in PI3K/AKT pathway-related proteins: Compared with blank group and sham operation group,the expression of TNF-α,Caspase-3,ERK and its mRNA in injury brain of model group were increased after modeling(P<0.05),reached the highest at 72h;the expression of AKT and its mRNA in injury brain of model group were decreased after operation(P<0.05),and reached the lowest at 72 h.while the expression of TNF-α,Caspase-3,ERK and its mRNA in injury brain of electroacupuncture treatment group 1 and electroacupuncture treatment group 2 were decreased after modeling(P<0.05),and reached the lowest at 7d in electroacupuncture treatment group 1;the expression of AKT and its mRNA in injury brain of electroacupuncture treatment group 1 and electroacupuncture treatment group 2 were increased after operation(P<0.05),and reached the highest at 7d in electroacupuncture treatment group 1.(6)The changes of brain edema-related protein AQP-4,TIMP-1,MMP-9 and its mRNA and the inhibition of apoptosis A20 mRNA: Compared with the blank group and sham operation group,the expression of MMP-9 and its mRNA in injury brain of model group were increased after modeling(P<0.05),reached the highest at 72h;the expression of AQP-4,TIMP-1 and its mRNA and the A20 mRNA in injury brain of model group were decreased after operation(P<0.05),TIMP-1 and A20 reached the lowest at 72 h,AQP-4 reached the lowest at 7d.while the expression of MMP-9 and its mRNA in injury brain of electroacupuncture treatment group 1 and electroacupuncture treatment group 2 were decreased after modeling(P<0.05),reached the lowest at 7d in electroacupuncture treatment group 1;the expression of AQP-4,TIMP-1 and its mRNA and the A20 mRNA in injury brain of electroacupuncture treatment group 1 and electroacupuncture treatment group 2 were increased after operation(P<0.05),TIMP-1 and A20 reached highest at 7d,AQP-4 reached the at 72 h.Conclusion(1)Electroacupuncture can effectively improve the neurological function,balance function,walking function and the retraction force of the right anterior and posterior limbs of rats with craniocerebral injury,reduce the bleeding,edema and inflammation of brain tissue,reduce the concentration of NSE in serum,inhibit the apoptosis of brain cells,and promote the recovery of brain function.(2)Electroacupuncture can significantly reduce the expression of TNF-α,Caspase-3,ERK protein and its mRNA related to inflammation and apoptosis in brain tissue of rats with brain injury,and increase the expression of AKT protein and its mRNA expression that related to cell survival and differentiation,and the earlier,the better the effect of electroacupuncture treatment.(3)Electroacupuncture can significantly inhibit the expression of MMP-9 and its mRNA that caused the brain edema in rats with brain injury,increase the expression of AQP-4,TIMP-1 proteins and its mRNA that related to elimination of brain cell edema,reduce inflammation,and cell survival and the inhibition of apoptosis A20 mRNA.Experimental study shows that: early electroacupuncture intervention is more effective in the prevention of brain edema and apoptosis. |
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