去泛素化酶A20在糖尿病肾病大鼠肾脏及脂多糖诱导的系膜细胞中的表达及变化

Changes of A20 expression in mesangial cells of LPS-induced diabetic nephropathy rat model

目的观察去泛素化酶A20在糖尿病肾病(DN)大鼠肾脏及脂多糖(LPS)诱导的系膜细胞中的表达变化及可能的作用机制。方法 (1)Wistar雄性大鼠30只,随机均分为2组,模型组给予链脲佐菌素(STZ)60 mg/kg腹腔注射建立早期DN大鼠模型;对照组给予60 mg/kg枸椽酸缓冲液腹腔注射。分析2组6、8周时血糖和尿微量白蛋白变化。HE染色分析2个时间点的肾脏病理变化。采用免疫组化检测A20在肾脏中的表达。(2)体外培养肾脏系膜细胞,采用不同时间(0、2、4、6、12、24、48、72 h)和不同浓度(0.1、1、10μg/L)的LPS处理细胞后,检测A20、核因子(NF)-κB、核因子κB抑制因子(IκB)、IκB激酶γ(IKKγ)、单核细胞趋化蛋白(MCP)-1蛋白及基因的表达变化。结果(1)模型组较对照组血糖和尿微量白蛋白含量均升高(均P<0.01)。模型组肾小管上皮细胞呈玻璃样变,8周时更明显。模型组较对照组A20蛋白在肾小管表达明显减弱,而肾小球中基本无表达,8周时表达更少。(2)IKKγ不同浓度与时间下差异均无统计学意义。与0 h相比,2、4 h时A20蛋白和基因水平呈高表达,6 h后表达降低(均P<0.05);各时间点NF-κB表达水平均较0 h增高,IκB表达均降低(均P<0.05)。A20和IκB的蛋白和基因表达呈浓度依赖性降低(均P<0.05),NF-κB蛋白和基因以及MCP-1基因表达呈浓度依赖性升高(均P<0.05)。结论A20可通过调节NF-κB信号通路来导致炎症信号通路的持续激活,可能在DN的发生发展中发挥作用。

Objective To observe the changes of A20 in mesangial cells of diabetic nephropathy（DN） rat model in-duced by lipopolysaccharide（LPS）- rat, and to explore its possible mechanism. Methods（1）Thirty health male Wistar ratswere randomly divided into two group. Model rats were given streptozotocin（STZ） at a dose of 60 mg/kg by intraperitoneal in-jection. Rats in the control group received the same volume of citrate buffer in the same way. Levels of blood glucose and uri-nary microalbumin were detected in two groups at the 6thand the 8thweek. Changes of renal pathology were observed by HEstaining. Changes of protein A20 were observed by immunohistochemistry.（2） Expression changes of gene and proteins A20,nuclear factor（NF）-κB, IκB, IKKγ and MCP-1 in renal cells treated with LPS were determined after treatment with differenttime points（0, 2, 4, 6, 12, 24, 48 and 72 h） and different concentrations（0.1, 1 and 10 μg/L）. Results（1） Levels of bloodglucose and urinary microalbumin were significantly increased in model group compared with those of control group（P 0.01）. HE stainig showed that hyaline degeneration in tubular epithelial cells was found in model group, especially at the 8th week. Results of immunohistochemistry showed that expression of protein A20 significantly decreased in kidney tubules andnearly disappeared in glomerulus in model group compared with that of control group, which expressed less at the 8thweek.（2） There was no significant difference in the expression of IKKγ between different concentrations and different times. Com-pared with 0 h, the expression of A20 protein was increased at 2 h and 4 h, except that the expression of A20 protein in-creased after 6 h（P 0.05）. Meanwhile NF-κB expression increased and IκB expression decreased in different time points（P 0.05）. In addition, the expressions of A20 and IκB were decreased concentration-dependently（P 0.05）. The expres-sion levels of NF-κB and MCP-1 were increased concentration-dependently（P 0.05）. Conclusion A20 may involve inthe development of diabetic nephropathy by regulating the NF-κB pathway.