山茱萸多糖通过上调Klotho表达和抑制PI3K/AKT通路对肝癌HepG2细胞增殖、凋亡的影响

Effect of Cornus officinalis polysaccharide on proliferation and apoptosis of hepatocellular carcinoma HepG2 cells by up-regulating Klotho expression and inhibiting PI3K/AKT pathway

目的探究山茱萸多糖通过上调Klotho表达和抑制PI3K/AKT通路对肝癌HepG2细胞增殖、凋亡的影响。方法分别以6.25、12.5、25 mg/mL山茱萸多糖作用于人肝癌细胞株HepG2,CCK8法检测细胞增殖能力,采用Hoechst 33258荧光染色以及流式AnnexinV-FITC/PI双染法检测细胞凋亡率;通过免疫印迹法检测增殖相关蛋白Ki67、凋亡相关蛋白Bax、Bcl-2、Caspase-3以及PI3K/Akt通路相关蛋白水平,并检测Klotho蛋白表达水平。结果与对照组相比,6.25、12.5、25 mg/mL山茱萸多糖组HepG2细胞克隆形成数显著下降(P<0.05),且随山茱萸多糖浓度升高克隆形成数显著下降;与对照组相比,6.25、12.5、25mg/mL山茱萸多糖组HepG2细胞凋亡率显著升高(P<0.05),且随山茱萸多糖浓度升高细胞凋亡率逐渐升高(P<0.05);与对照组相比,6.25、12.5、25mg/mL山茱萸多糖组HepG2细胞中Bax、cleaved-caspase-3、Klotho蛋白表达升高(P<0.05),Ki67、Bcl-2、p-PI3K/PI3K、p-Akt/Akt蛋白表达下降(P<0.05),且呈剂量相关性。结论山茱萸多糖可能通过上调Klotho表达抑制PI3K/AKT通路活化,抑制HepG2细胞增殖,促进HepG2细胞凋亡。

Objective To investigate the effect of Cornus officinalis polysaccharide on proliferation and apoptosis of hepatocellular carcinoma HepG2 cells by up-regulating Klotho expression and inhibiting PI3 K/AKT pathway. Methods Cornus officinalis polysaccharide at the concentrations of 6.25, 12.5, and 25 mg/mL were used to treat human hepatocellular carcinoma cell line HepG2. CCK8 assay was used to detect cell proliferation. Hoechst 33258 fluorescence staining and Annexin V-FITC/PI double staining were used to detect the apoptotic rate, and the levels of proliferation-related protein Ki67, apoptosis-related protein Bax, Bcl-2, Caspase-3, and PI3 K/Akt pathway-related protein were detected by Western blotting, and the expression of Klotho protein was detected. Results Compared with the control group, the numbers of HepG2 cell clone in 6.25, 12.5, and 25 mg/mL Cornus officinalis polysaccharide groups decreased significantly(P < 0.05), and the number of clones decreased significantly with the increase of Cornus officinalis polysaccharide(P < 0.05). Compared with the control group, the apoptotic rate in 6.25, 12.5, and 25 mg/mL Cornus officinalis polysaccharide groups increased significantly(P < 0.05), and the apoptotic rate increased significantly with the increase of cornus officinalis polysaccharide(P < 0.05). Compared with the control group, the expressions of Bax, cleaved-Caspase-3, and Klotho in HepG2 cells of 6.25, 12.5, and 25 mg/mL Cornus officinalis polysaccharide groups increased significantly(P < 0.05), but the expressions of Ki67, Bcl-2, p-PI3 K/PI3 K, and p-Akt/Akt decreased significantly(P < 0.05), and they had the dose-dependent. Conclusion Cornus officinalis polysaccharide may inhibit the activation of PI3 K/AKT pathway, inhibit the proliferation of HepG2 cells, and promote the apoptosis of HepG2 cells by up-regulating the expression of Klotho.