氟伐他汀抑制Rho激酶信号通路减少HK-2细胞纤维连接蛋白表达

Fluvastatin decreases the expression of fibronectin by inhibiting the signaling pathway of Rho-kinase in HK-2 cells

目的:探讨Rho激酶信号通路在糖尿病肾病(diabetic nephropathy,DN)肾间质纤维化中的作用,以及氟伐他汀在防治DN肾间质纤维化中的作用机制。方法:体外培养人近端肾小管上皮细胞(HK-2细胞),Rho激酶底物肌球蛋白磷酸酶目标亚单位-1(MYPT-1)的磷酸化作为Rho激酶活化的标志。高糖刺激HK-2细胞0、6、12、24h,不同浓度(10-7、10-6、10-5mol/L)氟伐他汀干预12h,Western blot检测p-MYPT-1和纤维连接蛋白(fibronectin,FN)的表达。后续实验中Western blot检测Rho激酶选择性激动剂(lysophosphatidic acid,LPA)对氟伐他汀调节HK-2细胞p-MYPT-1和FN表达的影响。结果:相对于0h,高糖刺激HK-2细胞6、12、24h均可以激活p-MYPT-1,其中12h到达高峰。高糖刺激FN的表达呈时间依赖性,氟伐他汀抑制高糖诱导的HK-2细胞p-MYPT-1的活化并抑制FN的表达,并呈浓度依赖性。LPA可以拮抗氟伐他汀的作用。结论:Rho激酶信号通路可能是DN肾间质纤维化发生的始动信号之一。氟伐他汀可能通过抑制Rho激酶磷酸化进而抑制FN的表达,从而起到一定的防治DN肾间质纤维化的作用。

Objective：To explore the effect of Rho-kinase signal pathway in renal interstitial fibrosis of diabetic nephropathy（DN） and the mechanism of fluvastatin in the prevention of renal interstitial fibrosis of DN. Methods：Human renal proximal tubular epithelial cells （HK-2 cells）were cultured in vitro. Rho-kinase activity was expressed as phosphorylation of myosin-phosphatase target-1 （p-MYPT-1）. The level of p-MYPT-1 and fibronectin（FN） stimulated by high glucose was determined by Western blot at the time of 0 h, 6 h,12 h and 24 h after treatment. Same markers were detected when HK-2 cells cultured with high glucose treated with different concentrations（10-7,10-6 and 10-5 mol / L）of fluvastatin for 12 h and treated by lysophosphatidic acid（LPA） further. Results：High glucose enhance the expression of p-MYPT-1 and FN in culured HK-2 cells at the time of 6 h,12 h and 24 h,compared with the time of 0 h. The increase of FN expression stimulated by high glucose was time-dependent and the increased level of p-MYPT-1 reached the peak at 12 h. Fluvastatin decreased the level of p-MYPT-1 and FN induced by high glucose in a dose-dependent manner. The inhibito ry effect of fluvastatin on up-regulation of p-MYPT-1 and FN stimulated by high glucose can be reversed by LPA. Conclusion：Rho-kinase may be one of the initiation signals of renal interstitial fibrosis of DN. Fluvastatin can prevent the development of renal interstitial fibrosis of DN by inhibiting Rho-kinase signaling pathway.