P38MAPK 信号通路在高糖导致人腹膜间皮细胞 PARP -1激活、细胞外基质聚集及转分化的作用

摘要

目的：探讨人腹膜间皮细胞株 HMrSV5在高糖刺激下，P38MAPK 信号通路的活化情况及 PARP -1的蛋白表达情况。探讨 P38MAPK 抑制剂对腹膜间皮细胞外基质聚集及细胞转分化的作用及对 PARP -1的活性变化。方法：无血清培养 HMrSV5细胞株16~24 h 后，分别用高糖（终浓度138 mmol/ L 葡萄糖）刺激0 m、5 m、30 m、1 h、2 h、8 h、24 h，并选择等渗的甘露醇作为对照用。Western Blot 分别检测不同时间总的 P38MAPK 及磷酸化的 P38MAPK 的蛋白表达水平。同时用Western blot 检测在不同时间点 PARP -1的蛋白表达情况。无血清培养 HPMCs16~24 h 后，分为4各组：（1）低糖组（5．6 mmol/ L 葡萄糖）；（2）刺激组（138 mmol/ L 葡萄糖）；（3）抑制剂组（138 mmol/ L 葡萄糖+ SB203580P38抑制剂，浓度为10μmol/ L）；（4）DMSO 对照组。在24 h 收取细胞用 Western blot 检测 P38MAPK、PARP -1、FN、PAI -1、E - cadherin 及α-SMA 的蛋白水平变化。结果：高糖以时间依赖的方式刺激 HPMCs 后，磷酸化的 P38MAPK 表达增加。PARP -1的表达在高糖刺激下也呈时间依赖性增高，24 h 已很明显。用 P38MAPK 抑制剂 SB203580后，PARP -1及 FN、PAI -1及α- SMA 也下调，E - cadherin 表达上调，差异有统计学意义（P <0．05）。结论：高糖可使得人腹膜间皮细胞 P38MAPK 通路的活化。运用P38的抑制剂，均能使得 PARP -1活性下调，同时逆转腹膜间皮细胞外基质聚集及 HPMCs 转分化。这提示在高糖刺激腹膜间皮细胞时，P38MAPK 参与了 PARP -1的活化，并参与腹膜间皮细胞外基质聚集及人腹间皮细胞转分化。%Objective:To investigate the expression of p38MAPK pathways and PARP - 1 under high glucose condition in human peritoneal mesothelial cells. To determine the effect of blockade P38MAPK on EMT and peritoneal fibrosis in mesothelial cells;to detect the effect of blockage P38MAPK on the activation of PARP - 1 in mesothelial cells. Methods:Human peritoneal mesothelial cells(HPMCs)were cultured in DMEM/ F12 with 10% FBS,100 μg/ ml streptomycin and penicillin(100 units/ ml)at 37 ℃ and 5%CO2 . Cells were cultured in serum - free media for 16 ~ 24 hours to arrest quiescent state then were stimulated by high glucose(final concentration of 138 mmol/ L glucose)for 0 m,5 m,30 m,1 h,2 h,8 h,24 h,mannitol was used as control. Then detected the protein expression of phospho - P38,total P38MAPF with the method of Western blot. The expression of PARP - 1 was also detected by Western blot. Arrested human peritoneal mesothelial cells for 16 ~ 24 h. Then the cells were randomly divided into four groups:(1)low glucose(5. 6 mmol/ L);(2)high glucose(138 mmol/ L);(3)high glucose(138 mmol/ L)+ SB203580(10 μmol/ L),re-spectively;(4)control group:Dimethyl Sulphoxide(DMSO). After 24 hours,cells were harvested to determine the level of PARP -1,FN,PAI - 1,E - cadherin,α - SMA with Western blot technique. Results:After stimulation with high glucose in HPMCs,the level of phospho - P38MAPK was increased. The level of PARP - 1 was also increased. Western Bloting showed that the protein ex-pressions of phospho - P38,PARP - 1,FN,PAI - 1,α - SMA were increased after high glucose stimulation while E - cadherin de-creased. However,by blockade the P38MAPK pathway with SB203580,expressions of PARP - 1,FN,PAI - 1,α - SMA were de-creased while E - cadherin increased. The difference was of significance. Conclusion:High glucose can activate the P38MAPK path-way,in human peritoneal mesothelial cells. By using both P38MAPK inhibitor,the expression of PARP - 1 is down - regulated,re-verses the process of EMT and the accumulation of ECM in human peritoneal mesothelial cells. It gives us some clues that P38MAPK maybe activates the PARP - 1directly,duo to its involvement in the EMT and the accumulation of ECM in peritoneal mesothelial cells. But it still needs further investigation.