全反式维甲酸对转化生长因子诱导的肾小球系膜细胞环氧化酶2表达的影响

The effects of all-trans retinoic acid on expression of cox-2 in rat glomerular mesangial cell treated by TGF-β

目的:研究全反式维甲酸(all-trans retinoic acid,ATRA)对转化生长因子β(transforming growth factor-β,TGF-β)刺激系膜细胞环氧化酶2(COX-2)及Smad3,Smad7蛋白表达的影响,探讨ATRA在TGF-β/Smad信号通路中对系膜细胞表达COX-2的影响作用。方法:取培养第4代的大鼠肾小球系膜细胞分组:(1)对照组;(2)TGF-β刺激组(5、10ng/ml);(3)TGF-β+NS398[COX-2抑制剂组(10μmol/LNS398+10ng/mlTGF-β)];(4)TGF-β+Staurosporine(Smad抑制剂)组(5nmol/ml Staurosporine+10ng/mlTGF-β);(5)药物组:ATRA(10-3、10-6、10-9mol/L)组,在不同的作用时间(4h,12h,24h),WesternBlot印迹法测定各组COX-2、Smad3、Smad7蛋白的表达变化。结果:(1)在外源性TGF-β刺激下,COX-2、Smad3、Smad7蛋白的表达明显增加,各组与对照组比较,差异有统计学意义(P<0.05)。(2)加入COX-2抑制剂NS-398和Smad抑制剂Staurosporine后,COX-2、Smad3、Smad7表达均减少,与TGF-β组比较,差异有统计学意义(P<0.05)。(3)ATRA各浓度组和TGF-β组比较COX-2、Smad3、Smad7蛋白的表达明显减少(P<0.05),呈时间和浓度依赖性。(4)COX-2蛋白的表达与Smad3、Smad7蛋白的表达均呈显著正相关(r=0.978,r=0.942,P<0.05)。结论:ATRA可能通过TGF-β/Smad信号通路抑制系膜细胞COX-2的表达,显示其具有良好的抗纤维化和抗细胞增殖作用。

Objective：To investigate the effects of all-trans retinoic acid （ATRA） on the protein expression of COX-2, Smad3 and Smad7 in mesangial ceils treated by TGF -β, and discuss the effects of TGF-β/Smad signaling pathway on the expression of COX-2 and ATRA influences. Methodology：The glomerular mesangial cells cultured in vitro were randomly divided into 5 groups： TGF-β group （treated with TGF-I3 5, 10 ng/ml）, TGF-β ＋ NS398 group （10 μmol/L NS398 ＋ 10 ng/ml TGF-β）, TGF-β ＋ Staurosporine group （5 nmol/ml Staurosporine ＋ 10 ng/mlTGF-β）, ATRA group （pre-treated with ATRA 10-3, 10 6 10-9 mol/L for 24h, then stimulated with TGF-13 10ng/ml for 4, 12, 24h periods） and the control group. The expression of COX-2, Smad3 and Smad7 were determined by Western blot analysis. Results ： （ 1 ） The protein expressions of COX-2, Smad3 and Smad7 were increased by stimulation of exogenous TGF-β. Compared with the control group, each group had a significant difference in both dose and time dependent ways （ P 〈 0. 05）. （2） The expression of COX-2 was increased after stimulating with TGF-13 （5, 10 ng/ml）. After treating with NS398 （ COX-2 inhibitor） and Staurosporine （ inhibitor of Smad） respectively, the expression of COX-2 showed a dose and time-dependent reduction （P 〈 0. 05 ）. （3） The protein expression of Smad3 and Smad7 were increased by stimulation of exogenous TGF-β（5, 10 ng/m｜）, but inhibited with dose and time-dependent manner by treatment with NS-398 and Staurosporine respectively. （4） After pre-treating with ATRA （10-3, 10-6, 10 9 mol/L） for 24h, the expression of COX-2,Smad3 and Smad 7 in ATRA groups were significantly reduced with dose and time- dependent manner （P 〈0. 05）. （5） The protein expression of COX-2 had a positive correlation with that of Srnad3 and Smad7 （r =0. 978, r = 0. 942, P 〈 0. 05 ）. Conclusion ： ATRA might repress the expression of COX-2 via the TGF-β/Smad signaling pathway, which may play an important role in inhibiting fibrosis and cell proliferation of ATRA.