基于Nogo/NgR及其下游Rho/ROCK信号通路探讨电针治疗脊髓损伤的作用机制

Based on the efficacy of acupunctue treating SCI(spinal cord injury) in clinic research, taking the important regulating role of the sigal pahway of Nogo/NgR and its following Rho/ROCK in the regeneration of neuraxon and remodeling of nerve function as keypoint, this research expect to find out the mechanism of electroacupuncture treating SCI.This research aims: To observe and compare the influence of the mRNA and proteinum express of Nogo-A、NgR、p75NTR 、 LINGO-1 and RhoA、ROCKⅡ、MLC P in injury place of SCI rats treated with electroacupuncuture, NEP1-40(inhibitor of NgR)、Y-27632(specific inhibitor of ROCK Ⅱ)，electroacupuncture combined with NEP1-40 and electroacupuncture combined with Y-27632，by using real time fluorescent quantitation PCR, Western blot and immunohistochemistry of hybridization in situ; To discuss the key effect component element of electro- acupuncture in influencing Nogo/NgR and its following Rho/ROCK signal pathway; To expect to confirm that electroacupunctue can produce key regulating role in restraining correlated cytokine of Nogo/NgR and its following Rho/ROCK signal transduction pathway and to provide theory evidence for electrocupuncture treating paraplegic patients caused by SCI.

基于针灸治疗脊髓损伤有效性的临床研究，本研究以Nogo/NgR及下游Rho/ROCK信号通路在神经轴突再生及功能重塑起关键调节作用为切入点，以冀探明电针治疗脊髓损伤的作用机制。本研究拟应用实时荧光定量PCR、Western blot和原位杂交免疫组织化学等方法观察和比较电针、NgR的拮抗剂NEP1-40、ROCKⅡ的特异性抑制剂Y-27632、电针加NEP1-40和电针加Y-27632分别治疗脊髓损伤大鼠脊髓损伤处Nogo-A、NgR、p75NTR、LINGO-1与RhoA、ROCKⅡ、MLC P mRNA及蛋白表达的影响，探讨电针影响Nogo/NgR及其下游Rho/ROCK信号通路的关键作用环节，拟证实电针抑制Nogo/NgR及其下游Rho/ROCK信号转导通路中相关细胞因子在改善脊髓损伤大鼠神经功能中起关键调节作用，为电针治疗脊髓损伤截瘫患者提供理论依据。

近年来随着交通、建筑和采矿业等的迅速发展，脊髓损伤患者数呈逐年上升趋势，且以中青年SCI最多见，给人们的生产、生活和国家的经济带来沉重负担。基于申请者的临床观察及相关临床报道，针灸治疗对于脊髓损伤所造成截瘫特别是不完全性截瘫的恢复有肯定的促进作用。实验研究表明，Nogo/NgR及下游Rho/ROCK信号通路在神经轴突再生及功能重塑起关键调节作用，电针能否通过影响这一信号通路而产生治疗作用？本研究在造模后7d介入电针治疗，治疗取大椎、腰阳关及双侧次髎、足三里；选择疏密波，持续时间20 min，每天1次。分别于治疗后7 d、14 d 提取损伤处脊髓组织，采用实时荧光定量PCR、Western blot和原位杂交免疫组织化学等方法观察和比较电针、NgR的拮抗剂NEP1-40、ROCKⅡ的特异性抑制剂Y-27632、电针加NEP1-40和电针加Y-27632分别治疗脊髓损伤大鼠脊髓损伤处Nogo-A、NgR、p75NTR、LINGO-1与RhoA、ROCKⅡ、MLCP mRNA及蛋白表达的影响，采用BBB评分评估大鼠后肢活动功能。BBB评分显示，电针治疗后各组评分均较模型组提高，且电针加阻断剂NEP(1-40)或Y-27632组BBB评分分别高于单纯电针治疗组和单纯阻断剂NEP(1-40)或Y-27632组(P<0.05)。脊髓损伤后，各治疗组与模型组比较，脊髓组织中Nogo-A、NgR、p75NTR、LINGO-1与RhoA、ROCKⅡ、MLC P mRNA及蛋白表达的表达均降低(P<0.05)。就p75NTR、MLCP 而言，电针组、阻断剂组和电针加阻断剂组之间无显著性差异(P>0.05)；LINGO-1电针+阻断剂阻组要低于电针组。. 本研究阐明了针灸治疗脊髓损伤的可能作用机制，即电针通过调控Nogo/NgR和下游Rho/ROCK信号通路中关键信息分子的表达，有效降低了对神经轴突生长的抑制作用。本研究为针灸治疗脊髓损伤截瘫患者提供了实验依据，为以“通督、通（肠）腑、通调膀胱”为取穴特点的“三通针法”治疗脊髓损伤的推广运用奠定了坚实的理论基础， 同时也可能为针灸治疗其它神经系统疾病提供借鉴。