STAT3抑制剂Stattic激活ERK通路诱导THP-1细胞IL-8的产生及细胞凋亡

STAT3 inhibitor Stattic induced IL-8 production and cell apotosis in THP-1 cells by activating ERK pathway

目的观察信号转导子与转录激活子3(STAT3)抑制剂Stattic对THP-1细胞产生白细胞介素8(IL-8)和细胞凋亡的影响,并探讨其机制。方法采用(0、1、5、10、15、20)μmol/L Stattic处理THP-1细胞0、1、3、6、12、24h,实时荧光定量PCR检测细胞IL-8、IL-6、IL-1β、肿瘤坏死因子α(TNF-α)的mRNA水平,ELISA检测细胞培养上清液IL-8蛋白水平,流式细胞术检测THP-1细胞的凋亡,Western blot法检测细胞STAT3、细胞外信号调节激酶(ERK)的蛋白磷酸化水平;采用(0、1、5、10)μmol/L的ERK通路选择性抑制剂U0126预处理THP-1细胞,反转录PCR检测U0126对Stattic诱导THP-1细胞表达IL-8 mRNA水平的影响。结果(10~20)μmol/LStattic显著上调IL-8在THP-1细胞中的mRNA和蛋白表达,仅(15、20)μmol/LStattic能诱导THP-1细胞凋亡;Stattic处理THP-1细胞1、3、6、12、24 h,均显著上调IL-8的mRNA水平,以3 h时最为明显,在6 h以后呈时间依赖性上调IL-8的蛋白水平并诱导THP-1细胞凋亡;Stattic呈浓度和时间依赖性抑制STAT3磷酸化,时间依赖性地诱导ERK磷酸化,在(1、5、10、15、20)μmol/L时均显著诱导ERK磷酸化。另外,U0126显著抑制Stattic诱导的IL-8 mRNA表达。结论STAT3抑制剂Stattic通过激活ERK信号通路诱导THP-1细胞凋亡和IL-8产生。

Objective To observe the effect of selectively inhibiting STAT3 on the production of IL-8 and cell apoptosis of THP-1 cells by Stattic,and explore the underlying mechanism.Methods THP-1 cells were treated with different concentrations of Stattic(0,1,5,10,15,20μmol/L)for 0,1,3,6,12,24 hours.Reverse transcription PCR or real-time PCR was performed to detect the mRNA expression of IL-8,IL-6,IL-1βand tumor necrosis factor-α(TNF-α);ELISA was used to detect the protein expression of IL-8;flow cytometry was applied to evaluate the apoptosis of THP-1 cells;and Western blot analysis was performed to detect the phosphorylation of STAT3 and extracellular signal-regulated kinase(ERK).Reverse transcription PCR was used to test the effect of U0126 at different concentrations(0,1,5,10μmol/L)on the mRNA expression of IL-8 induced by Stattic in THP-1 cells.Results Stattic significantly up-regulated the mRNA and protein expression of IL-8 in THP-1 cells in a concentration range of 10~20μmol/L,and induced cell apoptosis only at high concentration(15,20μmol/L).Treated with Stattic for 0,1,3,6,12,24 hours,IL-8 mRNA was significantly up-regulated,and after 6 hours,the expression of IL-8 protein and apoptosis of THP-1 cells were up-regulated in a time-dependent manner.STAT3 phosphorylation was inhibited in a time-and dose-dependent manner by Stattic.ERK phosphorylation was induced by different concentrations of Stattic in a time-dependent manner.In addition,U0126,a selective inhibitor of ERK pathway,inhibited Stattic-induced IL-8 expression in a concentration-dependent manner.Conclusion Stattic,a selective STAT3 inhibitor,can induce the apoptosis and IL-8 production by activating ERK signaling pathway in THP-1 cells.