褪黑素调控NLRP3/caspase-1通路减轻脂多糖诱导的人肺泡上皮细胞损伤

Effects of melatonin on lipopolysaccharide-induced human alveolar epithelial cells-A549 injury by regulating the nucleotide-binding oligomerization domain-like receptor protein 3/caspase-1 pathway

目的探讨褪黑素通过调节核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)/天冬氨酸特异性半胱氨酸蛋白酶-1(caspase-1)通路对脂多糖(LPS)诱导的人肺泡上皮细胞(A549)损伤的影响。方法体外培养A549细胞,分为对照组、LPS组(10 mg/L LPS)、LPS+褪黑素组(LPS+MT组,10 mg/L LPS+800μmol/L褪黑素)、LPS+抑制剂组(10 mg/L LPS+10μmol/L MCC950)。应用MTT法检测细胞增殖活性;流式细胞术检测细胞周期与细胞凋亡;酶联免疫吸附法(ELISA)检测细胞培养上清液中肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)水平;RT-qPCR法检测细胞中NLRP3、caspase-1 mRNA表达水平;Western blot法检测细胞中NLRP3、caspase-1蛋白表达水平。结果与对照组比较,LPS组A549细胞增殖活性、S期比例、G2/M期比例降低,细胞凋亡率、G0/G1期比例、TNF-α、IL-1β、IL-6水平和NLRP3、caspase-1 mRNA及蛋白表达水平显著升高,差异均有统计学意义(P<0.05);与LPS组比较,LPS+MT组与LPS+抑制剂组A549细胞增殖活性、S期比例、G2/M期比例显著升高,细胞凋亡率、G0/G1期比例、TNF-α、IL-1β、IL-6水平和NLRP3、caspase-1 mRNA及蛋白表达水平显著降低,差异均有统计学意义(P<0.05)。结论褪黑素可减轻LPS诱导的A549细胞损伤,其作用机制可能与抑制NLRP3/caspase-1信号通路有关。

Objective To investigate the of melatonin on lipopolysaccharide(LPS)-induced human alveolar epithelial cells(A549)injury by regulating the nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)/caspase-1 pathway.Methods A549 cells were cultured in vitro and divided into control group,LPS group(10mg/L LPS),LPS+melatonin group(LPS+MT group,10mg/L LPS+800μmol/L melatonin),LPS+inhibitor group(10mg/L LPS+10μmol/L MCC950).MTT method was used to detect cell proliferation activity;flow cytometry detected cell cycle and cell apoptosis;ELISA detected the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in the supernatant of cell culture;RT-qPCR detected the expression levels of NLRP3 and caspase-1 mRNA in cells;Western Blot detected the expression levels of NLRP3 and caspase-1 protein in the cells.Results Compared with those in control group,the cell proliferation activity,S phase ratio,and G2/M phase ratio in LPS group were significantly decreased,however,the apoptosis rate,G0/G1 phase ratio,and the expression levels of TNF-α,IL-1β,IL-6,NLRP3,caspase-1 mRNA and protein were increased significantly(P<0.05).Compared with those in LPS group,the cell proliferation activity,S phase ratio,and G2/M phase ratio in LPS+MT group and the LPS+inhibitor group were increased significantly,but,the cell apoptosis rate,G0/G1 phase ratio,and the expression levels of TNF-α,IL-1β,IL-6,NLRP3,caspase-1 mRNA and protein were decreased significantly(P<0.05).Conclusion Melatonin can relieve the damage of A549 cells induced by LPS,and its action mechanism may be related to the inhibition of NLRP3/caspase-1 signaling pathway.