附子含药血清对人肝癌细胞侵袭和迁移的影响及机制

Effect of aconite-containing serum on invasion and migration of human liver cancer cells and its mechanism

目的探讨中药材附子含药血清对人肝癌细胞侵袭、迁移的影响及机制。方法按50 g/L浓度生附子水煎液和0.9%生理盐水分别灌胃SD大鼠,一周后获得附子含药血清和空白血清;使用附子含药血清(5%、10%、15%及20%)干预肝癌SK-Hep-1、SMMC-7721细胞,筛选最佳含药血清浓度;取对数期生长期SK-Hep-1、SMMC-7721细胞,采用CCK-8法检测附子含药血清对肝癌细胞增殖能力的影响,流式细胞术检测对肝癌细胞凋亡的影响,Transwell实验和划痕实验来评估不同浓度的含药血清对细胞侵袭和迁移能力的影响;实时定量聚合酶链反应和蛋白印迹法检测细胞中钙黏蛋白1(CDH1)、钙黏蛋白2(CDH2)和核因子κB(NF-κB)mRNA和蛋白表达水平,酶联免疫吸附试验检测肿瘤坏死因子α(TNF-α)、转化生长因子β1(TGF-β1)、白细胞介素10(IL-10)含量变化。结果附子含药血清能显著抑制肝癌细胞的增殖并增加其凋亡率,同时降低细胞的迁移和侵袭能力,这些效应随含药血清浓度的提高而增强;附子含药血清能够增加两种肝癌细胞中CDH1的蛋白质和mRNA表达,而降低CDH2和NF-κB蛋白质和mRNA的表达;附子含药血清还使两种肝癌细胞中TNF-α和TGF-β1含量降低,而IL-10含量增加,并且显示出剂量效应,且具有统计学意义(P<0.05)。结论附子含药血清抑制人肝癌细胞的增殖、迁移和侵袭能力,促进其凋亡,其机制可能与调节CDH1、CDH2和NF-κB的表达有关。

Objective To investigate the effect of aconite-containing serum on invasion and migration of human liver cancer cells and its mechanism.Methods SD rats were orally administered with raw aconite decoction at a concentration of 50 g/L and 0.9%physiological saline,respectively.After one week,the aconite-containing serum and blank serum were obtained.Liver cancer cell lines SK-Hep-1 and SMMC-7721 were treated with different concentrations of aconite-containing serum(5%,10%,15%,and 20%)to screen optimal aconite-containing serum concentration.SK Hep-1 and SMMC-7721 cells at logarithmic phase growth were used for CCK8 assay to detect the effect of aconite-containing serum on liver cancer cell proliferation.Flow cytometry was used to measure the effect of aconite-containing serum on liver cancer cell apoptosis.Transwell assay and wound healing assay were applied to evaluate the effects of different concentrations of aconite-containing serum on cell invasion and migration ability.Real time quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to detect mRNA and protein expression levels of calmodulin 1(CDH1),CDH2,and nuclear factor-κB(NF-κB).Enzyme-linked immunosorbent assay(ELISA)was performed to measure content changes of tumor necrosis factor-α(TNF-α),transforming growth factor-β1(TGF-β1)and interleukin-10(IL-10).Results Aconite-containing serum significantly inhibited the proliferation of liver cancer cells,increased their apoptosis rates and reduced cell migration and invasion abilities.These effects were intensified with the increase of the concentration of aconite-containing serum.Aconite-containing serum increased the protein and mRNA expressions of CDH1,while decreased the protein and mRNA expressions of CDH2 and NF-κB.Aconite-containing serum also attenuated TNF-αand TGF-β,while augmented IL-10 in a dose-dependent manner(P<0.05).Conclusion Aconite-containing serum inhibits the proliferation,migrationand invasion ability of human liver cancer cells,promotes their apoptosis.Its mechanism may be related to regulating CDH1,CDH2,and NF-κB.