天津医药

天津医药 ›› 2019, Vol. 47 ›› Issue (12): 1205-1209.doi: 10.11958/20191656

• 细胞与分子生物学 • 上一篇    下一篇

生松素对AngⅡ诱导的大鼠肾小球系膜细胞细胞外基质合成的影响及其机制研究

王梦平1，王丽2，高利超1，肖文1，甘林望1，吴蔚桦1，刘建1,3△   

  1. 西南医科大学附属医院肾病内科（邮编646000）
  • 收稿日期:2019-06-05 修回日期:2019-08-19 出版日期:2019-12-15 发布日期:2019-12-15
  • 通讯作者: 刘建 E-mail:834300205@qq.com
  • 作者简介:王梦平（1992），女，硕士，住院医师，主要从事肾小球疾病与基础研究
  • 基金资助:
    生松素对大鼠糖尿病肾病的治疗作用及其机制研究;生松素调节血管紧张素Ⅱ诱导的系膜细胞细胞外基质合成的分子机制研究

The effect and mechanism of pinocembrin on synthesis of extracellular matrix in angiotensinⅡ-induced rat glomerular mesangial cells

WANG Meng-ping1, WANG Li2, GAO Li-chao1, XIAO Wen1, GAN Lin-wang1, WU Wei-hua1, LIU Jian1,3△   

  1. Department of Nephrology, the Affiliated Hospital of Southwest Medical University, Luzhou 646000,China
  • Received:2019-06-05 Revised:2019-08-19 Published:2019-12-15 Online:2019-12-15
  • Supported by:
     

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摘要： 目的 探讨生松素（Pb）对血管紧张素Ⅱ（AngⅡ）诱导的大鼠肾小球系膜细胞细胞外基质（ECM）合成的影响及其机制。方法 以大鼠肾小球系膜细胞为研究对象，将实验分为以下5组：正常对照（NC）组、1×10-6mol/L AngⅡ（Ng）组、1×10-6 mol/L AngⅡ+1‰ DMSO（Ng-D）组、 1×10-6 mol/L AngⅡ+30 μmol/L Pb（Ng-Pb）组、1×10-6 mol/L AngⅡ+10 μmol/L缬沙坦（Ng-Val）组。分别在干预12、24、48 h搜集细胞和上清液，采用Western blot检测Ⅳ型胶原（ColⅣ）、纤连蛋白（FN）、转化生长因子-β（ 1 TGF-β1 ）、Smad3、磷酸化Smad3（p-Smad3）、核因子κB（NF-κB）、磷酸化NF-κB（pNF-κB）的蛋白水平，酶联免疫吸附法（ELISA）检测上清液中肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）水平，实时荧光定量PCR（qPCR）检测细胞中ColⅣ、FN、TGF-β1、TNF-α、IL-6的mRNA水平。结果 （1）Pb对 AngⅡ诱导的系膜细胞合成ECM的影响：与NC组相比，Ng组细胞内ColⅣ和FN的蛋白质和mRNA 水平升高（P＜0.05），而Ng-Pb组和Ng-Val组细胞内ColⅣ和FN的蛋白质和mRNA水平均较Ng组降低（P＜0.05）。（2）Pb对TGF-β1/Smad3通路的 影响：与NC组相比，Ng组细胞内TGF-β1的蛋白质和mRNA水平增加，p-Smad3/Smad3水平增加（ P＜0.05），而Ng-Pb组和Ng-Val组细胞内TGF-β1的蛋白质和mRNA水平以及p-Smad3/Smad3水平均较Ng组降低（P＜0.05）。（3）Pb对NF-κb介导的促炎症因子产生的影响：与NC组相比，Ng组 p-NF-κb/NF-κb水平增加，IL-6和TNF-α的蛋白质和mRNA水平增加（P＜0.05），而Ng-Pb组和 Ng-Val组p-NF-κb/NF-κb水平以及IL-6和TNF- α的蛋白质和mRNA水平均较Ng组降低（P＜0.05）。结论 Pb可能通过抑制TGF-β1/Smad3信号通路和NF-κb介导的促炎症因子的产生， 从而抑制AngⅡ诱导的大鼠系膜细胞合成ECM。

关键词: 生松素血管, 紧张素Ⅱ, 细胞外基质, 转化生长因子-β1, 核因子-κB, 肾小球系膜细胞

Abstract: Objective To study the effect of pinocembrin (Pb) on extracellular matrix (ECM) synthesis in angiotensin Ⅱ (AngⅡ)-induced rat mesangial cells and its mechanism. Methods Rat mesangial cells were divided into the following groups: normal control group (NC), 1×10 -6 mol/L AngⅡ group (Ng), 1×10-6 mol/L AngⅡ+1‰ DMSO group (Ng-D), 1×10-6mol/L AngⅡ+30 μmol/L Pb group (Ng-Pb) and 1×10-6 mol/L AngⅡ+10 μmol/L valsartan group (Ng-Val). Cells and supernatants were collected at 12, 24 and 48 hours after intervention. CollagenⅣ (ColⅣ),fibronectin(FN),transforming growth factor β1 (TGF-β1), Smad3, p-Smad3, nuclear factor kappa B (NF-κb) and p-NF-κb were detected by Western blot assay. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)were detected by ELISA , and the RNA levels of Col Ⅳ, FN, TGF-β1, TNF-α and IL-6 were detected by qPCR.Results (1) The effect of Pb on synthesis of ECM in Ang Ⅱ-induced rat mesangial cells: after Ang Ⅱ stimulation, the protein and mRNA levels of ColⅣ and FN in rat mesangial cells were significantly increased(P＜0.05),and pre-stimulation with Pb could significantly reduce the protein and mRNA levels of ColⅣ and FN ( P＜0.05).(2) The effect of Pb on the TGF-β1 /Smad3pathway: AngⅡ increased the protein and mRNA levels of TGF - β1, and raised the level of p-Smad3 / smad3 (P＜0.05), while pre-stimulation with Pb could significantly reduce the protein and mRNA level of TGF - β1, and decrease the level of p-Smad3 / smad3 (P＜0.05). (3)Effects of Pb on NF-κB-mediated pro-inflammatory factor production: after AngⅡ stimulation, the level of p-NF-κB/NF-κB increased significantly, the protein and RNA levels of IL-6 and TNF -α increased significantly (P＜0.05).Prestimulation with Pb could significantly alleviate the above changes ( P＜0.05).Conclusion Pb may inhibit synthesis of ECM in AngⅡ-induced rat mesangial cells through inhibiting the TGF -β1 / Smad3 pathway and NF -κB-mediated proinflammatory factor production.

Key words: pinocembrin, angiotensin Ⅱ , extracellular matrix, transforming growth factor beta1, NF-kappa B, mesangial cells

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