艾灸对脑缺血再灌注损伤大鼠脑梗死区Nogo/神经营养素受体信号通路的影响

Effect of moxibustion on Nogo/neurotrophin receptor signaling pathway in rats with cerebral ischemia/reperfusion injury

目的:观察艾灸治疗对脑缺血再灌注损伤(CI/RI)大鼠脑梗死区Nogo-A、Nogo-A受体(NgR)、协同受体p75神经营养素受体(p75NTR)与亮氨酸富集重复片段和Ig域包含子(Lingo-1)表达的影响,探讨艾条悬灸治疗CI/RI的机制。方法:雄性SD大鼠按随机数字表法分为假手术组、模型组、阻断剂组和艾灸组,假手术组16只,其余每组17只。采用线栓法复制左侧大脑中动脉栓塞模型。造模后,艾灸组用艾条悬灸“百会”和右侧“曲池”“足三里”20 min,每日1次,7次为1个疗程,疗程中间休息2 d,共治疗2个疗程;阻断剂组硬膜下推注Nogo分子胞外段氨基端前40个残基多肽(NEP1-40)。干预前后对各组大鼠进行神经功能评分,干预后以Feeney走横木实验(BWT)评分对其精细运动功能恢复情况进行评估;用TTC染色法评估大鼠脑梗死体积;实时荧光定量PCR法检测梗死区脑组织Nogo-A、NgR、p75NTR、Lingo-1 mRNA表达;Western blot法检测梗死区脑组织Nogo-A、NgR、p75NTR、Lingo-1蛋白表达;免疫荧光双标法检测梗死侧脑组织NgR、Lingo-1的阳性表达。结果:与假手术组比较,模型组大鼠神经功能评分、脑梗死体积百分比均明显升高(P<0.01),BWT评分明显下降(P<0.01),梗死区脑组织Nogo-A、NgR、p75NTR、Lingo-1 mRNA及蛋白表达均明显升高(P<0.01),NgR和Lingo-1共表达增加(P<0.01)。与模型组比较,除阻断剂组Nogo-A蛋白表达外,两干预组大鼠上述其余指标都明显逆转(P<0.01)。与阻断剂组比较,艾灸组大鼠脑梗死体积百分比、梗死区脑组织Nogo-A、p75NTR mRNA表达,Nogo-A、NgR、Lingo-1、p75NTR蛋白表达及NgR和Lingo-1共表达均降低(P<0.01,P<0.05)。结论:艾灸能产生类似于阻断剂NEP1-40的作用,抑制脑梗死后Nogo/神经营养素受体信号通路相关分子的表达,这可能是艾灸治疗脑梗死的机制之一。

Objective To observe the effect of moxibustion treatment on the expression of Nogo-A,Nogo receptor(NgR),neurotrophin receptor p75(p75 NTR)and leucine rich repeat and Ig domain containing 1(Lingo-1)in brain tissue of rats with cerebral ischemia/reperfusion injury(CI/RI),so as to analyze its mechanism underlying improvement of CI/RI.Methods Male SD rats were randomly divided into sham operation group(16 rats),model group(17 rats),NEP1-40(extracellular peptide residues 1-40,a blocker targeting NgR)group(model+blocker,17 rats)and moxibustion group(model+moxibustion,17 rats).The CI/RI model was established by occlusion of the left middle cerebral artery(MCAO).Moxibustion was applied to“Baihui”(GV20),right“Quchi”(LI11)and“Zusanli”(ST36)for 20 min,once a day for 14 days,with 2 days’rest after the top 7 days’intervention.For rats of the NEP1-40 group,30μL PBS containing 18μg NEP 1-40 was injected into the epidural inferior vena(L5-S1)via a polyvinyl chloride conduit.The neurological deficit state in each group was evaluated by Longa’s 5-point scale and Feeney’s 7-point scale of beam walking test(BWT).The cerebral infarct volume was assessed by 2,3,5-triphenyltetrazole chloride staining.The brain tissue between the central anterior and posterior sulcus was taken for observing the expression of NgR and Lingo-1 by fluorescence double-label method,and for determining the expression levels of Nogo-A,NgR,p75 NTR and Lingo-1 mRNAs and proteins by real-time quantitative PCR and Western blot,respectively.Results After modeling,the Longa’s score,infarct volu-me percent,expression levels of Nogo-A,NgR,Lingo-1 and p75 NTR mRNAs and proteins were significantly increased(P<0.01)and BWT score was obviously decreased(P<0.01)in the model group relevant to the sham operation group.In comparison with the model group,the increase of Longa’s score,infarct volume percentage,expression levels of Nogo-A,NgR,Lingo-1 and p75 NTR mRNAs and proteins and decrease of BWT score in NEP1-40 and moxibustion groups were reversed(P<0.01)except Nogo-A protein in the NEP1-40 group.The effect of moxibustion was significantly superior to that of blocker NEP1-40 in redu-cing the infarct volume percentage,and down-regulating the expression of Nogo-A mRNA and protein,p75 NTR mRNA and protein,NgR and Lingo-1 proteins(P<0.01,P<0.05).Conclusion Moxibustion,similar to blocker NEP1-40 of NgR,can improve neurological dysfunction in CI/RI rats,which may be related to its functions in reducing cerebral infarction and down-regulating the activity of Nogo/neurotrophin receptor signaling pathway.