辛芍组方对氧糖剥夺损伤的神经PC12细胞凋亡的影响及其机制研究

Effects of Xinshao Formula on the Apoptosis of Oxygen-glucose Deprivation-induced Nerve PC12 Cells and Its Mechanism

目的:考察辛芍组方对氧糖剥夺(OGD)损伤的神经PC12细胞凋亡的影响,并探讨其作用机制。方法:采用连二亚硫酸钠培养PC12细胞建立OGD损伤模型,将OGD损伤的细胞分为模型组(空白培养基)、尼莫地平组(阳性药物,0.42 mg/L)和辛芍组方低、中、高浓度组[质量浓度分别为0.313、0.625、1.25 mg(生药)/L],并取正常细胞作为正常对照组(空白培养基)。各组细胞经药物作用4 h,再复糖复氧4 h后采用MTT法检测细胞存活率,比色法测定乳酸脱氢酶(LDH)的释放量,流式细胞术检测细胞凋亡率和线粒体膜电位(MMP),并采用Western blot法检测细胞凋亡相关基因Caspase-3、Bax、Bcl-2蛋白的表达。结果:与正常对照组比较,模型组细胞的存活率、MMP、Bcl-2蛋白表达水平以及Bcl-2/Bax比值显著降低,而LDH释放量、细胞凋亡率以及Caspase-3、Bax蛋白表达水平显著升高(P<0.05或P<0.01)。与模型组比较,辛芍组方可增加细胞的存活率和MMP,降低LDH的释放量和凋亡率,显著抑制Caspase-3和Bax蛋白表达水平,升高Bcl-2蛋白表达水平和Bcl-2/Bax比值;除辛芍组方低浓度组细胞的MMP、Bcl-2和Bax外,其余各给药组细胞上述指标差异均有统计学意义(P<0.05或P<0.01)。结论:辛芍组方对OGD损伤诱导的PC12细胞凋亡具有抑制作用,其机制可能与减少LDH释放、稳定细胞MMP、调节凋亡相关基因的表达有关。

OBJECTIVE： To investigate the effects of Xinshao formula on the apoptosis of oxygen-glucose deprivation （OGD）-induced PC12 cells, and to investigate its mechanism. METHODS： PC12 cells were cultured by Na2S2O4 to establish OGD model. OGD-induced cells were divided into model group （blank medium）, nimodipine group （positive drug, 0.42 mg/L） and Xinshao formula low-concentration, medium-concentration and high-concentration groups [0.313, 0.625, 1.25 mg （crude drug）/L]; normal cells were included in normal control group （blank medium）. MTT assay was used to determine the survival rate of PC12 cells after treated with drugs for 4 h and then oxygen-glucose restoration for 4 h. The release of LDH was detected by colorimetric method. The apoptosis rate and mitochondrial membrane potential （MMP） were detected by flow cytometry. The protein expression of Caspase-3, Bax and Bcl-2 were examined by Western blot, RESULTS： Compared with normal control group, the survival rate, MMP, the protein expression of Bcl-2 and the ratio of Bcl-2/Bax were decreased significantly in model group; while the release of LDH, apoptosis rate, protein expression of Caspase-3 and Bax were increased significantly （P〈0.05 or P〈0.01）. Compared with model group, the survival rate, MMP, the protein expression of Bcl-2 and the ratio of Bcl-2/Bax were increased in Xinshao formu- la groups; while the release of LDH, apoptosis rate, protein expression of Caspase-3 and Bax were decreased. The above indexes had significant differences except for MMP, Bcl-2 and Bax in Xinshao formula low-concentration group （P〈0.05 or P〈0.01）. CONCLUSIONS： Xinshao formula can inhibit the apoptosis of OGD-induced PC12 cells, which may be related to reducing the re- lease of LDH, keeping MMP stable and regulating the expression of apoptotic-related gene.