糖基化终产物对人肾小球系膜细胞氧化应激及MCP-1表达的影响及机制

Effect of advanced glycosylation end products on oxidative stress and MCP-1 in human renal mesangial cells

目的:探讨体外培养条件下糖基化终产物(AGEs)对人肾小球系膜细胞(HRMCs)中糖基化终产物受体(RAGE)、氧化应激及单核细胞趋化因子-1(MCP-1)表达的影响。方法:将HRMCs与不同浓度的糖化牛血清白蛋白(AGE-BSA)和牛血清白蛋白(BSA)共同培养,或与同一质量浓度的AGE-BSA和BSA共同培养不同时间,以中和抗RAGE抗体封闭细胞膜上RAGE;采用细胞免疫化学法检测AGEs对HRMCs中RAGE表达的影响,流式细胞术检测细胞内活性氧(ROS),半定量逆转录-聚合酶链反应(RT-PCR)法检测MCP-1 mRNA的表达。结果:在HRMCs中AGE-BSA能够促进RAGE的表达,并以时间和剂量依赖方式促进HRMCs中ROS及MCP-1的表达;ROS及MCP-1的表达水平在加入不同浓度(50、100、200、400 mg/L)的AGE-BSA作用48 h后以及加入质量浓度为200 mg/L的AGE-BSA作用不同时间(12、24、48、72 h)后,较相应质量浓度或时间的BSA组和对照组均明显升高(P<0.05);抗RAGE抗体干预后能够部分抑制AGE-BSA诱导ROS及MCP-1的表达,而人IgG没有这种作用。结论:AGEs通过RAGE激活氧化应激效应诱导MCP-1的表达上调,是糖尿病肾病发生发展的可能机制。

Objective： To investigate the effects of advanced glycosylation end products（AGEs） modified bovine serum albumin （AGE- BSA） on the expression of reactive oxygen species（ROS）and monocyte chemoauractant protein-1 （MCP-1）in human renal mesangial cells （HRMCs）. Methods： HRMCs were cultured/n v/tro with medium containing different doses of AGE-BSA or BSA（50,100,200,400 mg/L）for 48 hours,or with AGE-BSA （200 rng/L） for different times （12,24,48,72 h）. Immunocytochemistry assay was used to estimate the protein level of RAGE. The ROS in cells were measured by flow cytometry and the mRNA expression of MCP-1 were analyzed by semi-quantiative re- verse tmnscription-polymerase chain reaction （RT-PCR）after treatment with AGE-BSA or BSA. Results： The protein level of RAGE was up- regulated in the HRMCs with AGE-BSA . The expression of ROS and MCP-1 significantly enhanced by incubation of AGE-BSA in a time- and dose-dependent manner. The effects of AGE-BSA-induced up-regulation of ROS and MCP-1 level was significantly blocked by neutralizing anti- bodies to RAGE, while the expression of ROS and MCP-1 stood nearly unchanged after cultured with huamn IgG. Condusion： The expression of ROS and MCP-1 in HRMCs is induced by AGE-BSA through RAGE, which may have potential effects in the pathgenic mechanism of diabetic nephtopathy.