法舒地尔对高糖诱导人肾小管上皮细胞转分化的影响

Effect of fasudil on epithelial-myofibroblast transdifferentiation of human renal tubular epithelial cells induced by high glucose

目的探讨法舒地尔对高糖诱导的人肾小管上皮细胞（HK-2）转分化的影响及可能的作用机制。方法HK-2细胞分别加入葡萄糖5．5mmol·L^-1、葡萄糖5．5mmol·L^-1＋甘露醇54．5mmol·L^-1、葡萄糖60mmol·L^-1（高糖）以及葡萄糖60mmol·L^-1＋法舒地尔5，10和20ummol·L^-1。免疫共沉淀法检测葡萄糖60mmol·L^-1作用0—24h后磷酸化肌球蛋白磷酸酶目标亚单位1-苏氨酸696（p—MYPTl一Thr696）和P—MYPTl．Thr853的表达，以评估Rho相关的卷曲螺旋形成的蛋白激酶（ROCK）的活性；免疫细胞化学法检测a-平滑肌肌动蛋白（a-SMA）表达；Western蛋白质印迹法检测E-钙黏素、波形蛋白和结缔组织生长因子（CTGF）蛋白表达。结果与未加高糖刺激前比较，高糖培养3h后，细胞p—MYPTl-Thr696表达明显增加．积分吸光度（IA）值由1．08±0．09增加到2．4±0．09（P〈0．01）；与未加高糖刺激前比较，高糖培养7h后，细胞P—MYPT1-Th旧53表达明显增加，IA值由0．57±0．01增加到1．45±0．14（P〈0．01），表明高糖能导致HK-2细胞ROCK分子活化。与正常对照组相比，葡萄糖60mmol·L。组HK-2细胞培养72h后E·钙黏素表达减少（P〈0．01），oc—SMA、波形蛋白和CTGF表达增多（P〈0．01）；葡萄糖5．5mmol·L^-1＋甘露醇54．5mmol·L一组与正常对照组比较无明显变化。与葡萄糖60mmol·L^-1组相比，葡萄糖60mmol·L^-1＋法舒地尔5，10和20umol·L^-1组E-钙黏素表达增多（P〈0．01），a—SMA、波形蛋白和CTGF表达减少（P〈0．01），且法舒地尔20ummol·L^-1组改变更为明显，法舒地尔3个浓度组组间比较差异有显著性（P〈0．05）。结论法舒地尔能抑制高糖诱导的肾小管上皮细胞转分化，可能部分通过减少CTGF的表达而产生作用。

OBJECTIVE To investigate the effect of fasudil on the epithelial-myofibroblast transdif- ferentiation of human renal tubular epithelial （HK-2） cells induced by high glucose and to explore the mechanism. METHODS HK-2 cells were cultivated in glucose 5.5 mmol·L^-1, glucose 5.5 mmol·L^-1 ＋ rnannitol 54.5 mmol·L^-1 , high glucose （60 rnrnol.L-1 ） and high glucose ＋fasudil 5, 10 and 20 umol·L^-1, respectively, for 72 h. Changes in the p-MYPT1-Thr696 and p-MYPT1-Thr853 were detected with co-immunoprecipitation assay, a-Smooth muscle actin （a-SMA）, which reflected the phenotypic characteristics of rnyofibroblast cells, was detected by immunocytochemistry. Western blotting was used to detect the protein expression of E-cadherin, vimentin and connective tissue growth factor （CTGF）. RESULTS Compared with HK-2 cells without glucose 6.0 mmol·L^-1 , the expression of p-MYPT1- Thr696 was enhanced after 3 h exposure to high glucose E integrated absorbance （IA） from 1.08±0.09 to 2.4 ＋0.09, P〈0.01 ）, and that of p-MYPT1-Thr853 was enhanced after 7 h（ IA from 0.57 ±0.01 to 1.45 ±0.14, P〈0.01 ）, suggesting that the activity of Rho kinase could be activated by high glucose. Compared with glucose 5.5 mmol·L^-1 group, HK-2 cells cultured with glucose 60 mmol·L^-1 showed a decreased expression of E-cadherin （ P 〈 0.01 ）, increased expression of a-SMA, vimentin and CTGF （P〈0.01）. Compared with high glucose group, the high glucose ＋ fasudil 5, 10 and 20 umol·L^-1 groups showed an increased expression of E-cadherin（ P 〈0.01 ） , but decreased expression of a-SMA, vimentin and CTGF（P〈0.01 ）. The changes of fasudil 20 umol·L^-1 group were the most obvious. CONCLUSION Fasudil can inhibit high glucose-induced epithelial-mnyofibroblast transdifferention of renal tubular epithelial cells, possibly by reducing the expression of CTGF.