The Anti-cerebral Ischemia Reperfusion Injury Effect Of Electroacupuncture Preconditioning Via TRPV1

ObjectiveCerebral ischemia reperfusion injury model rats were selected as the research objects,and electroacupuncture preconditioning of "Baihui"(GV20),"Shenshu"(BL23)and "Sanyinjiao"(SP6)points of rats were used to observe the protective effect of electroacupuncture preconditioning on brain of rats with cerebral ischemia reperfusion injury.The relationship between the protective effect on brain reduced by electroacupuncture preconditioning and TRPV1 was further investigated by using AMG-517,an inhibitor of TRPV1,and Capsaicin,an agonist of TRPV1 respectively.Methods1.30 SD male rats were randomly divided into normal group(Control group),sham operation group(Sham group)and model group(MCAO group),with 10 rats in each group.The neurological function of rats was evaluated by modified neurological deficit score.The effect and stability of the rat model of focal cerebral ischemia reperfusion injurywere evaluated.The infarction of rat brain tissue was observed by TTC staining.2.60 rats were randomly divided into Control group,Sham group,MCAO group and electroacupuncture preconditioning group(EA+MCAO group)according to the random number table,with 15 rats in each group.Neurological deficit assessment was used to evaluate the neurological function of rats.The infarct volume of rats was detected by TTC staining and the percentage of infarct volume was calculated.The number and morphology of hippocampal neurons were observed by HE staining.The protein expression of TRPV1 in right hippocampal tissue was detected by Western Blot.3.80 rats were randomly divided into MCAO group,EA+MCAO group,electroacupuncture preconditioning + inhibitor group(AMG-517+EA+MCAO group),electroacupuncture preconditioning +agonist group(Capsicine + EA + MCAO group)according to the random number table.The neurological function was evaluated by neurological deficit score.The infarct volume of rats was observed by TTC staining and the percentage of infarct volume was calculated.The number and morphology of hippocampal neurons were observed by HE staining.The content of inflammation-associated cytokines IL-1? and TNF-a and oxidative stress related indicators SOD,MDA,GSH were detected by ELISA.Western Blot was used to detect the expression of TRPV1,p38 MAPK,p-p38 MAPK,ERK,p-ERK,JNK and p-JNK proteins in the right hippocampus of rats.Results1.Evaluation of the model(1)The neurological deficit score showed that the neurological deficitscores of the Control group and the Sham group were both 0,and no neurological impairment was observed.The neurological deficit score of rats in the MCAO group were varying degrees of neurological impairment after 6 hours of reperfusion.(2)TTC staining showed that the ischemic brain tissue in the MCAO group displayed typical staining loss,which was white,consistent with the middle cerebral artery innervation area,and the infarct was stable before and after the optic chiasm.There was no obvious abnormality in TTC staining of brain tissue of Control group and Sham group.2.Electroacupuncture preconditioning "Baihui","Shenshu" and "Sanyinjiao" exerted protective effects on brain in rats with cerebral ischemia reperfusion injury(1)The neurological deficit scores of the Control group and the Sham group were both 0,and no neurological impairment was observed.The MCAO group had obvious neurological deficitafter 6 hours of reperfusion.Compared with the MCAO group,the neurological deficit of the EA+MCAO group was significantly reduced.(2)The results of TTC staining showed that the brain tissues of the Control group and the Sham group were intact,with distinct layers and bright red,no ischemic lesions;In the MCAO group and EA+MCAO group,there were pale white infarcts with reddish edges in the blood supply area of the middle cerebral artery in the right brain tissue of the rats.Compared with the MCAO group,the cerebral infarction volume of rats in the EA+MCAO group decreased significantly.(3)HE staining of brain tissues of the Control group and Sham group showed that neurons in the CA3 region of hippocampus were closely arranged,with complete neuron morphology,clear structure,complete nuclear membrane,clear nucleoli and light coloring.In the MCAO group,the central area of cerebral infarction was stained lighter,the hippocampal neuron space was widened,the arrangement was disordered,the number was reduced,the nucleus was condensed,the boundary between the nuclear membrane and the surrounding was unclear,nucleoli became smaller,the morphology hippocampal neuron was irregular,and the nuclear staining was significantly deepened.Compared with the MCAO group,the morphological and pathological changes of the rats in the EA+MCAO group were significantly reduced,with more orderly arrangement and complete structure of neurons,mildly swollen cells,and more surviving neurons.(4)Western blot results showed that,compared with MCAO group,the expression of TRPV1 protein in EA+MCAO group was significantly reduced.3.The relationship between anti-cerebral ischemia reperfusion injury induced by electroacupuncture preconditioning and TRPV1(1)The neurological deficit score showed that,compared with the MCAO group,the neurological deficit score of the EA+MCAO group was significantly lower(P<0.05);compared with the EA+MCAO group,the neurological function score of AMG-517+EA+MCAO group was lightly lower,but the difference was not statistically significant,at the same time,the neurological function score of Capsicine+EA+MCAO group was significantly higher than the EA+MCAO group(P<0.05).(2)TTC staining results showed that brain tissue of four groups of MCAO modeled rats showed different degrees of cerebral infarction(stained white area),and the MCAO group had the largest cerebral infarction volume.Compared with MCAO group,the cerebral infarctionvolume of EA+MCAO group was significantly reduced(P<0.05).Compared with EA+MCAO group,the cerebral infarction volume of AMG-517+EA+MCAO group was further significantly reduced(P<0.05),while the cerebral infarction volume of the rats in Capsicine+EA+MCAO group was significantly increased(P<0.05).(3)HE staining showed that the morphology of the right hippocampal neurons in the four groups of rats modeled by MCAO showed different degrees of damage and the number of neurons changed to varying degrees.The hippocampal neurons in the MCAO group were the most severely damaged.Compared with the MCAO group,the damage of hippocampal neurons in the EA+MCAO group was alleviated.Compared with the EA+MCAO group,the damage of hippocampal neurons was further attenuatedthe in AMG-517+EA+MCAO group,while the damage the Capsicine+EA+MCAO group was aggravated.(4)Inflammation-associated cytokines: Compared with MCAO group,the content of TNF-a and IL-1? in the right hippocampus of EA+MCAO group were significantly decreased(P<0.05,P<0.05);compared with EA+MCAO group,the content of TNF-a in AMG-517+EA+MCAO group was significantly decreased(P<0.05,).Compared with EA+MCAO group,TNF-a and IL-1? in Capsicine+EA+MCAO group were increased significantly(P<0.05,P<0.05).(5)Oxidative stress-related indicators: Compared with the MCAO group,the content of MDA in the right hippocampus of the EA+MCAO group was significantly decreased(P<0.05),and the contents of GSH and SOD were significantly increased(P<0.01,P<0.01);Compared with EA+MCAO group,the content of MAD was decreased(P<0.05)and GSHand SOD were increased(P<0.05,P<0.05)in AMG-517+EA+MCAO group.Compared with EA+MCAO group,the content of MDA was increased overtly(P<0.05)and the contents of GSH and SOD were decreased significantly(P<0.05,P<0.05)in Capsicine+EA+MCAO group.(6)Western blot results showed that compared with MCAO group,the expression of TRPV1 protein in that of EA+MCAO group was significantly increased(P<0.05)and the ratio of p-p38 MAPK/p38 MAPK was significantly decreased(P<0.05).Compared with EA+MCAO group,the expression of TRPV1 protein was significantly down-regulated(P<0.01)and the ratio of p-p38 MAPK/p38 MAPK was significantly decreased(P<0.05).Unfortunately,There was no statistically significant difference in the ratios of p-ERK /ERK and p-JNK /JNK among four groups.Conclusion1.The modified Longa suture method can effectively embolize the middle cerebral artery of the rat in the blood vessel,causing infarction of the blood supply area of the middle cerebral artery and causing nerve dysfunction.Modeling can be replicated with some stability.2.Electroacupuncture preconditioning "Baihui","Shenshu" and "Sanyinjiao" points can significantly reduce the score of neurological deficit,reduce the infarct size of cerebral ischemia reperfusion rats,reduce the damage of neurons,and exert the effect of anti-ischemia reperfusion injury.Meanwhile,electroacupuncture preconditioning can inhibit the increase of TRPV1 protein expression in brain tissue of rats with cerebral ischemia reperfusion injury.3.TRPV1 is involved in the anti-cerebral ischemia reperfusioninjury effect of electroacupuncture preconditioning.Down-regulation of TRPV1 expression,inhibition of p38 MAPK phosphorylation,reduction of oxidative stress damage,and reduction of inflammatory response may be one of the mechanisms of electroacupuncture preconditioning against cerebral ischemia reperfusion injury.