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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 study the mechanism of intermittent high glucose on rat Schwann cell (SCs) injury in vitro and intervention of salvianolic acid B (Sal B). Methods: Primary cultured rat SCs were divided into normal group, stable high glucose group, intermittent high glucose group, osmotic pressure control group and intermittent high glucose group with different concentrations of Sal B (25, 50, 100μmol·L- 1) Processing group. Flow cytometry was used to detect apoptosis. DHE probe was used to detect intracellular reactive oxygen species (ROS), JC-1 probe was used to detect the change of mitochondrial membrane potential (MMP), and ELISA was used to detect 8-hydroxydeoxyguanosine )content. The expression of Bcl-2 and Bcl-2 mRNA was detected by real-time fluorescence quantitative PCR. The expressions of Bcl-2, Bax and cysteine were detected by Western blot Caspase-3 and PARP expression. RESULTS: Intermittent high glucose significantly increased the level of ROS in SCs (P <0.01), increased the production of 8-OHd G (P <0.01) and decreased the levels of MMPs (P (P <0.05, P <0.01), down-regulated the expression of Bcl-2 protein and mRNA (P <0.01), up-regulated the protein and mRNA expression of Bax in SCs (P < 0.01), and increased the apoptosis of SCs (P <0.01). Different concentrations of Sal B could decrease the concentration of ROS and 8-OHd G in SCs induced by intermittent high glucose (P <0.01), increase MMP (P <0.01) and up-regulate the expression of Bcl-2 protein and mRNA (P <0.05, P <0.01), downregulated the expression of Bax protein and mRNA (P <0.01), decreased the activation of PARP and caspase-3 (P <0.01) and inhibited the apoptosis of SCs (P <0.01). Conclusion: The effect of intermittent high glucose on SCs is more obvious than that of stable high glucose. Sal B can inhibit SCs injury induced by intermittent high glucose.