ERK1/2信号通路在高糖诱导的HK-2上皮间质转分化中的作用

The role of the ERK signaling pathway in high glucose-induced epithelial-mesenchymal transition of cultured human renal tubular epithelial cells

目的观察高糖诱导的细胞外信号调节激酶(ERK1/2)信号通路活化在上皮间质转分化中的作用,从而探讨延缓糖尿病肾病肾间质纤维化的发生机制。方法体外培养HK-2细胞,随机分为正常对照组、高糖组、ERK1/2通路抑制剂PD98059+高糖组和高渗组,处理72 h后收集细胞,应用免疫细胞化学方法检测α-SMA、CK18的表达;应用Western blot法检测磷酸化ERK1/2、总ERK1/2表达水平的变化。结果 (1)正常对照组胞质有大量CK18蛋白阳性表达,而α-SMA蛋白表达呈阴性;高糖组胞质中可见大量α-SMA蛋白强阳性染色,而CK18蛋白呈阴性表达;经PD98059处理后,CK18表达较高糖组染色深,而α-SMA表达较高糖组染色浅;高渗组胞质中CK18呈阳性表达,α-SMA表达呈阴性。(2)正常对照组可见少量总ERK1/2表达,p-ERK1/2蛋白仅有微量表达;经高糖刺激48 h后,总ERK1/2表达较正常对照组无明显差异,p-ERK1/2蛋白表达明显增加(P<0.05);而使用PD98059处理后,总ERK1/2的变化不大,但p-ERK1/2蛋白的表达却显著降低(P<0.05);高渗组与正常对照组无显著差异。结论 ERK1/2信号通路可能参与了高糖诱导的人近端肾小管上皮细胞(HK-2)转分化过程,阻断ERKl/2可部分抑制人近端肾小管上皮细胞的转分化,进而延缓肾小管间质纤维化的发生和发展。

Objective To observe the role of ERK signaling pathway in epithelial-mesenchymal transition induced by high glucose, and to investigate the mechanism of tubulointerstitial fibrosis associated with diabetic nephropathy. Methods The human proximal tubular epithelial cell line （HK-2） were randomly treated with normal glucose, high glucose, co-incubation of high glucose with specific ERK inhibitor PD98059 or D-mannitol for 72 h.The protein expression ofα-SMA and CK18 in all the cells was assessed by the method of immunocytochemistry. The protein expression of ERK and p-ERK was determined by Western blotting. Results （1）In control group, CK18 was highly expressed in the cytoplasm of tubular epithelial cells, but not α-SMA were observed. High glucose enhancedα-SMA expressed in the cytoplasm of tubular epithelial cells.Meanwhile, the CK18 expressed of scarcely. Combining with PD98059 and high glucose, the CK18 staining higher, butα-SMA staining shallower than that of treating with high glucose. In D-mannitol group, CK18 was expressed in the cytoplasm of tubular epithelial cells, but no α-SMA were observed. （2）In control group, only small amounts of total ERK1/2 were expressed, minute quantity phospho-ERK1/2 were observed.The protein of phospho-ERK1/2 increased remarkably in high glucose-treated cells（P〈0.05）, the expression of total ERK1/2 had no differences with control group. Co-incubation of high glucose with PD98059 reduced the activity of phospho-ERK1/2 compare to the high glucose group （P〈0.05）, while total ERK1/2 had no differences with control group. No significant differences between control group and D-mannitol group was observed. Conclusion This study suggests that ERK may play an important role in the high glucose-induced EMT in tubular epithelial cells, and interferring ERK may partly inhibit EMT in order to delay the occurrence and progress of tubulointerstitial fibrosis.