间断高浓度葡萄糖对雪旺细胞的损伤机制及丹参酚酸B的保护作用

Study on Mechanism and Inhibitory Effects of Salvianolic Acid B on Schwann Cells Injury Induced by Intermittent High Glucose

目的:研究间断高浓度葡萄糖对体外培养大鼠雪旺细胞(SCs)损伤机制及丹参酚酸B(Sal B)干预作用。方法:原代培养大鼠SCs,将其分为正常组、稳定性高糖组、间断高浓度葡萄糖组、渗透压对照组及间断高浓度葡萄糖加不同浓度Sal B(25,50,100μmol·L-1)处理组。流式细胞仪检测细胞凋亡,DHE探针检测细胞内活性氧(ROS)含量,JC-1探针检测线粒体膜电位(MMP)变化,ELISA法检测8-羟基脱氧鸟苷(8-OHd G)含量。实时荧光定量PCR检测B细胞淋巴瘤/白血病-2基因(Bcl-2)及Bcl-2相关X蛋白(Bax)mRNA表达;Western blot(蛋白印迹)法检测Bcl-2,Bax,半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)及多聚腺苷酸二磷酸核糖聚合酶(PARP)表达。结果:与正常组及稳定性高糖组相比,间断高浓度葡萄糖明显提高了SCs内ROS水平(P<0.01),增加了8-OHd G的生成(P<0.01),降低了MMP(P<0.05,P<0.01),下调SCs内Bcl-2蛋白及mRNA的表达(P<0.01),上调Bax蛋白及mRNA的表达(P<0.01),促进了Caspase-3及PARP的活化(P<0.01),并增加了SCs的凋亡(P<0.01)。不同浓度的Sal B可以降低间断高浓度葡萄糖所导致的SCs内ROS及8-OHd G浓度的增高(P<0.01),提高MMP(P<0.01),上调Bcl-2蛋白及mRNA的表达(P<0.05,P<0.01),下调Bax蛋白及其mRNA的表达(P<0.01),降低了PARP及caspase-3的活化(P<0.01),抑制了SCs凋亡(P<0.01)。结论:间断高浓度葡萄糖对SCs的损伤作用比稳定性高糖更为明显,Sal B可以抑制间断高浓度葡萄糖所致的SCs损伤。

Objective： To investigate the mechanism and inhibitory effects of salvianolic acid B（Sal B）on the intermittent high glucose（IHG）-induced Schwann cells（SCs） injury in vitro.Method： SCs were primarily cultured and were divided into various groups, such as normal, high glucose（HG）,IHG,osmotic controls,with IHG in the presence of 25,50,100μmol·L-1 Sal B for 48 hrs.Apoptosis,intracellular reactive oxygen species（ROS） generation and mitochondrial transmembrane potential were detected by flow cytometry analysis.The concentration of 8-hydroxy-2-deoxy guanosine（8-OHd G） was detected by enzyme-linked immune sorbent assay（ELISA）.Quantitative real-time reverse transcriptase PCR was performed to analyze the expression levels of B cell lymphoma / leukemia-2（Bcl-2） and Bcl-2 associated x protein（Bax）.Western blot were performed to analyze the expression levels of Bcl-2,Bax,Caspase-3 and PARP.Result： The relative levels of intracellular ROS and the percentages of depolarized cells in the IHG group was significantly increased than those in the control and HG group（P〈0.05,P〈0.01）.The concentrations of 8-OHd G showed a marked increase in SCs that were exposed to HG group compared with normal glucose exposure and further increased under IHG conditions（P〈0.01）.Compared to the normal and HG group,treatment with IHG down-regulated the Bcl-2expression of protein and mRNA（P〈0.01）,but up-regulated the Bax expression of protein and mRNA（P 0.01）.In addition,treatment with IHG increased the activation of Caspase-3 and the cleavage of PARP in SCs（P〈0.01）.The percentages of apoptotic cells was increased exposed to HG and substantially more in cells exposed to the IHG（P〈0.01）.Treatment with Sal B inhibited the IHG-induced oxidative stress by reducing ROS production,8-OHd G levels,mitochondrial depolarization,and apoptosis in SCs（P〈0.01）.Furthermore,treatment with Sal B mitigated the IHG-mediated up-regulation of Bax expression and down-regulation of Bcl-2 expression in SCs（P〈0.05,P〈0.01）.In addition,treatment with Sal B attenuated the IHG-induced activation of Caspase-3 andminimized the cleavage of PARP（P〈0.01）.Conclusion： The cytotoxic effect of IHG may be significantly more potent than that of HG and Sal B antagonized the IHG-induced injury of SCs.