吡格列酮对高糖环境中大鼠腹膜间皮细胞氧化应激及单核细胞趋化蛋白1表达的影响

Effects of pioglitazone on the oxidative stress and expression of monocyte chemoattractant protein 1 in rat peritoneal mesothelial cells cultured in high glucose

目的观察吡格列酮(降血糖药)对高糖培养条件下大鼠腹膜间皮细胞(RPMCs)活性氧及单核细胞趋化蛋白1(MCP-1)mRNA、蛋白表达的影响。方法用胰蛋白酶消化法分离培养大鼠腹膜间皮细胞;采用活性氧捕获剂双氢-乙酰乙酸二氯荧光黄(DCFH-DA)孵育细胞;通过流式细胞仪检测细胞内的荧光强度,测得细胞内活性氧(ROS)水平;分别用RT-PCR和ELISA法检测MCP-1 mRNA及蛋白含量。结果正常组和甘露醇组比较,ROS水平和MCP-1表达差异无统计学意义,高糖组ROS水平较正常组明显增高(P<0.01);采用吡格列酮和乙酰半胱氨酸干预后,ROS水平显著降低(P<0.01);高糖组MCP-1 mRNA及蛋白表达显著高于正常组(P<0.01);而小剂量吡格列酮干预组和乙酰半胱氨酸组的MCP-1 mRNA及蛋白表达显著低于高糖组(P<0.01);大剂量较小剂量吡格列酮干预组的ROS和MCP-1水平进一步降低。结论吡格列酮通过抑制氧化应激而降低高糖所诱导的MCP-1表达,这可能是吡格列酮保护腹膜功能的作用机制之一。

Objective To investigate the effects of pioglitazone on the synthesis of reactive oxygen species（ROS） and the expression of monocyte chemoattractant protein 1 induced by high glucose in rat peritoneal mesothelial cells （RPMCs）.Methods RPMCs were isolated and subcultured by enzymatic disaggregation.The level of ROS was assessed indirectly by measuring the fluorescent product from the oxidation of an oxidant-sensitive 2 ,7 -dichlorodihydrofluorescein diacetate（DCFH-DA）by using flow cytometry.The mRNA and the protein expression of MCP-1 were semi-quantitatively determined with RT-PCR and ELISA respectively. Results The level of ROS and MCP-1 were no significant differences between normal glucose group and mannitol group. High glucose increased the intracellular ROS （ P 〈 0. 01 ） and the effect was prevented by pioglitazone （ 5 μmol · L^- 1 ） and acetylcysteine （ NAC ） respectively. The expression of MCP-1 mRNA and protein was upregulated significantly following stimulation with 2. 5% glucose （P 〈 0. 01 ）. Both pioglitazone （5 μmol · L^-1 ）and NAC decreased the expression of MCP-1 mRNA and protein（ P 〈0. 01 ）. The ROS and MCP-1 （mRNA and protein） level in high dose pioglitazone group were lower than that in low dose pioglitazone group. Conclusion Pioglitazone may suppress high glucose induced MCP-1 expression by reducing the level of ROS ,which may be one of the mechanisms that pioglitazone protects peritoneal mesothelial cell and peritoneal function.