吡格列酮对肾小管上皮细胞转分化及CTGF表达的影响

Effects of Pioglitazone on the Transdifferentiation and CTGF Expression of Renal Tubular Epithelial-myofibroblast in vitro

目的探讨吡格列酮能否负性调节转化生长因子β(TGF-β)诱导的肾小管上皮细胞转分化为肌成纤维细胞(TEMT),以及抑制TGF-β下游介质结缔组织生长因子(CTGF)表达,以阐明吡格列酮抗肾纤维化的作用及机制。方法将体外培养的正常大鼠肾小管上皮细胞(NRK52E)分为空白对照组、TGF-β3 ng/mL诱导组、TGF-β3 ng/mL+DMSO组、不同剂量吡格列酮阻断组。应用倒置相差显微镜观察细胞形态,免疫组织化学、RT-PCR方法定性及定量检测肌成纤维细胞特异性标志物α-平滑肌肌动蛋白(α-SMA);RT-PCR方法定量检测CTGFmRNA的表达;ELISA法测定培养细胞上清液中Ⅲ型胶原的含量。结果①TGF-β诱导组细胞从原来典型的上皮细胞形态转变为长梭形肌成纤维细胞形态;免疫组织化学染色见大量α-SMA阳性细胞,与对照组相比,α-SMAmRNA表达量增加(P<0.05);CTGFmRNA表达量增加(P<0.05);细胞培养上清液中Ⅲ型胶原的含量增加(P<0.05)。②吡格列酮阻断组明显抑制TGF-β诱导的NRK52E细胞形态学改变;各剂量组均能抑制TGF-β诱导的α-SMAmRNA、CTGFmRNA表达及Ⅲ型胶原分泌(P<0.05),且呈剂量依赖(P<0.05);③相关分析显示,CTGFmRNA与α-SMAmRNA、Ⅲ型胶原的分泌呈正相关(r=0.975,0.988,P均<0.01),α-SMAmRNA与Ⅲ型胶原的分泌呈正相关(r=0.952,P<0.01)。结论吡格列酮能够抑制TGF-β诱导的TEMT及Ⅲ型胶原的分泌,对肾间质纤维化具有负性调节作用;吡格列酮抑制TGF-β诱导的CTGF表达可能是其负性调节TEMT的机制之一。

Objective To investigate whether pioglitazone can inhibit TGF-β-induced phenotypic remodeling of renal tubular epithelial cell and the expression of CTGF, so to illuminate the anti-fibrotie mechanism of pioglitazone. Methods Cultured NRK52E cells were divided into control group, TGF-β-treated（3 ng/mL） group, TGF-β ng/mL）and DMSO-treated group, TGF-β and pioglitazone-treated groups with different doses. The morphological change of cultured tubular cells was observed under phase-contrast microscopy. Immunohistochemistry were used to assess the α-SMA＋ cells, and the expression of α-SMA mRNA, CTGF mRNA was measured by RT-PCR. The level of collagen type Ⅲ in the culture supernatant was measured by ELISA. Results （1） Confluent NRK52E cells cultured in medium with 5% FCS showed a normal epithelial morphological state and cobblestone pattern. All cells almost had no expression of α-SMA and CTGF. After the NRK52E cells were cultured with TGF-β3 ng/mL for 5 days, many cells had strong cytoplasmic staining for a-SMA, and α-SMA＋ cells showed marked hypertrophy and elongated shape. The levels of α-SMA mRNA and CTGF mRNA in TGF-β- treated group cells were higher than those of normal control group（P〈0.05）. Compared with control group, there were significant increases of collagen typeⅢ content in supernatant of TGF-β-treated groups （P〈0.05）. （2）The phenotypie changes induced by TGF-β were prevented by incubation with pioglitazone. Pioglitazone inhibited the expression of a-SMA mRNA and CTGF mRNA of NRK52E cells, and the supernatant content of collagen type Ⅲ in a dose-dependent manner（P〈0.05）. （3） CTGF mRNA expression of NRK52E cells was significantly associated with ocSMA mRNA expression（r=0. 975, P〈0.01）, and the content of collagen type Ⅲ （r=0. 988, P〈0.01）. The expression of a-SMA mRNA was correlated with the content of collagen type Ⅲ （r= 0. 952, P〈0.01）. Conclusion Pioglitazone may negatively regulate TEMT through inhibiting the expression of CTGF.