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

doi:10.3877/cma.j.issn.2095-3216.2016.01.006

目的

观察Intermedin(IMD)对血管紧张素Ⅱ(Ang Ⅱ)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、Ang Ⅱ组(10^－6mol/L)、转染IMD质粒＋Ang Ⅱ组、转染空质粒＋Ang Ⅱ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Ang Ⅱ(10^－6mol/L)干预24 h。用real- time RT- PCR检测各组细胞中Ⅰ型胶原(Col Ⅰ)的表达；Western印迹法检测Col Ⅰ、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SPSS 17.0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，Ang Ⅱ组Col Ⅰ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Col Ⅰ的表达较Ang Ⅱ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Col Ⅰ、E-钙黏蛋白的表达与Ang Ⅱ无明显差别(Col Ⅰ mRNA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，Ang Ⅱ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Ang Ⅱ组相比，IMD质粒＋Ang Ⅱ组的浓度进一步增加(eNOS t＝10.682，P<0.001；cGMP t＝18.974，P<0.001)；而转染空质粒组＋Ang Ⅱ组eNOS及cGMP的浓度与Ang Ⅱ组无明显差别(eNOS t＝2.039，P＝0.111；cGMP t＝1.469，P＝0.216)。

结论

IMD对Ang Ⅱ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Ang Ⅱ的作用实现的。

关键词: 血管紧张素Ⅱ, 纤维化, Intermedin, 内皮型一氧化氮合酶, 环磷酸鸟苷

Objective

To investigate the effect of intermedin (IMD) on angiotensin Ⅱ- induced rat tubular cell (NRK- 52E) fibrosis and its possible mechanisms.

Methods

NRK- 52E cells were cultured and randomly allotted to the following 4 groups: control group, Ang Ⅱ (10^-6 mol/L) group, IMD+ Ang Ⅱ group, 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 cGMP 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.

Key words: Angiotensin Ⅱ, Fibrosis, Intermedin, eNOS, cGMP

表1 Real- time RT- PCR引物序列

基因	引物序列
Col I	5′- GACATGTTCAGCTTTGTGGACCTC- 3′
Col I	5′- AGGGACCCTTAGGCCATTGTGTA- 3′
β- actin	5′- CCCATCTATGAGGGTTACGG- 3′
β- actin	5′- TTTAATGTCACGCACGATTC- 3′

表1 Real- time RT- PCR引物序列

基因	引物序列
Col I	5′- GACATGTTCAGCTTTGTGGACCTC- 3′
Col I	5′- AGGGACCCTTAGGCCATTGTGTA- 3′
β- actin	5′- CCCATCTATGAGGGTTACGG- 3′
β- actin	5′- TTTAATGTCACGCACGATTC- 3′

图1 NRK－52E细胞转染流式图

图2 各组NRK- 52E细胞Ⅰ型胶原的表达

图3 各组NRK- 52E细胞E-钙黏蛋白的表达

表2 各组细胞培养上清液eNOS、cGMP的浓度(±s，n＝5)

分组	eNOS(ng/L)	cGMP(nmol/L)
正常对照组	732.70±11.64	16.17±0.66
Ang Ⅱ组	1247.31±28.76^a	33.03±3.44^a
IMD质粒＋Ang Ⅱ组	1532.12±36.13^ab	71.98±0.89^ab
空质粒＋Ang Ⅱ组	1319.65±54.29^a	37.04±3.24^a

表2 各组细胞培养上清液eNOS、cGMP的浓度(±s，n＝5)

分组	eNOS(ng/L)	cGMP(nmol/L)
正常对照组	732.70±11.64	16.17±0.66
Ang Ⅱ组	1247.31±28.76^a	33.03±3.44^a
IMD质粒＋Ang Ⅱ组	1532.12±36.13^ab	71.98±0.89^ab
空质粒＋Ang Ⅱ组	1319.65±54.29^a	37.04±3.24^a

图4 各组NRK- 52E细胞上清液eNOS、cGMP浓度

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

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