Intermedin对血管紧张素Ⅱ诱导的NRK-52E细胞纤维化的影响及机制研究

Effect and mechanism of intermedin in NRK-52E cellular fibrosis induced by angiotensin Ⅱ

目的观察Intermedin(IMD)对血管紧张素Ⅱ(AngⅡ)诱导的大鼠肾小管上皮细胞系NRK-52E纤维化的影响,并探讨其机制。方法体外培养的NRK-52E细胞,随机分为4组:正常对照组、AngⅡ组(10^(-6)mol/L)、转染IMD质粒+AngⅡ组、转染空质粒+AngⅡ组。采用Fugene HD转染试剂,将pIRES2-EGFP空质粒和pIRES2-IMD真核表达质粒分别转染至NRK-52E细胞,应用流式细胞仪检测转染效率,转染成功48 h后用AngⅡ(10^(-6)mol/L)干预24 h。用real-time RT-PCR检测各组细胞中Ⅰ型胶原(ColⅠ)的表达;Western印迹法检测ColⅠ、E-钙黏蛋白(E-cadherin)的表达;ELISA法检测细胞培养上清液内皮型一氧化氮合酶(eNOS)、环磷酸鸟苷(cGMP)的浓度。采用SPSS17.0软件包进行统计学处理,以P<0.05为差异有统计学意义。结果与正常对照组相比,AngⅡ组ColⅠ的表达显著上调(mRNA水平t=4.835,P=0.008;蛋白质水平P<0.001),E-钙黏蛋白的表达显著下降(t=4.284,P=0.013);转染IMD质粒后ColⅠ的表达较AngⅡ组明显下降(mRNA水平t=3.032,P=0.039;蛋白质水平P<0.001),E-钙黏蛋白的表达明显上调(t=6.054,P=0.004);而转染空质粒后ColⅠ、E-钙黏蛋白的表达与AngⅡ无明显差别(ColⅠmRNA水平t=0.951,P=0.395;蛋白质水平t=1.208,P=0.294;E-钙黏蛋白蛋白质水平t=1.613,P=0.182)。与正常对照组相比,AngⅡ组细胞上清液eNOS、cGMP的浓度显著增加(eNOS t=20.718,P<0.001;cGMP t=8.324,P=0.001);与AngⅡ组相比,IMD质粒+AngⅡ组的浓度进一步增加(eNOSt=10.682,P<0.001;cGMP t=18.974,P<0.001);而转染空质粒组+AngⅡ组eNOS及cGMP的浓度与AngⅡ组无明显差别(eNOSt=2.039,P=0.111;cGMP t=1.469,P=0.216)。结论 IMD对AngⅡ诱导的肾小管上皮细胞纤维化有抑制作用,可能通过eNOS-cGMP途径阻断AngⅡ的作用实现的。

Objective To investigate the effect of intermedin（ IMD） on angiotensin Ⅱ-induced rat tubular cell（ NRK-52E） fibrosis and its possible mechanisms. Methods NRK-52 E cells were cultured and randomly allotted to the following 4 groups： control group,Ang Ⅱ（ 10-（-6）mol / L） group,IMD ＋ Ang Ⅱgroup,and empty plasmid ＋ Ang Ⅱ group. Cells in IMD ＋ Ang Ⅱ group and empty plasmid ＋ Ang Ⅱ group were transfected with pIRES2-IMD or pIRES2-empty vector, respectively, by transfection complex comprising optimal proportion of plasmid and Fugene HD reagents. The transfer efficiency was detected by flow cytometry. The mRNA expression of collagen I（ Col I） was detected by real-time RT-PCR. Protein levels of Col I and E-cadherin were examined by Western blot analysis. The contents of eNOS and c GMP in the supernatant were determined by ELISA. Results Compared with the control group,the expression of Col I was significantly increased while E-cadherin markedly decreased in the Ang Ⅱ group（ Col I mRNA level t = 4. 835,P = 0. 008; Col I protein level P 0. 001; E-cadherin protein level t = 4. 284,P = 0. 013）;eNOS and cGMP levels in the supernatant of Ang Ⅱ-stimulated cells were significantly higher than those in the control cells（ eNOS t = 20. 718,P 0. 001; cGMP t = 8. 324,P = 0. 001）. IMD gene transfer reversed the above changes（ Col I mRNA level t = 3. 302,P = 0. 039; Col I protein level P 0. 001; E-cadherin protein level t = 6. 045,P = 0. 004; eNOS t = 10. 682,P 0. 001; cGMP t = 18. 974,P 0. 001）,while the empty plasmid had no effects on them（ Col I mRNA level t = 0. 951,P = 0. 395; Col I protein level t =1. 208,P = 0. 294; E-cadherin protein level t = 1. 613,P = 0. 182; eNOS t = 2. 039,P = 0. 111; cGMP t =1. 469,P = 0. 216）. Compared with the Ang Ⅱ group,the eNOS and cGMP levels of the IMD plasmid ＋Ang Ⅱ group were further increased（ eNOS t = 10. 682,P 0. 001; cGMP t = 18. 974,P 0. 001）; and in the empty plasmid ＋ Ang Ⅱ group,the eNOS and cGMP levels were not significantly different from those in the Ang Ⅱ group（eNOS t = 2. 039,P = 0. 111; cGMP t = 1. 469,P = 0. 216）. Conclusion IMD inhibited the Ang Ⅱ-induced tubular cell fibrosis,which might be achieved,at least partly,by blocking the effects of Ang Ⅱ through the eNOS-cGMP pathway.