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

作　　者：高琼[1] 刘昱昕 陈冬梅 尚元元[1] 司佳薇 施惠娟[1] GAO Qiong;LIU Yu-xin;CHEN Dong-mei;SHANG Yuan-yuan;SI Jia-wei;SHI Hui-juan(Department of Dermatology,General Hospital of Ningxia Medical University,Yinchuan 750004,Ningxia,China;不详)

机构地区：[1]宁夏医科大学总医院皮肤科,宁夏银川750004 [2]银川市妇幼保健院皮肤科,宁夏银川750001 [3]宁夏医科大学,宁夏银川750004

出　　处：《广东医学》2022年第1期40-45,共6页Guangdong Medical Journal

基　　金：宁夏自然科学基金项目(2018AAC03259)。

摘　　要：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-1βconte

关 键 词：氧化苦参碱 叉头框转录因子O亚族1 肿瘤坏死因子 HACAT细胞 炎症因子 

分 类 号：R758.63[医药卫生—皮肤病学与性病学] R751.05[医药卫生—临床医学]