蒺藜皂苷抑制肿瘤坏死因子α和干扰素γ诱导HaCaT细胞的炎症反应

Gross Saponins of Tribulus Terrestris Inhibits TNF-α/IFN-γ-induced Inflammatory Reaction in HaCaT Cells

目的探讨蒺藜皂苷(gross saponins of tribulus terrestris,GSTT)对肿瘤坏死因子α(TNF-α)和干扰素γ(IFN-γ)刺激后HaCaT细胞的炎症相关因子产生的影响及可能机制。方法采用CCK-8细胞活力测定法检测不同浓度蒺藜皂苷对HaCaT细胞增殖的影响,ELISA法检测蒺藜皂苷对TNF-α和IFN-γ刺激后HaCaT细胞趋化因子产生的影响; Western印迹法对诱导型一氧化氮合酶(inducible nitric oxidesynthase,i NOS)、环氧合酶-2 (Cyclooxygenase-2,COX-2)、丝聚合蛋白(Filaggrin,FLG)、外皮蛋白(Involucrin,IVL)以及NF-κB通路和MAPK通路的相关蛋白进行了检测;免疫荧光法检测了TNF-α和IFN-γ刺激后HaCaT细胞核因子κB(NF-κB)蛋白核转位。结果在所选浓度内,蒺藜皂苷对HaCaT细胞增殖无明显影响,下调了TNF-α和IFN-γ诱导的HaCaT细胞培养上清中胸腺和活化调节趋化因子(TARC),人巨噬细胞来源的趋化因子(MDC),调节激活正常T细胞表达分泌因子(RANTES)和白介素-8(IL-8)的表达。蒺藜皂苷能剂量依赖性抑制TNF-α和IFN-γ诱导的HaCaT细胞i NOS和COX2的表达,促进FLG和IVL蛋白的表达;并降低TNF-α和IFN-γ诱导的HaCaT细胞胞核内p65的水平,抑制p65的核转位,提高胞浆内IκB的水平;蒺藜皂苷能剂量依赖性抑制TNF-α和IFN-γ诱导的HaCaT细胞MAPK通路中p38蛋白和ERK蛋白的磷酸化。结论蒺藜皂苷可能通过调节NF-κB通路和p38/ERK MAPK通路的活化抑制了TNF-α和IFN-γ刺激后HaCaT炎症相关因子产生,促进皮肤屏障功能的恢复。

Objective To investigate the influence of gross saponins of tribulus terrestris(GSTT)on inflammatory response of HaCaT cells induced by TNF-α/IFN-γ stimulation,and to provide insight into the possible mechanisms.Methods CCK-8 assay was applied to determine the effect of different concentrations of GSTT on the proliferation of HaCaT cells.ELISA assay was used to examine the effect of GSTT on the level of chemokines in HaCaT cells induced by TNF-α/IFN-γ stimulation.Western blot was used to examine the expression of iNOS,COX-2,filaggrin(FLG)and involucrin(IVL),related NF-κB signal pathway and MAPK signal pathway.The nuclear translocation of NF-κB p65 was detected by immunofluorescence in HaCaT cells induced by TNF-α/IFN-γ stimulation.Results In the selected concentration,GSTT had no distinct influence on the growth of HaCaT cells.GSTT significantly decreased the level of thymus and activation-regulated chemokines(TARC),human macrophage-derived chemokine(MDC),regulated upon activation normal T cell expressed and secreted(RANTES)and interleukin(IL)-8 in supernatants of HaCaT cells induced by TNF-α/IFN-γ stimulation.GSTT suppressed the expression of iNOS and COX-2 in HaCaT cells induced by TNF-α/IFN-γ stimulation in a dose-dependent manner,and promoted the expression of FLG and IVL proteins.In addition,GSTT reduced the level of p65 in the nucleus in a dose-dependent manner,inhibited the nuclear translocation of p65,and increased the level of IκB in cytoplasm in HaCaT cells induced by TNF-α/IFN-γ stimulation.In the meanwhile,GSTT suppressed the phosphorylation of p38 and ERK proteins in the MARK pathway of HaCaT cells in a dose-dependent manner.Conclusion It is possible that GSTT inhibits inflammation-associated cytokines in HaCaT cells induced by TNF-α/IFN-γ stimulation by regulating the activation of the NF-κB pathway and the MAPK pathway,promoting the recovery of skin barrier function.