锌指蛋白A20对脂多糖诱导的大鼠腹膜间皮细胞炎症效应的影响

The role of zinc finger protein A20 on LPS-induced inflammation in rat peritoneal mesothelial cells

目的探讨锌指蛋白A20(zinc finger protein A20)对脂多糖(LPS)诱导的大鼠腹膜间皮细胞(RPMCs)炎症效应的影响及可能机制。方法分离及培养RPMCs,常规传代及鉴定。取第2代细胞用于实验研究。将细胞随机分成对照组、LPS组、转染A20组、空载体组。脂质体转染A20质粒(pGEM-Teasy-A20)至RPMCs12h后分别在LPS刺激不同时间点收获细胞提取蛋白及细胞上清液。用Western blotting检测细胞TLR4、p-IκBα、IκBα蛋白的表达;用ELISA法检测培养上清液IL-18蛋白水平。结果 LPS刺激8h后,转染A20组RPMCsTLR4蛋白表达水平无明显增高,与对照组相比,P=0.223;与LPS组及空载体组相比,差异有显著性(P=0.003,0.002)。LPS刺激1h后,转染A20组RPMCsp-IκBα蛋白无明显降解,p-IκBα/IκBα比值与对照组相比,P=0.553。与LPS组及空载体组相比,差异有显著性(P=0.001,0.001)。在LPS刺激12h后,转染A20组RPMCsIL-18蛋白分泌水平(479.12±85.79)pg/ml高于对照组(274.34±47.21)pg/ml(P=0.012),但明显低于LPS组(1049.45±185.01)pg/ml及空载体组(1028.77±192.90)pg/ml(P=0.011,0.015)。结论 A20通过对LPS信号通路中多个相关功能蛋白的负性调控作用,阻抑LPS诱导的RPMCs炎症效应。

Objective To investigate the role and the mechanism of zinc finger protein A20 （A20） on LPS-induced inflammation in cultured peritoneal mesothelial cells from Sprague-Dawley rats （RPMCs）. Method The second passage of cultured RPMCs were used in the study and randomly assigned into control group, LPS group, A20 plasmid transfection group, and parental plasmid transfection group. RPMCs were transfected with pGEM-T-easy-A20 plasmid using liposome for 12 hours, and were then stimulated with LPS. The cells and cell supernatant were harvested at different time after LPS stimulation. TLR4, p-IkBctand IkBct expressions in cells were determined by western blotting, and IL-18 in cell supernatant was determined by ELISA. Results In the A20 plasmid transfection group after LPS stimulation for 8h, the change of TLR4 expression was insignificant as compared with that in the control group （P=0.223）, but was statistically different as compared with that in the LPS group and the parental plasmid transfection group （P=0.003 and 0.002, respectively）. In the A20 plasmid transfection group after LPS stimulation for 1 h, p-IκBα expression changed insignificantly. The change ofp- IκBα/IkBet ratio was insignificant as compared with that in the control group （p=0.553）, but was statistically different as compared with that in the LPS group and parental plasmid transfection group （p=0.001 and 0.001, respectively）. In A20 plasmid transfection group after LPS stimulation for 12 h, IL-18 secretion was higher （479.12 ± 85.79 pg/ml） than that in the control group （274.34± 47.21 pg/ml, P=-0.012）, but was lower than that in the LPS group （1049.45 ± 185.01 pg/ml, P=0.011） and in the parental plasmid transfection group （1028.77 ±192.90 pg/ml, P=0.015）. Conclusions A20 inhibits the LPS induced inflammation in cultured RPMCs probably through negative regulation to several relevant proteins in the LPS signaling pathway.