氧化苦参碱调控FOXO1表达对TNF-α诱导HaCaT细胞炎症因子分泌的影响

Effect of oxymatrine-regulated FOXO1 expression on TNF-α-induced inflammatory factor secretion in HaCaT cells

目的探讨氧化苦参碱调控叉头框转录因子O亚族1(FOXO1)表达对肿瘤坏死因子-α(TNF-α)诱导的HaCaT细胞炎症因子分泌的影响。方法细胞计数试剂盒-8(CCK-8)检测不同浓度TNF-α对HaCaT细胞增殖活性的影响,筛选TNF-α诱导浓度和时间。将诱导后的HaCaT细胞分为模型组(25μg/L TNF-α)、氧化苦参碱组(25μg/L TNF-α和40 mg/mL氧化苦参碱,未转染)、氧化苦参碱+LV-NC-GFP组(25μg/L TNF-α和40 mg/mL氧化苦参碱,转染空载体)、氧化苦参碱+LV-CA-FOXO1组(25μg/L TNF-α和40 mg/mL氧化苦参碱,转染FOXO1过表达载体),另取正常培养的HaCaT细胞为对照组。实时荧光定量聚合酶链式反应(qRT-PCR)法检测细胞中FOXO1表达水平;CCK-8法检测细胞增殖活性;流式细胞术检测细胞凋亡率;ELISA法检测细胞中炎性因子:肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)含量;蛋白免疫印迹(Western blot)法检测细胞中AKT、TLR-4和FOXO1蛋白表达水平。结果本研究采用25μg/L TNF-α处理24 h诱导HaCaT细胞。与对照组相比,模型组HaCaT细胞FOXO1 mRNA表达水平、细胞增殖活性、TNF-α、IL-6、IL-1β含量以及TLR-4、FOXO1蛋白表达和AKT磷酸化水平显著升高(P<0.05),细胞凋亡率显著降低(P<0.05)。与模型组相比,氧化苦参碱组和氧化苦参碱+LV-NC-GFP组HaCaT细胞FOXO1 mRNA表达水平、TNF-α、IL-6、IL-1β含量以及TLR-4、FOXO1蛋白表达和AKT磷酸化水平显著降低(P<0.05),细胞凋亡率显著升高(P<0.05)。与氧化苦参碱+LV-NC-GFP组相比,氧化苦参碱+LV-CA-FOXO1组HaCaT细胞FOXO1 mRNA表达水平、细胞增殖活性、TNF-α、IL-6、IL-1β含量以及TLR-4、FOXO1蛋白表达和AKT磷酸化水平显著升高(P<0.05),细胞凋亡率显著降低(P<0.05)。结论氧化苦参碱可能通过靶向抑制FOXO1表达降低炎性因子分泌,并可促进HaCaT细胞凋亡,抑制细胞增殖。

Objective To investigate the effect of oxymatrine-regulated forkhead box transcription factor O1(FOXO1)expression on the secretion of inflammatory factors in HaCaT cells induced by tumor necrosis factor-α(TNF-α).Methods Cell counting kit-8(CCK-8)was used to detect the effect of different concentrations of TNF-αon the proliferation of HaCaT cells,so thus to screen the concentration and time of TNF-αinduction.The induced HaCaT cells were divided into model group(25μg/L TNF-α),oxymatrine group(25μg/L TNF-αand 40 mg/mL oxymatrine,not transfected),oxymatrine+LV-NC-GFP group(25μg/L TNF-αand 40 mg/mL oxymatrine,transfected with empty vector),oxymatrine+LV-CA-FOXO1 group(25μg/L TNF-αand 40 mg/mL oxymatrine,transfected with FOXO1 overexpression vector),and another HaCaT cells in normal culture were taken as control group.The expression of FOXO1 was assessed by real-time fluorescence quantitative PCR(qRT-PCR).CCK-8 method was used to assess cell proliferation.Flow cytometry was used to assess apoptosis rate.The levels of TNF-α,interleukin-6(IL-6)and interleukin-1β(IL-1β)were assessed by ELISA.Western blot was used to assess the protein expression levels of Akt,TLR-4 and FOXO1.Results In this study,HaCaT cells were induced by 25μg/L TNF-αfor 24 h.Compared with those in the control group,the FOXO1 mRNA expression level,cell proliferation activity,TNF-α,IL-6 and IL-1βcontents,TLR-4 and FOXO1 protein expression and Akt phosphorylation level of HaCaT cells in model group were significantly higher(P<0.05);and the apoptosis rate was significantly lower(P<0.05).Compared with those in the model group,the FOXO1 mRNA expression level,TNF-α,IL-6 and IL-1βcontents,TLR-4 and FOXO1 protein expression and Akt phosphorylation level of HaCaT cells in oxymatrine group and oxymatrine+LV-NC-GFP group were significantly lower(P<0.05);and the apoptosis rate was significantly higher(P<0.05).Compared with those in oxymatrine+LV-NC-GFP group,the FOXO1 mRNA expression level,cell proliferation activity,TNF-α,IL-6 and IL-1βcontents,TLR-4 and FOXO1 protein expression and Akt phosphorylation level of HaCaT cells in oxymatrine+LV-CA-FOXO1 group were significantly higher(P<0.05);and the apoptosis rate was significantly lower(P<0.05).ConclusionOxymatrine may reduce the secretion of inflammatory factors,promote the apoptosis of HaCaT cells and inhibit the proliferation of HaCaT cells by targeted inhibiting the expression of FOXO1.