电针调控胞浆型磷脂酶A2（cPLA2）及相关信号通路治疗脊髓损伤作用机制研究

Our previsious research find out that electro-acupuncture could relieve growth inhibiting of neuroal axon in treating spinal cord injury(SCI), and this effect could be attained by electro-acupuncture mediating RhoA/Rho Kinase(Rho/ROCK)signaling pathway. Cytosolic phospholipase A2(cPLA2), as the effector molecule of Rho/ROCK and extracellular signal regulated kinases (ERK) signaling pathway, plays important role in the regulation of inflammatory reaction and apoptosis. Taking Rho/ROCK and ERK mediates inflammatory reaction and apoptosis through regulating cPLA2 activation in the secondary injury of spinal cord as key point, this research expects to reveal the mechanism of electro-acupuncture treating SCI. Random and contrast method was employed, and spinal cord injury animal experiment in vivo and cell culture in vitro research method was used, this research aims: ①To investigated the relationship between Rho/ROCK, ERK signaling pathway and cPLA2, and further demonstrate the activation of cPLA2 influencing inflammatory reaction and apoptosis in the secondary injury of spinal cord, by evaluating motor function of rats post limbs and change of histomorphology pre- and post-treatment, detecting the changes of important molecular in Rho/ROCK and ERK signaling pathway, inflammatory reaction and apoptosis,②To expect to confirm that electro-acupuncture can produce key regulating role in improving never remodeling of SCI rat, ③To provide theory evidence for electro-acupuncture treating paraplegic patients caused by SCI.

课题组前期研究发现，电针能够减轻神经轴突生长抑制，而这一作用是通过对Rho/ROCK信号通路的调控来实现的。胞浆型磷脂酶A2（cPLA2）作为Rho/ROCK及ERK信号通路中的效应分子，在炎性反应和神经细胞凋亡中发挥重要作用。本研究以Rho/ROCK及ERK信号通路调控cPLA2介导炎症反应及细胞凋亡作用为切入点，以冀探明电针治疗脊髓损伤的作用机制。本研究采用随机对照方法，利用脊髓损伤动物模型和细胞培养，从组织形态学、细胞学、分子生物学角度，探讨脊髓损伤后Rho/ROCK及ERK信号通路对cPLA2的调控，并深入探讨cPLA2激活对脊髓损伤后的炎性反应以及神经细胞凋亡的影响，通过评估治疗前后运动功能及脊髓组织形态学改变、检测Rho/ROCK及ERK信号通路和凋亡、炎性关键分子的变化，拟证实电针在改善脊髓损伤大鼠神经功能重塑中的关键作用，为电针治疗脊髓损伤截瘫患者提供理论依据。

脊髓损伤（SCI）是一种外伤所致的中枢神经系统疾病，近年来随着交通、建筑、采矿业的发展，SCI发病率呈逐年升高趋势，且中青年患者最多见。SCI容易造成严重性伤残且没有有效治疗的药物和方法，医疗费用高昂，给社会和家庭带来沉重的负担。有研究表明胞浆型磷脂酶A2(cPLA2)是介导脊髓继发性损伤炎症反应和细胞凋亡等多种关键机制的中心或交叉汇合的重要分子；该细胞因子受RhoA/Rho kinase 和MEK/ERK信号通路的调控。电针能否通过调控RhoA/Rho kinase和MEK/ERK信号通路相关因子，抑制cPLA2的活性，减轻炎性反应和降低神经细胞凋亡而发挥治疗脊髓损伤的作用？本研究项目以Allen重物打击法制备T10脊髓损伤动物模型，分成相应治疗组。电针治疗组取大椎、腰阳关及双侧次髎、足三里进行电针治疗，同时设立ERK阻断剂U0126治疗组、ROCK2阻断剂Y27632治疗组、cPLA2阻断剂AACOCF3治疗组、模型组和假手术组(sham)对照；并采用细胞实验相结合的方法进行研究。干预结束后收集相关组织和细胞进行相关指标检测。研究结果显示：SCI后大鼠BBB评分明显低于sham组；脊髓组织和血清PGE2和PAF水平，脊髓组织阳性细胞数明显高于sham组(P < 0.01)；脊髓组织Bcl-2、RhoA、ROCKⅡ蛋白及基因表达水平明显低于sham组(P < 0.01)，而Bax、Casepase-3、MEK、ERK1/2、蛋白及基因表达水平明显高于sham组(P < 0.01)；脊髓组织p-cPLA2蛋白和cPLA2基因表达明显高于sham组(P < 0.01)。经治疗后，上述各指标都有不程度地逆转。细胞实验显示，RhoA、ROCKⅡ、MEK、ERK1/2与cPLA2表达存在正相关。.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Rho/ROCK和MEK/ERK信号通路相关因子表达，抑制胞浆型磷脂酶A2的活性，减少脊髓神经细胞凋亡，减轻脊髓损伤炎症反应，最终达到治疗脊髓损伤的作用。本研究为针灸治疗脊髓损伤截瘫患者进一步提供了实验依据，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再一次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。