解毒化瘀方对凝血酶合并缺氧诱导PC12细胞损伤ERK1/2信号通路表达的影响

Effect of Jiedu Huayu Fang on the Expression of ERK1/2 Signaling Pathway in PC12 Cells Injuried by Thrombin and Hy Poxia

目的探讨急性缺血性中风(AIS)瘀毒病机的分子机制及解毒化瘀方对凝血酶合并缺氧损伤的保护作用。方法(1)用凝血酶合并缺氧损伤PC12细胞,模拟急性缺血性中风的体外细胞模型。(2)将细胞分为空白组,模型组,解毒化瘀方含药血浆组和PD98059组(MEK抑制剂组),应用荧光定量实时RT-PCR方法检测MEK1、PAR-1及ERK1/2的m RNA表达,应用免疫荧光法检测ERK1/2、P-ERK1/2的蛋白表达。结果 PCR结果提示:模型组MEK1、PAR-1及ERK1/2的m RNA表达与空白对照组相比显著升高(P<0.01),解毒化瘀方组和PD98059组MEK1、PAR-1及ERK1/2的m RNA表达较模型组显著降低(P<0.01),免疫荧光结果提示:解毒化瘀方组和PD98059组ERK1/2、P-ERK1/2的蛋白表达较模型组显著降低(P<0.01)。结论解毒化瘀方以凝血酶为作用的分子靶点,能够显著降低ERK1/2信号通路在缺血性中风体外细胞模型中的表达。

Objective To investigate the molecular mechanism of cerebral vascular occlusion and the mechanism of brain cell damage induced by acute ischemic stroke（AIS） and the protective effect of Jiedu Huayu Fang. Methods（1） PC12 cells were used to establish acute ischemic stroke in vitro.（2） The cells were divided into blank group, model group, Jiedu Huayu Fang serum group and PD98059 group（MEK inhibitor group）. The m RNA expression of MEK1, PAR-1 and ERK1/2was detected by Real-time PCR, and the protein expression of P-ERK1/2 and ERK1/2 was detected by immunofluorescence assay. Results PCR results indicated that the expression of MEK1, PAR-1 and ERK1/2 m RNA in the model group was significantly higher than that in the blank control group（P〈0.01）. The MEK1, PAR-1 and ERK1/2 m RNA expression in Jiedu Huayu Fang group and PD98059 group was much lower than that of model group（P〈0.01）. Immunofluorescence results show that： the ERK1/2, p-ERK1/2 protein expression of Jie Du Hua Yu Fang group and PD98059 group was much lower than that of model group（P〈0.01）. Conclusion Jiedu Huayu Fang, as the molecular targets of thrombin, can significantly reduce the expression of ERK1/2 signaling pathway in acute ischemic stroke.