复方苦参注射液对人肝癌SMMC-7221细胞体外增殖、转移和侵袭的抑制作用及其机制

Inhibitory effects of compound Kushen injection on human liver cancer SMMC-7221 cells in vitro proliferation,metastasis and invasion and its mechanism

目的:探究复方苦参注射液对人肝癌SMMC-7221细胞体外增殖、转移和侵袭的抑制作用及其机制。方法:体外培养的SMMC-7221细胞分别加入终浓度为0、20、40、80、160μl/ml复方苦参注射液,CCK-8法检测细胞活力,筛选最适实验浓度。然后将SMMC-7221细胞分为对照组、复方苦参注射液组和复方苦参注射液+LY294002组,复方苦参注射液组和复方苦参注射液+LY294002组细胞均加入终浓度80μl/ml复方苦参注射液,复方苦参注射液+LY294002组细胞另加入终浓度2μg/ml LY294002。分别通过CCK-8法、克隆形成实验、流式细胞术、细胞划痕和Transwell实验检测细胞活力、增殖、凋亡、迁移和侵袭水平。通过qPCR检测PI3K、AKT mRNA表达水平,Western blot检测PI3K、AKT蛋白和磷酸化水平。结果:复方苦参注射液浓度为80μl/ml时SMMC-7221细胞活力降低为49.67%,选为最适实验浓度。复方苦参注射液显著抑制SMMC-7221细胞的细胞活力、增殖、迁移、侵袭能力,并促进细胞凋亡(P<0.05);使用LY294002抑制PI3K/AKT通路后,复方苦参注射液对肝癌细胞的生物学行为的抑制作用不显著(P>0.05)。复方苦参注射液可显著抑制PI3K、AKT mRNA表达和蛋白磷酸化(P<0.05),加入LY294002后对PI3K、AKT mRNA影响不明显,但会进一步抑制蛋白磷酸化(P<0.05)。结论:复方苦参注射液具有调控肝癌细胞生物学行为的作用,包括抑制增殖、迁移、侵袭和促进凋亡,这种作用可能与其抑制PI3K/AKT通路的作用有关。

Objective:To investigate the inhibitory effect of compound Kushen injection on the proliferation,metastasis and invasion of human liver cancer SMMC-7221 cells in vitro and its mechanism.Methods:First,SMMC-7221 cells were cultured at a final concentration of compound Kushen injection of 0,20,40,8 and 160μl/ml.Cell viability was measured using the CCK-8 method.The cells were then divided into the control group,the compound Kushen injection group and the compound Kushen injection+LY294002 group.The final concentration of compound Kushen injection was 80μl/ml.The final concentration of LY294002 was 2μg/ml.Cell viability,proliferation,apoptosis,migration and invasion levels were measured by CCK-8 method,clone formation experiment,flow cytometry,cell scratch and Transwell experiment,respectively.The mRNA and protein of PI3K and AKT were detected by qPCR and Western blot.Results:When the compound Kushen injection was 80μl/ml,the liver cancer cell viability decreased to 49.67%.Compound Kushen injection could significantly inhibit the cell viability,proliferation,migration,invasion and angiogenesis of liver cancer cells,and promote cell apoptosis(P<0.05).However,after using LY294002 to inhibit the PI3K/AKT pathway,the compound Kushen injection has no significant inhibitory effect on the biological behavior of liver cancer cells(P>0.05).Compound Kushen injection could significantly inhibit the mRNA and protein phosphorylation levels of PI3K and AKT(P<0.05).The addition of LY294002 further inhibited the level of protein phosphorylation(P<0.05),but had no significant effect on PI3K and AKT mRNA.Conclusion:Compound Kushen Injection can regulate the biological behavior of hepatocellular carcinoma cells,including inhibiting proliferation,migration and invasion,and promoting apoptosis,it may be related to its inhibitory effect on PI3K/AKT pathway.