“三通针法”电针调控Rho/ROCK(Rho kinase)及MEK/ERK信号通路对脊髓损伤大鼠胞浆型磷脂酶A2的影响

Effects of Santong electroacupuncture on the activity of cytoplasmic phospholipase A2 in rats with spinal cord injury via the Rho/Rho kinase and MEK/ERK signaling pathways

背景:胞浆型磷脂酶A2(cytosolic phospholipase A2,cPLA2)是治疗脊髓损伤的新型策略和靶标,其受RhoA/Rho kinase及MEK/ERK信号通路的调控。而电针可通过调控RhoA/Rho kinase和MEK/ERK信号通路治疗脊髓损伤。目的:探讨电针调控Rho/ROCK(Rho kinase)及MEK/ERK信号通路对脊髓损伤后cPLA2的影响。方法:90只雌性SD大鼠,随机取72只制备脊髓损伤模型,BBB评分后随机分为脊髓损伤组、电针治疗组、U0126治疗组(ERK阻断剂)和Y27632治疗组(ROCK阻断剂),另18只设为假手术组(只进行椎板咬除,但不进行脊髓撞击)。电针治疗组大鼠取大椎、腰阳关以及双侧次髎、足三里进行电针治疗,每天1次,每次20 min,共14次;U0126治疗组和Y27632治疗组分别予以U0126和Y27362隔天1次硬膜下腔注射。治疗结束经BBB评分后处死大鼠,收集脊髓组织,ELISA检测脊髓组织前列腺素E2、血小板活化因子的水平;TUNEL法检测脊髓神经细胞凋亡率;Western blot检测脊髓RhoA、ROCKⅡ、MEK、ERK1/2、p-ERK1/2、cPLA2、p-cPLA2蛋白的表达;qRT-PCR检测脊髓RhoA、ROCKⅡ、MEK、ERK1/2与cPLA2的基因表达。结果与结论:(1)与假手术组比较,脊髓损伤组大鼠BBB评分明显下降(P<0.01),脊髓组织细胞凋亡阳性细胞数、前列腺素E2和血小板活化因子水平、p-cPLA2蛋白和cPLA2基因的表达均明显升高(P<0.01);除电针治疗组大鼠cPLA2基因表达降低差异无显著性意义外,各治疗组大鼠上述指标均明显逆转(P<0.01);(2)与假手术组比较,脊髓损伤组大鼠脊髓组织RhoA及ROCKⅡ蛋白和基因的表达、MEK和ERK1/2基因的表达、MEK和p-ERK1/2蛋白的表达均明显升高(P<0.01),电针治疗组及其他两治疗组大鼠上述指标均明显降低(P<0.01或P<0.05);(3)结果说明,电针能下调Rho/ROCK和MEK/ERK信号通路相关因子表达,抑制cPLA2的活性,减少神经细胞凋亡、减轻炎症反应,最终达到治疗脊髓损伤的作用。

BACKGROUND:Cytoplasmic phospholipase A2(cPLA2)is a novel strategy and target for the treatment of spinal cord injury.cPLA2 is regulated by RhoA/Rho kinase and mitogen-activated protein kinase(MEK)/extracellular regulated protein kinases(ERK)signaling pathways.Electroacupuncture can treat spinal cord injury by regulating the RhoA/Rho kinase and MEK/ERK signaling pathways.OBJECTIVE:To investigate the effects of electroacupuncture on the activity of cPLA2 after spinal cord injury by regulating Rho/ROCK(Rho kinase)and MEK/ERK signaling pathways.METHODS:After spinal cord injury modeling and Basso,Beattie,Bresnahan scoring,72 female Sprague-Dawley rats were randomly divided into model group,electroacupuncture group,U0126 treatment group and Y27632 treatment group,with 18 rats in each group.Another 18 rats were used as sham operation group,in which only laminectomy was performed without spinal cord impingement.In the electroacupuncture group,Dazhui(GV 14),Yaoyangguan(GV 3),bilateral Ciliao(BL 32)and Zusanli(ST 36)were treated with electroacupuncture,once a day,20 minutes each time,14 times in total.Rats in the U0126 and Y27632 treatment groups were given U0126 and Y27362 subdural injection every other day,respectively.At the end of treatment,the rats were sacrificed after Basso,Beattie,Bresnahan scoring,and the spinal cord tissue was collected to detect the content of prostaglandin E2 and platelet activating factor by ELISA.TUNEL assay was used to detect the apoptosis rate of spinal cord nerve cells.Western blot was used to detect the protein expression of RhoA,ROCKⅡ,MEK,ERK1/2,p-ERK1/2,cPLA2 and p-cPLA2 in the spinal cord.qRT-PCR was used to detect the mRNA expression of RhoA,ROCKⅡ,MEK,ERK1/2 and cPLA2 in the spinal cord.RESULTS AND CONCLUSION:Compared with the sham operation group,Basso,Beattie,Bresnahan score was significantly decreased in the model group(P<0.01),while the number of apoptotic cells,the levels of prostaglandin E2 and platelet activating factor,and the expression of p-cPLA2 protein and cPLA2 mRNA in spinal cord tissue were significantly increased in the model group(P<0.01).After treatment,the above indexes were significantly reversed in all the treatment groups except for the mRNA expression of cPLA2 in the electroacupuncture group(P<0.01).Compared with the sham operation group,the expression of RhoA,ROCKⅡ,and MEK protein and gene,the mRNA expression of ERK1/2,and the protein expression of p-ERK1/2 were significantly increased in the model group(P<0.01).After treatment,the above indexes were significantly decreased in the three treatment groups(P<0.01 or P<0.05).To conclude,electroacupuncture can down-regulate the expression of factors related to Rho/ROCK and MEK/ERK signaling pathways,inhibit the activity of cPLA2,reduce the apoptosis of nerve cells,alleviate inflammatory responses,and finally achieve the therapeutic effect on spinal cord injury.