白花蛇舌草提取物对表皮生长因子诱导的HaCaT细胞增殖、凋亡和TNF-α诱导的炎症因子影响  

Effects of Hedyotis diffusa Extract on Epidermal Growth Factor Induced Proliferation,Apoptosis,and TNF-α Induced Inflammatory Factors of HaCaT Cells

作　　者：付丹丹[1] 宋向凤[2] 李占国[1] 李敏[1] 刘冬[1] 夏永华[1] 田中伟[1] 

机构地区：[1]河南省新乡医学院第一附属医院皮肤性病科,河南453100 [2]河南省新乡医学院免疫学教研室,河南453000

出　　处：《中国中西医结合杂志》2016年第8期975-980,共6页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：河南省教育厅科学技术研究重点项目(No.13A320852);新乡市科技发展计划项目(No.15SF26);河南省卫生科技创新型人才工程(No.201004159);河南省医学科技攻关计划项目(No.20150216)

摘　　要：目的观察白花蛇舌草提取物(Hedyotis diffusa extract,HDE)对HaCaT细胞增殖、凋亡和炎症因子的影响,并探究可能的分子机理。方法实验分为3组:空白组不加表皮生长因子(epidermal growth factor,EGF)和HDE处理,对照组只加EGF而不加HDE处理,白花蛇舌草组分别用25、50、100mg/mL HDE和EGF共培养。CCK-8法检测HaCaT细胞增殖情况,流式细胞仪检测HaCaT细胞生长周期和凋亡率,Western blot法测定Ki67、Bcl-xL、cIAP1、procaspase-3、cleaved caspase-3的蛋白表达,ELISA法检测HaCaT细胞培养基上清液中IL-6、IL-8、IL-10的浓度,Western blot检测NF-κB p65亚基磷酸化(p-p65)水平。结果与空白组比较,对照组HaCaT细胞吸光度及Ki67蛋白表达升高(P<0.05,P<0.01),p-p65、IL-6、IL-8水平升高(P<0.05,P<0.01),IL-10水平降低(P<0.01);与对照组比较,白花舌草组25、50、100mg/mL浓度时HaCaT细胞吸光度及Ki67蛋白表达降低(P<0.05,P<0.01),p-p65、IL-6、IL-8水平降低,IL-10水平升高(P<0.05,P<0.01);同时白花蛇舌草组各浓度组凋亡率较对照组增加(P<0.05,P<0.01)。对照组处于G1期的细胞数量百分比为52.85%,白花蛇舌草组25、50、100mg/mL浓度时分别是58.51%、73.12%和79.95%,与对照组比较差异有统计学意义(P<0.05,P<0.01)。与白花蛇舌草组25mg/mL比较,白花蛇舌草组50和100mg/mL处于G1期的细胞数量百分比及凋亡率升高(P<0.01),且白花蛇舌草组100mg/mL时处于G1期的细胞数量百分比及凋亡率高于白花蛇舌草组50mg/mL(P<0.05)。与对照组比较,白花蛇舌草组100mg/mL时Bcl-xL和cIAP1蛋白表达降低(P<0.01,);caspase-3总量变化差异无统计学意义(P>0.05),cleaved caspase-3表达升高,差异有统计学意义(P<0.01)。结论 HDE可能通过将HaCaT细胞阻滞在G1期而抑制其增殖,通过抑制Bcl-xL和cIAP1蛋白表达、提高cleaved caspase-3蛋白表达而促进HaCaT细胞凋亡,通过调控NF-κB而抑制HaCaT细胞炎症反应。Objective Io observe the effects of Hedyotis diiiusa extract(HDL) on the proliferation,apoptosis,and inflammatory factors of HaCaT cells,and to explore its possible molecular mechanisms.Methods HaCaT cells were divided into 3 groups,the vehicle group,the control group,and 3 dose HDE groups.No epidermal growth factor(EGF) or HDE was added in the vehicle group.EGF was added with no HDE treatment in the control group.HDE(25,50,100 mg/mL) and EGF were added in the 3 HDE groups to coculture HaCaT cells.The effects of HDE on EGF-induced proliferation of HaCaT cells were detected using CCK-8 assay.The effects of HDE on the growth cycle and apoptosis rate of HaCaT cells were measured using flow cytometry.Moreover,protein expression levels of Ki67,Bcl-xL,cIAP1,procaspase-3,and cleaved caspase-3 were determined using Western blot.In addition,levels of IL-6,IL-8,and IL-1 0 in the supernatant were detected using ELISA.The level of phosphoration of NF-κB p65(p-p65) was measured using Western blot.Results Compared with the vehicle group,the absorbance of HaCaT cells and the expression level of Ki67 increased(P<0.05,P<0.01);levels of p-p65,IL-6,and IL-8 were elevated(P<0.05,P<0.01);IL-1 0 level was lowered(P<0.0 1) in the control group.Compared with the control group,the absorbance of HaCaT cells and the expression level of Ki67 decreased(P< 0.05,P< 0.0 1);levels of p-p65,IL-6,and IL-8 were reduced(P<0.05,P<0.0 1);IL-1 0 level was elevated(P<0.05,P<0.01) in the 3dose HDE groups.Meanwhile,the apoptosis rate of HaCaT cells increased more in the 3 dose HDE groups than in the control group(P<0.05,P<0.01).The percentage of HaCaT cells at G1 phase was 58.51%,73.12%,and 79.95%in 25,50,and 100 mg/mL HDE groups,respectively,showing statistical difference when compared with that in the control group(52.85%;P< 0.05,P<0.01).The percentage and apoptosis rate of HaCaT cells at G1 phase were elevated more in 50 and 100 mg/mL HDE groups than in 25 mg/mL HDE group(P< 0.0 1).Besides,the percentage and apoptosis rate of HaCaT cells at G1 phase w

关 键 词：白花蛇舌草提取物 人永生化表皮细胞 增殖 凋亡 炎性因子 

分 类 号：R285[医药卫生—中药学]