人参皂甙Rb1通过调控缝隙连接蛋白40治疗脑缺血再灌注损伤的机制

Regulation of Cx40 By GS-Rb1 In The Treatment For Cerebral Ischemia-Reperfusion Injury

目的观察人参皂甙Rb1(GS-Rb1)通过调控缝隙连接蛋白40(Cx40)表达在治疗脑缺血再灌注损伤(Cerebral ischemia and reperfusion injury,Cerebral I/R injury)的相关性机制。方法选取100只体重350～450 g的4月龄的SD雄性大鼠随机分为5组,每组20只,脑缺血再灌注模型建立为采用夹闭颈总动脉法。具体分组为:(1)假手术(sham)组;(2) GS-Rb1(I/R+GS-Rb1)治疗组;(3) GS-Rb+H-89阻断(I/R+GS-Rb1+H-89)组、(4)溶媒(I/R+DMSO)组及;(5)损伤(I/R)组,每组20只。实验时间均设定为I/R后8 h。每组取10只大鼠进行行为学检测和脑组织含水量测定,另10只则获取海马区皮质通过蛋白免疫印记法(Western Blot,WB)测定神经元Cx40蛋白的变化、采用酶联免疫荧光(ELFA)及酶联免疫吸附(ELISA)检测神经元氧化应激因子NAPDH氧化酶活性、炎性因子IL-1β、IL-6及TNF-a等含量的变化。结果实验结果显示,治疗组的NSS评分、脑水肿程度、NAPDH氧化酶活性、炎性因子及海马皮质Cx40表达均较溶媒组及损伤组明显下降(P <0. 05)。而H-89可明显抑制上述GS-Rb1对Cx40蛋白表达下调、降低NAPDH氧化酶活性及缓解炎性因子产生的作用(P <0. 05)。结论 GS-Rb1可能通过激活PKA途径而对Cx40蛋白表达下调,从而产生抑制损伤性物质的释放及传递,以达到脑损伤缓解作用。

Objective To investigate the mechanism of protective effects of GS-Rb1 against Cerebral ischemia-repufusion injury in super acute phase with the expression of protein Cx40. Methods Cerebral ischemia-repufusion injury model were established by Sprague-Dawley rats at 3 months age,weight at 300 ~ 500 g,with clipping bilateral CCA and intraperitoneal injection of GS-Rb1 or GS-Rb1 + PKA inhibitor H-89. One hunderd rats Rats were randomly divided into 5 groups,100 rats in total,20 rats in each group: sham operation,treatment group( I/R8 hour + GS-Rb1),inhibition group( I/R8 hour + GS-Rb1 + H-89),vehicle group( I/R8 hour + DMSO) and injury group( I/R 8 hour). Ten rats were selected for testing behavioral disorder by NSS and cerebral edema by measuring brain water content. Another ten rats in each group were sacrificed and glial tissue in hippocampus were obtained for detecting the level of Cx40 by western Western blot,NAPDH oxidase activity by EFLA,IL-1 β,IL-10 and TNF-α by ELISA. Result NSS,brain water content,NAPDH oxidase activity,IL-1 β,IL-10,TNF-α and Cx40 in GS-Rb1 treatment group were lower than in vehicle group and injury group,PKA inhibitor H-89 was proved that it can could inhibit above protective effect of GS-Rb1. Conclusion Protective effect of GSRb1 to cerebral I/R injury is involved in PKA pathway to reduce the expression of Cx40,by which can reduce the oxidative stress,inflammatory factors and alleviate the injuries of form and function of brain after cerebral ischemia-reperfusion injury.