艾叶提取物对HepG2.2.15细胞凋亡和乙型病毒性肝炎病毒复制的影响

Effect of extract of folium artemisiae argyi on HepG2.2.15 cell apoptosis and hepatitis B virus replication

目的研究艾叶提取物对HepG2.2.15细胞凋亡和乙型病毒性肝炎(乙肝)病毒复制的影响。方法取人肝癌HepG2.2.15细胞,以0,25,50,100和200μg·mL^(-1)艾叶提取物处理48 h后,用噻唑蓝(MTT)法测定细胞活力。另取HepG2.2.15细胞随机分为对照组和艾叶提取物组,以Annexin V-FITC/PI双染流式细胞术检测细胞凋亡,以蛋白质印迹(Western Blot)法检测各组HepG2.2.15细胞中B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bax)、乙肝病毒X蛋白(HBx)蛋白表达水平,用酶联免疫吸附法检测细胞上清液中乙肝表面抗原和e抗原。选取细胞BRL、HepaRG和HepG2.2.15,以Western Blot法检测各组细胞中β-catenin、c-Myc和survivin蛋白表达水平。将HepG2.2.15细胞分为艾叶提取物组和艾叶提取物+LiCl组,以Annexin V-FITC/PI双染流式细胞术检测细胞凋亡,以Western Blot检测各组HepG2.2.15细胞中Bax、Bcl-2和HBx蛋白表达水平,用酶联免疫吸附法检测细胞上清液中乙肝表面抗原和e抗原。结果0,25,50,100和200μg·mL^(-1)艾叶提取物组细胞活力分别为0.81±0.09,0.76±0.07,0.69±0.08,0.57±0.06和0.43±0.04。对照组和艾叶提取物组细胞凋亡率分别为(3.89±0.75)%和(19.07±1.82)%;Bax蛋白表达分别为0.23±0.04和0.79±0.08;Bcl-2蛋白表达分别为0.82±0.13和0.36±0.05,HBx蛋白表达分别为1.13±0.15和0.42±0.06,β-catenin蛋白表达分别为0.91±0.12和0.42±0.05,c-Myc蛋白表达分别为1.07±0.13和0.49±0.07,survivin蛋白表达分别为0.83±0.09和0.35±0.04,差异均有统计学意义(均P<0.05)。艾叶提取物组与艾叶提取物+LiCl组细胞活力分别为0.40±0.05和0.68±0.09;细胞凋亡率分别为(22.19±3.12)%和(7.85±0.93)%;Bax蛋白表达分别为0.85±0.08和0.41±0.06;Bcl-2蛋白表达分别为0.30±0.05和0.64±0.07;HBx蛋白表达值分别0.40±0.06和0.87±0.09,差异均有统计学意义(均P<0.05)。结论艾叶提取物可通过抑制Wnt/β-catenin信号通路激活进而诱导HepG2.2.15细胞凋亡和抑制乙肝病毒的复制。

Objective To study the effects of the extract of folium artemisiae argyi(FFA)on HepG2.2.15 cell apoptosis and hepatitis B virus replication.Methods Human liver cancer HepG2.2.15 cells were treated with 0,25,50,100 and 200μg·mL^(-1) extract of FFA for 48 h,and the cell viability was determined by MTT method.In addition,HepG2.2.15 cells were randomly divided into control group and extract of FFA group,cell apoptosis was detected by Annexin V-FITC/PI double staining flow cytometry,B-cell lymphoma-2(Bcl-2),Bcl2-associated X protein(Bax),hepatitis B virus X protein(HBx)protein expression levels in HepG2.2.15 cells of each group were detected by Western Blot,enzyme-linked immunoassay was used to detect hepatitis B surface antigen and e antigen in cell supernatant.The expression levels ofβ-catenin,c-Myc and survivin in each group of BRL,HepaRG and HepG2.2.15 cells was detected by Western Blot.The HepG2.2.15 cells were divided into extract of FFA group and extract of FFA+LiCl group,apoptosis was detected by Annexin V-FITC/PI double staining flow cytometry,the expression of Bax,Bcl-2 and HBx protein were detected by Western Blot,enzyme-linked immunoassay was used to detect the hepatitis B surface antigen and e antigen in cell supernatant.Results The cell viability of 0,25,50,100 and 200μg·mL^(-1) extract of FFA groups were 0.81±0.09,0.76±0.07,0.69±0.08,0.57±0.06,0.43±0.04.The apoptosis rates of control group and extract of FFA group were(3.89±0.75)%,(19.07±1.82)%,the expression of Bax protein were 0.23±0.04,0.79±0.08;the expression of Bcl-2 protein were 0.82±0.13,0.36±0.05;HBx protein expression were 1.13±0.15,0.42±0.06;the expression ofβ-catenin were 0.91±0.12,0.42±0.05;the expression of c-Myc were 1.07±0.13,0.49±0.07;the expression of survivin were 0.83±0.09,0.35±0.04,all with significant difference(all P<0.05).The cell viability of the extract of FFA group and extract of FFA+LiCl group were 0.40±0.05,0.68±0.09;the apoptosis rates were(22.19±3.12)%,(7.85±0.93)%;the Bax protein expression were 0.85±0.08,0.41±0.06;Bcl-2 protein expression were 0.30±0.05,0.64±0.07;HBx protein expression value were 0.40±0.06,0.87±0.09,all with significant differences(all P<0.05).Conclusion Extract of FFA can induce HepG2.2.15 cell apoptosis and inhibit hepatitis B virus replication by inhibiting the activation of Wnt/β-catenin signaling pathway.