白细胞介素17通过激活p38MAPK信号通路调控人牙周膜成纤维细胞RANKL和OPG的表达

IL-17 regulates the expression of RANKL and OPG in human periodontal ligament fibroblasts by activating p38MAPK signaling pathway

目的使用p38丝裂原活化蛋白激酶(p38MAPK)信号通路抑制剂确定白细胞介素17(IL-17)是否通过该通路参与调控人牙周膜成纤维细胞(HPDLF)核因子κB受体激活蛋白配体(RANKL)和护骨因子(OPG)的表达。方法组织块法分离培养HPDLF,20 ng/mL IL-17分别刺激0、20、40、60、80 min,Western blot法检测HPDLF磷酸化的p38MAPK(p-p38MAPK)蛋白水平。HPDLF随机分为空白对照组、二甲基亚砜(DMSO)组、p38MAPK抑制剂SB203580组、IL-17组、IL-17联合DMSO组、IL-17联合SB203580组。IL-17及联合处理组,分别添加10μmol/L SB203580、20 ng/mL IL-17。实时定量PCR检测HPDLF的RANKL、OPG mRNA水平,Western blot法检测RANKL蛋白水平、ELISA检测OPG蛋白含量。结果p-p38MAPK蛋白水平在IL-17刺激后的0~60 min内随时间增加,在60 min时达到最高,在80 min时下降。IL-17可上调HPDLF中RANKL mRNA及蛋白表达,下调OPG mRNA和蛋白表达。较单纯使用IL-17刺激,添加SB203580通路抑制剂后,RANKL mRNA和蛋白水平均降低,OPG的mRNA水平升高。结论IL-17通过激活p38MAPK信号通路,上调HPDLF的RANKL表达,抑制OPG mRNA表达。

Objective To illuminate whether IL-17 regulates receptor activator of NF-κB ligand(RANKL)and osteoprotegerin(OPG)expression in human periodontal ligament fibroblasts(HPDLFs)via p38MAPK signaling pathway.Methods HPDLFs were incubated in the presence of 20 ng/mL IL-17 for 0,20,40,60 and 80 minutes.HPDLFs were divided randomly into 6 groups:control group,dimethyl sulfoxide(DMSO)group,p38MAPK pathway inhibitor SB203580 group,IL-17 group,IL-17 combined with DMSO group and IL-17 combined with SB203580 group.SB203580(10μmol/L)and IL-17(20 ng/mL)were added to the corresponding groups.Real-time quantitative PCR was used to detect the expression of RANKL and OPG mRNAs in HPDLFs.The levels of phospho-p38MAPK(p-p38MAPK)and RANKL protein were measured using Western blot analysis.The protein level of OPG was detected by ELISA.Results After IL-17 stimulation,the expression level of p-p38MAPK protein gradually increased starting from 0 minute and reached its highest level at 60 minutes.It started to decline at 80 minutes.Stimulation with IL-17 could increase the mRNA and protein expression level of RANKL but decrease the mRNA and protein expression level of OPG.Nevertheless,unlike the IL-17 group,IL-17 combined with inhibitor SB203580 decreased the expression of RANKL mRNA and protein and increased OPG mRNA.Conclusion IL-17 can enhance the expression of RANKL in human periodontal fibroblasts and inhibit the expression of OPG mRNA through the p38MAPK signal transduction pathways.