阿魏酸对人肝癌HepG2细胞增殖、侵袭和凋亡的影响

Effects of Ferulic Acid on the Proliferation,Invasion and Apoptosis of HepG2 Hepatocellular Carcinoma Cells

目的:研究阿魏酸对人肝癌HepG2细胞增殖、侵袭和凋亡的影响。方法:采用CCK-8法筛选阿魏酸的给药浓度;采用Western blot法筛选白细胞介素6(IL-6)诱导磷酸化信号转导蛋白及转录激活子3(p-STAT3)蛋白高表达的HepG2细胞模型的最佳造模浓度。将HepG2细胞分为空白对照组、模型组、阿魏酸组(0.5 mmol/L)和阳性对照组(p-STAT3抑制剂C188-9,10μmol/L),除空白对照组外,模型组加入IL-6、各给药组加入IL-6和相应药物分别进行培养。采用CCK-8法、Transwell法和Annexin V-FITC/PI双染色法分别检测各组细胞存活率、侵袭数和早期、晚期凋亡率;采用Western blot法检测各组细胞中p-STAT3、胱天蛋白酶3(caspase-3)、锌指蛋白(ZBP-89)和波形蛋白(vimentin)的表达水平。基于PDB蛋白数据库,以STAT3高度相似晶体结构1BG1为对接模板,以Tyr705周围的区域为假定的结合口袋,进行阿魏酸与STAT3蛋白的分子对接分析。结果:选择以0.5 mmol/L阿魏酸干预HepG2细胞48 h作为后续实验条件,以50 ng/mL IL-6为造模条件。与空白对照组比较,模型组细胞侵袭数和p-STAT3/STAT3比值、vimentin蛋白表达水平均显著增加或升高(P<0.05或P<0.01),细胞晚期凋亡率和caspase-3蛋白表达水平均显著降低(P<0.05或P<0.01)。与模型组比较,阿魏酸组和阳性对照组细胞存活率、侵袭数、p-STAT3/STAT3比值和vimentin蛋白表达水平均显著减少或降低(P<0.05或P<0.01),细胞早期凋亡率(阿魏酸组除外)、晚期凋亡率和caspase-3、ZBP-89(阳性对照组除外)蛋白表达水平均显著升高(P<0.05或P<0.01)。分子对接结果表明,阿魏酸的羧酸基团与Asn581、Lys591分别存在1.9?、2.0?的氢键作用,结合能为-4.4 kcal/mol。结论:阿魏酸可能通过与STAT3磷酸化位点直接结合来抑制p-STAT3的活性;并可通过STAT3依赖性途径上调caspase-3蛋白表达,或可通过STAT3非依赖性途径上调ZBP-89蛋白表达,进而下调vimentin蛋白表达,从而抑制HepG2细胞的增殖、侵袭与凋亡。

OBJECTIVE:To study the effects of ferulic acid on the proliferation,invasion and apoptosis of HepG2 hepatocelluar carcinoma cells.METHODS:CCK-8 assay was used to screen the concentration of ferulic acid.Western blot assay was adopted to screen the optimal concentration of interleukin 6(IL-6)to induce HepG2 cell model with high expression of phosphorylated signal transduction protein and activator 3(p-STAT3)protein.HepG2 cell were divided into blank control group,model group,ferulic acid group(0.5 mmol/L)and positive control group(p-STAT3 inhibitor C188-9,10μmol/L).Except for blank control group,model group treated with IL-6,while administration groups were treated with IL-6 and relevant drugs.Cell survival rate,invasion and apoptosis rate in early and late stage were detected by CCK-8 assay,Transwell assay and Annexin V-FITC/PI double staining,respectively.Western blot assay was used to detect the expression of p-STAT3,caspase-3,ZBP-89 and vimentin proteins in each group.On the basis of the PDB protein database,using 1 BG1,a highly similar crystal structure of STAT3,as docking template,using the region around Tyr705 as the putative binding pocket,the docking analysis of ferulic acid with STAT3 protein was carried out.RESULTS:It is selected to use 0.5 mmol/L ferulic acid intervention for 48 h as the follow-up experimental condition;50 ng/mL IL-6 was selected as the modeling condition.Compared with blank control group,the number of cell invasion,p-STAT3/STAT3 ratio and protein expression of vimentin were increased significantly in model group(P<0.05 or P<0.01),while late apoptosis rate and protein expression of caspase-3 were decreased significantly(P<0.05 or P<0.01).Compared with model group,cell survival rate,the number of cell invasion,p-STAT3/STAT3 ratio and protein expression of vimentin were decreased significantly in ferulic acid group and positive control group(P<0.05 or P<0.01);early apoptotic rate(except for ferulic acid group),late apoptotic rate,the protein expression of caspase-3 and ZBP-89(except for positive control group)were increased significantly(P<0.05 or P<0.01).The results of molecular docking showed that the carboxylic groups of ferulic acid could interact with 1.9?hydrogen bond of Asn581 and 2.0?hydrogen bond of Lys591,with binding energy of-4.4 kcal/mol.CONCLUSIONS:Ferulic acid may inhibit the activity of p-STAT3 by directly binding to the phosphorylation site of STAT3;it may up-regulate the protein expression of caspase-3 via STAT3 dependent pathway,or up-regulate the protein expression of ZBP-89 via STAT3 independent pathway and then down-regulate the protein expression of vimentin,so as to inhibit the proliferation,invasion and apoptosis of HepG2 cells.