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目的观察不同浓度缬沙坦体外干预高糖环境下小鼠足细胞间充质细胞转分化及转化生长因子-β1(TGF-β)/Smad信号通路的调节作用。方法复苏和传代小鼠足细胞,用Rand A 1.0软件完全随机分组法将培养成熟的小鼠足细胞随机分为5 mmol·L~(-1)葡萄糖培养液干预组(A组)和25 mmol·L~(-1)葡萄糖培养液干预组(B组),在25 mmol·L~(-1)葡萄糖培养液中分别加入2×10~(-7)mol·L~(-1)(C组),2×10~(-6)mol·L~(-1)(D组)和2×10~(-5)mol·L~(-1)(E组)缬沙坦。体外培养48 h后,倒置显微镜下观察足细胞形态变化,用实时定量-聚合酶链式反应(RT-PCR)和western-blot技术分别检测各组足细胞肾小球足细胞相关蛋白1(NEPH1)、desmin、TGF-β1和Smad7的mRNA和蛋白表达的变化。结果与A组相比,B组足细胞形态和数量显著变化;C、D、E组足细胞形态和数量均不同程度的接近A组,以E组恢复程度最为明显。A组足细胞NEPH1mRNA为0.75±0.08,NEPH1蛋白为2.30±0.14;Smad7 mRNA为1.97±0.14,Smad7蛋白为1.88±0.16;desmin mRNA为0.54±0.08,desmin蛋白为1.52±0.11;TGF-β1mRNA为0.35±0.08,TGF-β1蛋白为0.22±0.10。B组足细胞NEPH1 mRNA为0.58±0.14,NEPH1蛋白为1.91±0.12;Smad7 mRNA为0.34±0.11,Smad7蛋白为0.34±0.12;desmin mRNA为0.670±0.059,desmin蛋白为2.02±0.17;TGF-β1mRNA为1.69±0.10,TGF-β1蛋白为1.60±0.13,与A组比较,差异有统计学意义(P<0.01)。C组NEPH1 mRNA为0.60±0.15,NEPH1蛋白为1.92±0.13;Smad7 mRNA为0.37±0.16,Smad7蛋白为0.49±0.11;desmin mRNA为0.66±0.13,desmin蛋白为2.01±0.09;TGF-β1mRNA为1.53±0.09,TGF-β1蛋白为1.44±0.11,与A组比较,差异有统计学意义(P<0.01)。D组NEPH1 mRNA为0.63±0.08,NEPH1蛋白为1.95±0.09;Smad7为mRNA 0.66±0.13,Smad7蛋白为0.72±0.12;desmin mRNA为0.62±0.16,desmin为1.96±0.13;TGF-β1mRNA为0.98±0.08,TGF-β1蛋白为0.87±0.11,与B组比较,差异有统计学意义(P<0.01)。E组NEPH1 mRNA为0.72±0.14,NEPH1蛋白为2.13±0.16;Smad7 mRNA为1.41±0.12,Smad7蛋白为1.65±0.12;desmin mRNA为0.56±0.17,desmin蛋白为1.55±0.12;TGF-β1mRNA为0.54±0.12,TGF-β1蛋白为0.52±0.14,与B、D组比较,差异有统计学意义(P<0.01)。结论缬沙坦通过抑制TGF-β/Smad信号通路缓解高糖诱导的足细胞转分化,是其保护受损足细胞的可能机制。
Objective To investigate the effects of valsartan on the regulation of transdifferentiation of podocytes and TGF-β / Smad signaling pathway in vitro under high glucose conditions. Methods The mouse podocytes were resuscitated and passaged. The mature mouse podocytes were randomly divided into 5 mmol·L -1 glucose medium (group A) and 25 mmol · L ~ (-1) glucose medium intervention group (B), 25 × L ~ (-1) glucose medium were added 2 × 10 ~ (-7) mol·L ~ (-1) C group), 2 × 10 -6 mol·L -1 (D group) and 2 × 10 -5 mol·L -1 (group E) valsartan. After cultured in vitro for 48 h, the morphological changes of podocytes were observed under inverted microscope. The expressions of NEPH1 ), Desmin, TGF-β1 and Smad7 mRNA and protein expression changes. Results Compared with group A, the morphology and number of podocytes in group B were significantly changed. The morphology and number of podocytes in group C, D and E were all close to those in group A, and the recovery was the most obvious in group E. In group A, the numbers of NEPH1 mRNA in podocyte were 0.75 ± 0.08, in NEPH1 protein was 2.30 ± 0.14, in Smad7 mRNA was 1.97 ± 0.14, in Smad7 protein was 1.88 ± 0.16, in desmin mRNA was 0.54 ± 0.08, and in desmin protein was 1.52 ± 0.11; ± 0.08, and TGF-β1 protein was 0.22 ± 0.10. NEPH1 mRNA of group B was 0.58 ± 0.14, NEPH1 was 1.91 ± 0.12, Smad7 mRNA was 0.34 ± 0.11, Smad7 protein was 0.34 ± 0.12, desmin mRNA was 0.670 ± 0.059, desmin protein was 2.02 ± 0.17, TGF-β1 mRNA was 1.69 ± 0.10, and TGF-β1 protein was 1.60 ± 0.13, which was significantly different from that of group A (P <0.01). The NEPH1 mRNA of group C was 0.60 ± 0.15, the value of NEPH1 was 1.92 ± 0.13, that of Smad7 mRNA was 0.37 ± 0.16, that of Smad7 was 0.49 ± 0.11, that of desmin mRNA was 0.66 ± 0.13, that of desmin was 2.01 ± 0.09, and that of TGF-β1 was 1.53 ± 0.09, and TGF-β1 protein was 1.44 ± 0.11, which was significantly different from that of group A (P <0.01). The NEPH1 mRNA of group D was 0.63 ± 0.08, the value of NEPH1 was 1.95 ± 0.09, the level of mRNA of Smad7 was 0.66 ± 0.13, the value of Smad7 was 0.72 ± 0.12, the value of desmin mRNA was 0.62 ± 0.16, the value of desmin was 1.96 ± 0.13 and that of TGF-β1 was 0.98 ± 0.08 , TGF-β1 protein was 0.87 ± 0.11, compared with the B group, the difference was statistically significant (P <0.01). The NEPH1 mRNA of group E was 0.72 ± 0.14, the value of NEPH1 protein was 2.13 ± 0.16, the value of Smad7 mRNA was 1.41 ± 0.12, the value of Smad7 protein was 1.65 ± 0.12, the value of desmin mRNA was 0.56 ± 0.17, the value of desmin protein was 1.55 ± 0.12 and that of TGF-β1 mRNA was 0.54 ± 0.12, and TGF-β1 protein was 0.52 ± 0.14. There was significant difference between group B and group D (P <0.01). Conclusion Valsartan can relieve the podocyte transdifferentiation induced by high glucose by inhibiting the TGF-β / Smad signaling pathway, which is a possible mechanism of protecting the damaged podocytes.