骨膜素通过p38 MAPK信号通路抑制缺氧诱导的人牙周膜成纤维细胞的氧化应激和细胞凋亡

Periostin inhibits hypoxia-induced oxidative stress and apoptosis in human periodontal ligament fibroblasts via p38 MAPK signaling pathway

目的探讨骨膜素对缺氧诱导的人牙周膜成纤维细胞氧化应激与细胞凋亡的影响及其分子机制。方法体外培养人牙周膜成纤维细胞,厌氧产气袋内缺氧处理48 h。采用不同浓度(25、50、100 ng/mL)的骨膜素处理细胞,随机分为空白组、缺氧组、骨膜素低浓度组、骨膜素中浓度组、骨膜素高浓度组。甲基噻唑基四唑(MTT)检测细胞存活率;流式细胞术检测细胞凋亡率;夹心酶联免疫吸附试验(ELISA)测定炎症因子白细胞介素-1β(IL-1β)、白细胞介素6(IL-6)、肿瘤坏死因子-α(TNF-α)的含量;应用荧光酶标仪检测细胞活性氧(ROS)水平;ELISA检测细胞内超氧化物歧化酶(SOD)活性;蛋白免疫印迹法(Western blot)检测HIF-1α、P21、CyclinD1、Bax、Cleaved caspase-3、Bcl-2、P38MAPK、p-p38 MAPK蛋白表达量。结果缺氧处理后可明显降低细胞存活率(P<0.05),提高P21、Bax、Cleaved caspase-3蛋白水平(P<0.05),促进细胞凋亡(P<0.05),降低CyclinD1、Bcl-2蛋白水平(P<0.05);与缺氧组相比,骨膜素不同浓度组细胞存活率升高(P<0.05),细胞凋亡率降低(P<0.05),P21、Bax、Cleaved caspase-3蛋白水平降低(P<0.05),CyclinD1、Bcl-2蛋白水平升高(P<0.05)。与空白组相比,缺氧组HIF-1α、p-p38 MAPK蛋白表达水平升高(P<0.05),IL-1β、IL-6、TNF-α、ROS水平升高(P<0.05),SOD活性降低(P<0.05);与缺氧组相比,骨膜素不同浓度组HIF-1α、p-p38 MAPK蛋白水平降低(P<0.05),IL-1β、IL-6、TNF-α、ROS水平降低(P<0.05),SOD活性升高(P<0.05)。结论骨膜素可促进缺氧诱导的人牙周膜成纤维细胞增殖,抑制细胞凋亡,增强细胞抗氧化能力,减轻炎症损伤,其可能通过抑制p38 MAPK信号通路的活化而发挥作用。

Objective To investigate the effect of periostin on hypoxia-induced oxidative stress and apoptosis in human periodontal ligament fibroblasts and the molecular mechanism involved.Methods In vitro cultured human periodontal ligament fibroblasts were placed in an anaerobic gas-producing bag for hypoxia treatment for 48 h followed by treatment with periostin at low(25 ng/mL),moderate(50 ng/mL)or high(100 ng/mL)doses.MTT assay was used to measure the cell viability,and the cell apoptosis rate was determined using flow cytometry.The contents of IL-1β,IL-6 and TNF-αin the cells were determined with ELISA,and ROS levels were measured using a fluorescent plate reader.The intracellular SOD activity was detected using ELISA.The expressions of HIF-1α,P21,cyclin D1,Bax,cleaved caspase-3,Bcl-2,P38MAPK and p-p38 MAPK proteins in the cells were detected with Western blotting.Results Hypoxia treatment significantly reduced the cell viability(P<0.05),increased P21,Bax,and cleaved caspase-3 protein levels(P<0.05),promoted cell apoptosis(P<0.05),and decreased cyclin D1 and Bcl-2 protein levels(P<0.05)in the cells.Compared with the hypoxic group,the cells treated with periostin at different concentrations showed significantly increased cell viability(P<0.05)with significantly lowered apoptotic rates(P<0.05)and decreased expression levels of Bax and cleaved caspase-3(P<0.05)but significantly increased expression levels of cyclin D1 and Bcl-2(P<0.05).Hypoxic exposure of the cells resulted in significantly increased expression levels of HIF-1αand p-p38 MAPK(P<0.05)and increased levels of IL-1β,IL-6,TNF-αand ROS(P<0.05)but decreased SOD activity(P<0.05).Periostin treatment at different concentrations significantly lowered the expression levels of HIF-1αand p-p38 MAPK(P<0.05)and the levels of IL-1β,IL-6,TNF-αand ROS(P<0.05)and significantly increased SOD activity in the hypoxic cells(P<0.05).Conclusion Periostin promotes the proliferation,inhibits apoptosis,enhances cellular antioxidant capacity,and reduces inflammatory damage in human periodontal ligament fibroblasts exposed to hypoxia possibly by inhibiting the activation of the p38 MAPK signaling pathway.