天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (8): 978-983.doi: 10.11958/20160149

• 细胞与分子生物学 • 上一篇    下一篇

白藜芦醇对H2O2 及TGF-β2诱导人小梁网细胞的影响及机制探讨

齐艳1,2 ， 赵秀娟2 ， 徐琳琪1,2 ， 吴旭东2△， 汪建涛1△   

  1. 1天津医科大学眼科医院， 天津医科大学眼科研究所， 天津医科大学眼视光学院 （邮编300384）； 2天津医科大学基础医学院细胞生物学系
  • 收稿日期:2016-03-11 修回日期:2016-05-02 出版日期:2016-08-15 发布日期:2016-08-22
  • 通讯作者: 吴旭东 E-mail： wuxudong@tijmu.edu.cn； 汪建涛 E-mail： wangjiantao65@126.com E-mail:qiyantianjin@sina.com
  • 作者简介:齐艳 （1990）， 女， 硕士在读， 主要从事眼视光学、 青光眼研究
  • 基金资助:
    国家自然科学基金 （81270994， 31570774， 81500170）； 天津市科技计划项目 （10ZCKFSY08400）； 天津市教育委员会基金 （093-201301）

The role and mechanism of resveratrol on trabecular meshwork cells induced by H2O2 and TGF-β2

QI Yan1,2 , ZHAO Xiujuan2 , XU Linqi 1,2 , WU Xudong2△, WANG Jiantao1△   

  1. 1 Tianjin Medical University Eye Hospital, School of Optometry and Ophthalmology, Eye Institute, Tianjin 300384,China； 2 Deparment of Cell Biology, College of Basic Medicine, Tianjin Medical University
  • Received:2016-03-11 Revised:2016-05-02 Published:2016-08-15 Online:2016-08-22
  • Contact: WU Xudong E-mail: wuxudong@tijmu.edu.cn; WANG Jiantao E-mail: wangjiantao65@126.com E-mail:qiyantianjin@sina.com

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摘要： 摘要： 目的 观察过氧化氢 （H2O2 ） 及转化生长因子 （TGF） -β2 诱导人小梁网细胞 （HTMCs） 后对纤维连接蛋白（FN）、 胶原蛋白 1 型 （COL1）、 核因子 （NF） -κB P65 蛋白和白细胞介素 （IL） -1β基因表达的影响及白藜芦醇 （RSV） 的干预作用。方法 （1） 选取汇合度 70%～80%的 HTMCs 分为 5 组。实验组于无血清培养基中分别加入浓度为 150、 300、 450、 800 μmol/L 的 H2O2 处理， 对照组的培养基中不加 H2O2。Western blot 法检测各组 FN、 COL1、 NF-κB P65、 NF-κB P65 磷酸化 （P-NF-κB P65） 蛋白的表达， 实时定量 PCR 法检测 IL-1β基因的表达。（2） HTMCs 细胞分为 3 组。对照组以不含 H2O2 及 RSV 的无血清培基处理， H2O2 组以 300 μmol/L 的 H2O2 处理， H2O2+RSV 组同时加入 300 μmol/L 的 H2O2 及 25 μmol/L 的 RSV 处理。检测各组上述蛋白和基因的表达情况。免疫荧光检测各组 NF-κB P65 在 HTMCs 中的定位。（3） HTMCs 细胞分为 3 组。对照组以不含 TGF-β2 及 RSV 的无血清培基处理， TGF-β2 组以 5 μg/L 的 TGF-β2 处理， TGF-β2+RSV 组为同时加入 5 μg/L 的 TGF-β2 及 25 μmol/L 的 RSV 处理。检测各组上述蛋白和基因的表达情况。结果 （1） 与对照组比较， 150、 300、 450、 800 μmol/L 组 FN 和 P-NF-κB P65 蛋白表达水平均增高， 300、 450、 800 μmol/L 组 COL1 蛋白和 IL-1β基因表达水平增高 （P < 0.05）， 其他指标比较差异均无统计学意义。（2） H2O2 组较对照组 FN、 COL1、 P-NF-κB P65 蛋白和 IL-1β基因表达水平均增高， 而 H2O2+RSV 组较 H2O2 组上述指标均降低， H2O2+RSV 组较对照组仅 IL-1β降低 （P < 0.05）。对照组仅细胞质表达 NF-κB P65， H2O2 组细胞胞质及核中均有 NF-κB P65 表达， 且核中表达较多； H2O2+RSV 组细胞胞质中表达 NF-κB P65 较核中多。（3） TGF-β2 组较对照组 FN、 COL1、 P-NF-κB P65 的蛋白和 IL-1β基因水平表达均增高 （P < 0.05）， TGF-β2+RSV 组较 TGF-β2 组上述指标均降低 （P < 0.05）。 结论 H2O2 和 TGF-β2 能上调 HTMCs 的 FN、 COL1、 P-NF-κB P65 蛋白及 IL-1β基因的表达， 可能参与青光眼的发生发展过程。RSV 能抑制H2O2和 TGF-β2对HTMCs 的影响， 对青光眼发挥一定的保护作用。

关键词: 青光眼, 小梁网, 过氧化氢, 转化生长因子β2, 细胞外基质, 核因子-κB, 白细胞介素 1β, 纤维连接蛋白, 胶原蛋白 1型, 白藜芦醇

Abstract: Abstract: Objective To investigate hydrogen peroxide (H2O2) and transforming growth factor-β2 (TGF-β2) induced fibronectin (FN), collagen 1 (COL1), nuclear factor (NF) -κB P65 proteins and interlukin (IL) -1β gene expression in human trabecular meshwork cells (HTMCs), and the interventional mechanism of resveratrol (RSV). Methods (1) HTMCs with 70 to 80% confluency were divided into 5 groups. The experimental groups were treated with serum-free medium and with H2O2 at concentrations of 150, 300, 450 and 800 μmol/L. The control group was treated with 0 μmol/L H2O2. The protein levels of FN, COL1, NF- κB P65 and NF- κB P65 phosphorylation (P- NF- κB P65) were measured by Western blot assay. The expression of IL-1β gene was measured by qPCR. (2) HTMCs were divided into 3 groups. The control group was treated with serum-free medium and without H2O2 and RSV. The H2O2 group was treated with 300 μmol/L H2O2. The H2O2 +RSV group was treated with 300 μmol/L H2O2 and 25 μmol/L resveratrol (RSV). The expressions of proteins and genes mentioned above were detected in three groups. NF-κB P65 nuclear translocation was assessed by immunofluorescence technique. (3) HTMCs were divided into 3 groups. The control group was treated with serum-free medium and without TGF-β2 and RSV. The TGF- β2 group was treated with 5 μg/L TGF-β2. The TGF-β2 +RSV group was treated with 5 μg/L TGF-β2 and 25 μmol/L RSV. The expressions of proteins and genes mentioned above were detected in three groups. Results (1) Compared with control group, the protein levels of FN and P-NF-κB P65 were significantly increased in 150, 300, 450 and 800 μmol/L groups， the expression levels of COL1 protein and IL-1β gene were significantly increased in 300, 450 and 800 μmol/L groups (P < 0.05). There were no statistical significances between other indicators. (2) The expression levels of FN, COL1, P-NF-κB P65 proteins and IL-1β gene were significantly higher in H2O2 group than those in control group, and which were significantly lower in H2O2 +RSV group than those in H2O2 group. Compared with control group, only the expression of IL-1β gene was decreased in H2O2 +RSV group (P < 0.05). NF- κB P65 was only expressed in cytoplasm in control group, while it was expressed in both cytoplasm and nucleus in H2O2 group. Compared with H2O2 group, NF-κB P65 was mainly expressed in cytoplasm. (3) Compared with control group, the expressions of FN, COL1, P-NF-κB P65 proteins and IL-1β gene were significantly increased in TGF-β2 group (P < 0.05). Compared with TGF-β2 group, the indicators mentioned above were significantly decreased in TGF-β2+RSV group (P < 0.05). Conclusion H2O2 and TGF-β2 can upregulate the expression of FN, COL1, P-NF-κB P65 proteins and IL-1β gene in HTMCs, which may be involved in the development and progression of glaucoma. RSV can inhibit the influence of H O and TGF-β in HTMCs and exert a protective effect on glaucoma.

Key words:  glaucoma, trabecular meshwork, hydrogen peroxide, transforming growth factor beta2, extracellular matrix, NF-kappa B, interleukin-1beta, protein fiber connection； collagen 1； Resveratrol