山奈酚与肽聚糖联合对HepG2细胞增殖和凋亡的影响

Effects of Kaempferol and Peptidoglycan on the Proliferation and Apoptosis of HepG2 Cells

目的:探讨罗汉果山奈酚(kaempferol,KF)联合枯草芽胞杆菌肽聚糖(peptidoglycan,PG)对肝癌细胞HepG2生物学活性的影响。方法:以两因素(KF和PG)六水平(0、10、20、40、60、80μmol/L)设计KF和PG正交实验,采用MTT法筛选出抑制HepG2增殖的最佳KF和PG组合浓度。将HepG2细胞分为对照组、PG组(40μg/mL)、KF组(40μmol/L)、联合组(40μg/mL PG+40μmol/L KF),分别干预48 h后,采用倒置显微镜观察细胞形态与分布;划痕试验检测细胞迁移率;通过流式细胞实验检测HepG2细胞凋亡率;用Western blot法检测HepG2细胞的Bax、Bcl-2、Akt和p-Akt水平;qPCR检测Bax和Bcl-2 mRNA水平。结果:HepG2细胞增殖抑制率随PG和KF浓度的升高而明显增加(P<0.05),二者最佳组合浓度为40μg/mL+40μmol/L;在此浓度作用下,与PG组和KF组相比,联合用药对HepG2细胞增殖呈现出明显的抑制效果,细胞形态逐渐皱缩,迁移能力明显下降(P<0.05),凋亡率显著提高(P<0.05);Bax蛋白表达水平显著升高(P<0.05),Bcl-2、p-Akt蛋白表达显著下调(P<0.05);Bax mRNA水平显著升高(P<0.05),Bcl-2 mRNA水平显著降低(P<0.05)。结论:KF与PG联合能有效增强单独应用KF或PG对HepG2细胞增殖的抑制作用和凋亡效果,其机制可能与调节Bax、Bcl-2、p-Akt表达水平有关。

Objective:To investigate the effect of Siraitia grosvenori kaempferol(KF)combined with Bacillus subtilis peptidoglycan(PG)on the biological activity of HepG2 cells in hepatocellular carcinoma.Methods:A two-factor(KF and PG)six-level(0,10,20,40,60,80μmol/L)orthogonal experiment was designed to screen the best concentration of the combination of KF and PG for inhibiting HepG2 proliferation by MTT method.HepG2 cells were assigned to the control group,the PG group(40μg/mL PG),the KF group(40μmol/L KF)and the combined group(40μg/mL PG+40μmol/L KF),and the intervention was performed for 48 h.After the intervention,cell morphology and distribution were observed by inverted microscope.Cell migration rate was detected by the scratch assay.The apoptosis rate of HepG2 cells was detected by flow cytometry.Bax,Bcl-2,Akt and p-Akt levels in HepG2 cells were detected by Western blot.Bax and Bcl-2 mRNA levels were detected by qPCR.Results:The inhibition rate of HepG2 cell proliferation significantly increased with the increase of PG and KF concentrations(P<0.05).The optimal combination of PG and KF was 40μg/mL PG+40μmol/L KF.At this concentration,compared with the PG group and the KF group,the combined group showed significant inhibitory effect on the proliferation of HepG2 cells,gradually shrinking cell morphology and decreased migration ability(P<0.05);in the combined group,apoptosis rate significantly increased(P<0.05),Bax protein expression level significantly increased(P<0.05),Bcl-2 and p-Akt protein expressions were significantly down-regulated(P<0.05),Bax mRNA level significantly increased(P<0.05),and Bcl-2 mRNA level significantly decreased(P<0.05).Conclusion:KF combined with PG can effectively enhance the inhibitory effect of KF or PG on the proliferation and apoptosis of HepG2 cells,and its mechanism may be related to the regulation of Bax,Bcl-2 and p-Akt expression levels.