天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (4): 444-448.doi: 10.11958/20150120

• 实验研究 • 上一篇    下一篇

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

梁雅灵， 陈娇， 龙洋， 李衍辉， 黎秋晗， 范芳， 徐勇△   

  1. 西南医科大学附属医院内分泌科 （邮编646000）
  • 收稿日期:2015-08-19 修回日期:2015-11-13 出版日期:2016-04-15 发布日期:2016-05-20
  • 通讯作者: △通讯作者 E-mail： xywyll@aliyun.com E-mail:xywyll@aliyun.com
  • 作者简介:梁雅灵 （1989）， 女， 硕士在读， 主要从事糖尿病肾病研究
  • 基金资助:
    四川省教育厅重点项目 （10ZA036）； 四川省国际合作项目 （14GH0003）

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

LIANG Yaling, CHEN Jiao, LONG Yang, LI Yanhui, LI Qiuhan, FAN Fang, XU Yong△   

  1. Department of Endocrinology, the Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000， China
  • Received:2015-08-19 Revised:2015-11-13 Published:2016-04-15 Online:2016-05-20
  • Contact: △Corresponding Autor E-mail: xywyll@aliyun.com E-mail:xywyll@aliyun.com

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摘要： 摘要: 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、 10 μg/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。

关键词: 糖尿病肾病, 肾小球系膜细胞, 脂多糖类, 血糖, NF-κB, 尿微量白蛋白, A20 基因

Abstract: Abstract：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 rats were 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 6th and the 8th week. Changes of renal pathology were observed by HE staining. 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 different time 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 blood glucose 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 and nearly disappeared in glomerulus in model group compared with that of control group, which expressed less at the 8th week. (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 in the development of diabetic nephropathy by regulating the NF-κB pathway.

Key words: diabetic nephropathy, mesangial cells, lipopolysaccharides, blood glucose, NF-kappa B, micro albuminuria, A20 gene