Department of Nephrology, Affiliated Hospital of Nantong University, Nantong 226001, PR China.
Exp Biol Med (Maywood). 2014 Mar;239(3):272-83. doi: 10.1177/1535370213519216. Epub 2014 Feb 5.
All-trans retinoic acid (ATRA) has been used for the treatment of acute promyelocytic leukemia. It remains unclear, however, whether ATRA affects cyclooxygenase-2 (COX-2; an enzyme involved in prostaglandin production), PGE2, and thromboxane A2 (TXA2) (metabolic products of COX-2) by a transforming growth factor-β/Smad-signaling pathway, which plays important roles in mesangial-cell proliferation and renal fibrosis. In this study, the mRNA and protein of Smad3, Smad7, and COX-2 were detected by reverse transcription-polymerase chain reaction and Western blot, respectively, in mesangial cells stimulated by transforming growth factor-β (TGF-β) and treated with ATRA at various concentrations and times. The protein level of PGE2 and TXA2 was also measured by enzyme-linked immunosorbent assay. The localization of Smad3 and Smand7 was observed by confocal microscope. Cell proliferation was detected by MTT assay, while apoptosis was determined using Hoechest staining. The expression of Smad3, Smad7, and COX-2 mRNA and protein was increased by exogenous TGF-β, but inhibited by pretreatment of ATRA, in dose and time-dependent manners. In addition, the expression of Smad3 and Smad7 was significantly reduced not only by staurosporine, an inhibitor of threonine/serine protein kinases as well as smad, but also by NS-398, an inhibitor of COX-2. PGE2 and TXA2 were raised by TGF-β, but also decreased by ATRA, staurosporine, and NS-398. Moreover, ATRA reversed the translocation of Smad3 and Smad7 induced by TGF-β. Compared with the control, TGF-β also significantly enhanced proliferation and inhibited apoptosis of mesangial cells. ATRA dose-dependently inhibited TGF-β-induced cell proliferation, but had no significant effect on apoptosis in rat mesangial cells. Therefore, ATRA repressed COX-2, PGE2, and TXA2 via the TGF-β/Smad-signaling pathway and inhibited mesangial-cell proliferation, which might subsequently prevent renal fibrosis.
全反式维甲酸（ATRA）已被用于治疗急性早幼粒细胞白血病。然而，目前尚不清楚 ATRA 是否通过转化生长因子-β/Smad 信号通路影响环氧化酶-2（COX-2；参与前列腺素产生的酶）、PGE2 和血栓烷 A2（TXA2；COX-2 的代谢产物），该通路在系膜细胞增殖和肾纤维化中发挥重要作用。在这项研究中，通过逆转录-聚合酶链反应和 Western blot 分别检测转化生长因子-β（TGF-β）刺激和不同浓度和时间 ATRA 处理后系膜细胞中 Smad3、Smad7 和 COX-2 的 mRNA 和蛋白，通过酶联免疫吸附试验测量 PGE2 和 TXA2 的蛋白水平。通过共聚焦显微镜观察 Smad3 和 Smand7 的定位。通过 MTT 测定检测细胞增殖，通过 Hoechest 染色测定细胞凋亡。外源性 TGF-β可增加 Smad3、Smad7 和 COX-2 mRNA 和蛋白的表达，而 ATRA 预处理呈剂量和时间依赖性抑制其表达。此外，不仅用丝氨酸/苏氨酸蛋白激酶和 Smad 的抑制剂 staurosporine，而且用 COX-2 抑制剂 NS-398 预处理均可显著降低 Smad3 和 Smad7 的表达。PGE2 和 TXA2 由 TGF-β 上调，但也被 ATRA、staurosporine 和 NS-398 下调。此外，ATRA 逆转了 TGF-β诱导的 Smad3 和 Smad7 的易位。与对照组相比，TGF-β还显著增强了系膜细胞的增殖并抑制了其凋亡。ATRA 呈剂量依赖性抑制 TGF-β诱导的系膜细胞增殖，但对大鼠系膜细胞的凋亡无明显影响。因此，ATRA 通过 TGF-β/Smad 信号通路抑制 COX-2、PGE2 和 TXA2，抑制系膜细胞增殖，从而可能预防肾纤维化。
