| ObjectiveIn this study,we studied the regulatory effect of electroacupuncture on neuronal apoptosis in the brain of a rat model of traumatic brain injury(TBI)using electroacupuncture treatment as an intervention.And the association between this effect and the PI3K/AKT signaling pathway.In order to reveal the partial effect mechanism of electroacupuncture in the treatment of TBI and provide a scientific basis for electroacupuncture treatment of TBI.MethodA total of 160 SD rats(SPF grade)were used in the experiment.They were randomly divided into blank group(10 rats),sham operation group(30 rats),model group(30 rats),blocker group(30 rats),electroacupuncture + blocking group(30 rats),and electroacupuncture group(30 rats)by random number table.PI3K/AKT signaling pathway blockers(LY294002)were applied in the blocker group and electroacupuncture + blocking group.Electroacupuncture treatment(electroacupuncture group,electroacupuncture + blocking group)was started 24 hours after modeling(modeling using free-fall impact method),and acupoints were selected: "Baihui,Shuigou,Quchi,Neiguan,Zusanli,and Yongquan" once a day for a total of 14 days.(1)To evaluate the behavioral function(balance,walking,and neurological function,etc.)of TBI rat model before modeling and 3 days,7 days,and 14 days after electroacupuncture treatment,to observe the pathomorphological(HE staining)changes and brain apoptosis(TUNEL assay)of injured brain tissue of rats at each evaluation time point,and to observe the NSE(neuron-specific enolase)concentration in the serum of TBI rats at each study time point,to comprehensively evaluate the therapeutic effect of electroacupuncture intervention on TBI rats,and the relationship between PI3K/AKT pathway and this therapeutic effect.(2)Immunofluorescence and Western-blot were used to observe the expression of apoptotic factors(pro-apoptotic: Bad,bak,bax;anti-apoptotic: bcl-xl,NF-k B,Bcl-2)in the injured brain tissue of TBI rats after electroacupuncture treatment from different levels and angles through a series of experiments to investigate the regulatory effect of electroacupuncture treatment on neuronal apoptosis in the brain of TBI rats,and the relationship between the changes of apoptotic factors and PI3K/AKT signaling pathway after electroacupuncture intervention.(3)Immunofluorescence,Western-blot,real-time quantitative PCR(real-time PCR)experimental techniques were used to detect the expression of PI3 K,PDK1,AKT,pAKT(phosphorylation level of AKT)and their m RNA,which are key proteins of PI3K/AKT pathway,in the injured brain tissue of TBI rats after electroacupuncture treatment from different levels and perspectives,and to analyze the association between the changes in the expression of key proteins of this pathway and pathway blockers(LY294002)and electroacupuncture intervention.Result(1)Behavioral evaluation results: Before modeling,the behavioral function scores of rats in each group were compared,P > 0.05;at 3 days,7 days,and 14 days after electroacupuncture treatment,the behavioral function scores of rats at the corresponding evaluation time points,between the sham operation group and the blank group,P > 0.05;at 3 evaluation time points after electroacupuncture treatment,the behavioral function score results of the sham operation group were better than those of the model group(P < 0.01),while the behavioral function evaluation results of the model group were better than those of the blocking group(P < 0.05);at 3 days after electroacupuncture treatment,the balance and walking function behavioral evaluation results of rats in the electroacupuncture group were compared with those of the model group,P > 0.05,while the other behavioral evaluation results(neurological function,limb retraction force)were compared between the two groups,P < 0.05;at 7 days and 14 days after electroacupuncture treatment,the behavioral function evaluation results of the electroacupuncture group were better than those of the model group(P < 0.05);The evaluation results of electroacupuncture group were better than those of electroacupuncture + blocker group(P < 0.05);the comparison between electroacupuncture + blocker group was 3 days and 7 days after electroacupuncture treatment,P > 0.05,and 14 days after electroacupuncture treatment,P < 0.05.(2)HE staining results: HE staining results of pathological sections of injured brain tissue of TBI rats: At 3 days,7 days,and 14 days after electroacupuncture treatment,there was no significant difference between sham operation group and blank group rats,and the structure was normal;at 3 days after electroacupuncture treatment,the inflammation,edema,and nuclear pyknosis of injured brain tissue of TBI rats in the model group were more obvious;the pathological morphological structure changes of injured brain tissue of TBI rats in the blocker group were the most obvious,with inflammatory cell infiltration,exudation,interstitial hemorrhage and edema at the damaged site,significant tissue vacuole-like changes,and necrotic areas,normal morphology of necrotic area cells disappeared,irregularly arranged,nuclear pyknosis and lysis were obvious;the morphological structure of brain tissue of TBI rats in the electroacupuncture group was not significantly different from that in the model group,but superior to the electroacupuncture + blocker group,the blocker + blocker group was not significantly different;Inflammatory cell infiltration at the damaged site increased than before,exudation decreased than before,interstitial edema was severe,tissue vacuole-like changes were more obvious than before,the necrotic area became larger,and the normal morphology of cells in the necrotic area disappeared,nuclear pyknosis and lysis increased than before;in TBI rats in the electroacupuncture group,the morphological structure of brain tissue showed significantly better than that in the model group,electroacupuncture + blocker group,and differences were observed between the blocker electroacupuncture + blocker group.(3)NSE concentration in serum: NSE(neuron specific enolase)concentration change in rat serum: 3 days,7 days,14 days after electroacupuncture treatment,compared between the sham operation group,P > 0.05,NSE concentration in the model group was higher than that in the sham operation group(P < 0.01),NSE concentration in the model group was lower than that in the blocker group(P < 0.05);3 days after electroacupuncture treatment,NSE concentration in the electroacupuncture group was compared with that in the model group,P > 0.05;7 days and 14 days after electroacupuncture treatment,NSE concentration in the electroacupuncture group was lower than that in the model group,P < 0.01;at the three detection time points after electroacupuncture treatment,NSE concentration in the electroacupuncture group was lower than that in the electroacupuncture + blocker group(P < 0.01);In addition,with the advancement of treatment time,the NSE concentration in the electroacupuncture group showed a decreasing trend.(4)Brain cell apoptosis: TUNEL apoptosis detection results(expressed as the average optical density value of fluorescence image of apoptosis detection): 3 days,7 days,14 days after electroacupuncture treatment,compared with the sham operation group,P > 0.05,the detection results of the model group were higher than those of the sham operation group(P < 0.01),and the detection results of the blocker group were higher than those of the model group(P < 0.01);3 days after electroacupuncture treatment,compared with the electroacupuncture model group,P > 0.05,the detection results of the electroacupuncture group were lower than those of the electroacupuncture + blocker group(P < 0.05);7 days and 14 days after electroacupuncture treatment,the detection results of the electroacupuncture group were lower than those of the model group(P < 0.01),and lower than those of the electroacupuncture + blocker group(P < 0.05);(5)Changes of pro-apoptotic proteins: IF and Western-blot were used to detect the expression of Bad,bak,and bax in the injured brain tissue of TBI rats in each group: at the corresponding time points of 3 days,7 days,and 14 days after electroacupuncture treatment,P > 0.05 was compared between the sham group;at the corresponding time points of model group,P < 0.05 was compared,and the expression level of model group was lower than that of blocker group(P < 0.05);at 3 days after electroacupuncture treatment,P > 0.05 was compared between the electroacupuncture model group,while the expression level of electroacupuncture group was lower than that of electroacupuncture + blocker group(P < 0.05);at the corresponding detection time points of 7 days and 14 days after electroacupuncture treatment,the expression level of electroacupuncture group was lower than that of model group(P < 0.05),and lower than that of electroacupuncture + blocker group(P < 0.05);The results of Bad and bak were P > 0.05 and bax were P < 0.05 at 7 days after electroacupuncture treatment,and the results of Bad,bak,and bax were P < 0.05 at 14 days after electroacupuncture treatment.(6)Changes of anti-apoptotic proteins: IF and Western-blot were used to detect the expression of bcl-2,NF-k B,and bcl-xl in the injured brain tissue of TBI rats in each group: at each corresponding detection time point of 3 days,7 days,and 14 days after electroacupuncture treatment,P > 0.05 was compared between the sham group and P < 0.05 was compared between the model group and the sham group,while the expression level in the model group was higher than that in the blocker group(P < 0.05);at 3 days after electroacupuncture treatment,P > 0.05 was compared between the electroacupuncture model group,while the expression level in the electroacupuncture group was higher than that in the electroacupuncture + blocker group(P < 0.05);at each corresponding detection time point of 7 days and 14 days after electroacupuncture treatment,the expression level in the electroacupuncture group was higher than that in the model group(P < 0.05),and higher than that in the electroacupuncture + blocker group(P < 0.01);However,bcl-2 and NF-k B were at 3 days and 7 days after electroacupuncture treatment,P > 0.05,and at 14 days,P < 0.05.(7)Changes of PI3 K,PDK1,AKT,p-AKT IF test results: Immunofluorescence(IF)was used to detect the expression of key proteins of PI3K/AKT pathway in the injured brain tissue of TBI rats in each group: at the corresponding time points of 3 days,7 days,and 14 days after electroacupuncture treatment,the expression levels of each index in the sham group were compared with those in the blank group,P > 0.05,the expression levels of each index in the model group were compared with those in the sham group,P < 0.05,and the expression levels of PI3 K,PDK1,and AKT in the model group were higher than those in the model group at 7 days and 14 days after electroacupuncture treatment(P < 0.05);P > 0.05.At each corresponding detection time point after electroacupuncture,the phosphorylation level of AKT(p-AKT)expression in the electroacupuncture group was higher than that in the model group(P < 0.05)and higher than that in the electroacupuncture + blocker group(P < 0.01),in addition,compared between the blocker electroacupuncture + blocker group,P > 0.05.(8)Changes of PI3 K,PDK1,AKT,p-AKT Western-blot(W-b)test results: W-b detected the expression of key proteins of PI3K/AKT pathway in the injured brain tissue of TBI rats in each group: at the corresponding time points of 3 days,7 days,and 14 days after electroacupuncture treatment,the expression levels of each index in the sham group were compared with those in the blank group,P > 0.05,the expression levels of each index in the model group were compared with those in the sham group,P < 0.05;at 3 days after electroacupuncture treatment,the expression levels of PI3 K and PDK1 in the electroacupuncture group were higher than those in the model group(P < 0.05);at 7 days after electroacupuncture treatment,the expression levels of PI3 K,PDK1,and AKT in the electroacupuncture group were higher than those in the model group(P < 0.05);The expression level of AKT in the electroacupuncture group was P > 0.05 compared with the model group;at the three corresponding detection time points,the expression levels of PI3 K,PDK1,and AKT in the electroacupuncture group were higher than those in the electroacupuncture + blocker group(P < 0.01),and between the blocker electroacupuncture + blocker group,P > 0.05;at each corresponding detection time point after electroacupuncture,the phosphorylation level of AKT(pAKT)in the electroacupuncture group was higher than that in the model group(P < 0.05),and higher than that in the electroacupuncture + blocker group(P < 0.01),In addition,P > 0.05 for the comparison between the blocker electroacupuncture + blocker group.(9)Changes of PI3 K,PDK1,and AKT m RNA Real-time PCR assay results: Real-time PCR was used to detect the expression of PI3K/AKT pathway key protein m RNA in the injured brain tissue of TBI rats in each group: at the corresponding time points of 3 days,7 days,and 14 days after electroacupuncture treatment,each index in the sham group was compared with that in the blank group,P > 0.05,the expression of each index in the model group was compared with that in the sham group,P < 0.05,and each index in the model group was compared with that in the blocker group,P < 0.05;at 3 days after electroacupuncture treatment,the expression of PI3 K,PDK1,and AKT m RNA in the electroacupuncture group was higher than that in the model group(P < 0.05);at 7 days and 14 days after electroacupuncture treatment,the expression of PI3 K,PDK1,and AKT m RNA in the electroacupuncture group was higher than that in the model group(P < 0.01).P > 0.05.Conclusion(1)Electroacupuncture can effectively improve the behavioral function of TBI rats,reduce the exudation,inflammation,edema of injured brain tissue,reduce the concentration of NSE in the serum of TBI rats,and promote the recovery of TBI.(2)Electroacupuncture can down-regulate the expression of Bad,bak and bax,upregulate the expression of Bcl-2,NF-k B and bcl-xl in the injured brain tissue of TBI rats,thereby inhibiting the apoptosis of brain nerve cells.(3)Electroacupuncture intervention could up-regulate the expression of PI3 K,PDK1,AKT,p-AKT and their m RNA in the injured brain tissue of TBI rats,and this effect was affected by the inhibitor LY294002.(4)The therapeutic effects of electroacupuncture on the improvement of behavioral function and pathomorphology of TBI rats,as well as the inhibition of brain neuronal apoptosis in TBI rats are related to the PI3K/AKT signaling pathway. |
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