p38丝裂素活化蛋白激酶信号转录对IL-1β诱导肾小管细胞转分化的影响

目的　探讨 p38MAPK信号转导途径对IL 1β诱导肾小管上皮细胞转分化的影响及其导致的功能改变。方法　以培养的人近端肾小管上皮细胞 (HK 2 )为研究对象 ,给予IL 1β(10ng/ml)刺激 2 4h ,在阻断实验中加入SB2 0 35 80阻断 p38通路。采用蛋白印记杂交法检测 p38MAPK的磷酸化程度 ;细胞表型特征检测包括细胞形态变化、标志蛋白 (角蛋白、α SMA)的表达及分布、细胞增殖、黏附和移行能力。结果　 (1)IL 1β刺激 6～ 4 8h可使α SMA表达上调 ,刺激 2 4h可使角蛋白表达减弱、α SMA分布紊乱 ,但细胞形态变化不明显 ;(2 )IL 1β在 5min时可使p38激活达高峰 ,较基础水平升高 2～ 3倍 (P <0 0 5 ) ;至 12 0min时 p38则呈持续激活状态 ;(3)p38阻断剂 (SB2 0 35 80 )对HK 2细胞α SMA的基础表达有抑制作用 ,抑制率 37 13% (P <0 0 1) ;同时可明显抑制IL 1β刺激的α SMA表达 ,抑制率 4 7 0 7% (P <0 0 0 1) ;(4)IL 1β及 p38阻断剂并不影响HK 2细胞之间的黏附能力 ,但IL 1β可使细胞的移行能力增强 (P <0 0 1) ,而 p38阻断剂可明显抑制其移行能力。IL 1β并不影响HK 2细胞增殖。结论　IL 1β可以激活肾小管上皮细胞p38/MAPK ,并可使其发生表型转化 ,这一作用部分通过 p38信号通路所介导。

Interleukin-1beta-induced transdifferentiation of renal proximal tubular cells is mediated by p38 mitogen-activated protein kinase phosphorylation

OBJECTIVE: To investigate the role of p38MAPK signaling pathway in interleukin (IL)-1beta induced transdifferentiation and its functional influences in human renal proximal tubular cell line HK-2 cells. METHODS: Human renal proximal tubular cells, cell line HK-2, were cultured and then co-incubated with IL-1beta (10 ng/ml) for 24 hours. The expression and distribution of cytokeratin and alpha-smooth muscle actin (SMA), markers of transdifferentiation of renal proximal tubular cells, were detected by immunofluorescence staining and confocal microscopy. Western blot technique was used to detect the expression of alpha-SMA 2, 4, 6, 12, 24, and 48 hours after IL-1beta stimulation. The morphology of cells was monitored from the 1st to the 5th day. Expression of alpha-SMA, phosphorylation of p38MAPK was assayed by western blot. Specific p38MAPK inhibitor SB203580 was added into the culture of HK-2 cells, 24 hours later IL-1beta was added for 24 hours Cell-cell adhesion and migration assay were performed. Inverted microscopy was used to examine the morphology of cells after stimulation of IL-1beta for 24 hours. Western blot technique was used to detected total and phosphorylated p38MAPK after stimulation of IL-1beta for 2, 5, 15, 30, 60 and 120 min. RESULTS: The phosphorylation of p38MAPK was increased after treatment of IL-1beta, and reached the level of 1.7 times the basic level 5 - 30 minutes after stimulation (P < 0.05). Almost no expression of alpha-SMA was shown in the control group. Two hours after IL-1beta stimulation, the expression of alpha-SMA was increased a little followed by an up-regulated expression of alpha-SMA 6 to 48 hours after the stimulation. The expression of alpha-SMA was down-regulated by 40% in the SB203580 group in comparison with that in the control (P < 0.01) and by 50% in comparison with that in the IL-1beta group (P < 0.001). The cell number was not significantly different between the IL-1beta group and control group (P > 0.05). A decreased expression of cytokeratin and disordered distribution of alpha-SMA were shown in the cells 24 hours after stimulation of IL-1beta. The morphology of cells remained unchanged 1 to 5 days after IL-1beta stimulation. The cell-cell adhesion was almost identical in the groups with IL-1beta and/or SB203580 no in comparison with that in the control group (P > 0.05). The migration ability of HK-2 cells was 2.2 times that in the control group in the IL-1beta group (P < 0.05) and decreased by 35% in the SB203580 group (P < 0.01). SB203580 uppercased the IL-1beta-induced expression of alpha-SMA and the enhancement of cell migration ability were suppressed by 51% (P < 0.05). CONCLUSION: IL-1beta induces transdifferentiation of renal proximal tubular cells characterized by alpha-SMA expression and enhanced ability of cell migration. These effects are mediated, at least in part, through the activation of p38 MAPK signaling pathway.