一种肠道微生物代谢产物混合物对肝癌的影响

A metabolite cocktail of gut microbiota affects hepatocellular carcinoma

摘要:

【目的】探究肠道微生物代谢产物的混合物——吲哚-3-丙酸(indole-3-propionic acid, IPA)、丁酸钠(sodium butyrate, SB)和戊酸(valeric acid, VA)对肝癌细胞增殖的影响。【方法】体外培养人肝癌HepG2细胞系，分别给予不同浓度的混合物(1×、2×、3×、4×和5×)处理细胞，用总胆固醇和甘油三酯检测试剂盒检测细胞总胆固醇(total cholesterol, TC)和甘油三酯(triglycerides, TG)含量，用细胞增殖-毒性检测试剂盒(Cell Counting Kit-8, CCK-8)及克隆形成检测细胞增殖。12只BALB/c无胸腺裸鼠随机分为对照(Ctrl)组和处理(Treat)组，皮下注射HepG2细胞。每3 d测量肿瘤大小并计算肿瘤体积和抑瘤率。当肿瘤体积达到100 mm³时，Ctrl组小鼠每天给予无菌水灌胃，Treat组每天给予混合物灌胃直至麻醉处死。治疗27 d后，分别称量两组小鼠的体重，分离肿瘤并称量瘤重，计算瘤重/体重比。通过免疫组织化学(immunohistochemical, IHC)染色比较肿瘤Ki-67蛋白的含量，通过油红O染色比较肿瘤组织内的脂质积累。【结果】IPA、SB、VA的混合物降低了肝癌HepG2细胞内的TC和TG，对HepG2细胞的增殖有抑制作用，CCK-8和克隆形成结果都表明，混合物以剂量依赖的方式抑制HepG2细胞的增殖。在体内，混合物灌胃阻断了肝癌细胞的生长，表现为更小的肿瘤体积，更轻的肿瘤质量和更低的瘤重/体重比(Ctrl组: 723 mm³, 0.47 g, 22.23%; Treat组: 526 mm³, 0.32 g, 16.65%)。IHC和油红O染色进一步证明Ki-67的表达和脂质积聚在混合物处理的小鼠中减少。【结论】IPA、SB、VA的混合物可以抑制肝癌细胞增殖，抑制脂质合成。

Abstract:

[Objective] To investigate the effects of a metabolite cocktail composed of indole-3-propionic acid (IPA), sodium butyrate (SB), and valeric acid (VA) of gut microbiota on the proliferation of hepatocellular carcinoma cells. [Methods] The human hepatocellular carcinoma HepG2 cells were cultured in vitro and treated with the cocktail at different concentrations (1×, 2×, 3×, 4×, and 5×). The total cholesterol (TC) and triglyceride (TG) levels in the cells were determined by the total cholesterol and triglyceride assay kits. The Cell Counting Kit-8 (CCK-8) and colony formation assays were employed to examine the cell proliferation. Twelve BALB/c athymic nude mice were randomized into a control (Ctrl) group and a treatment (Treat) group and then subjected to subcutaneous injections of HepG2 cells. The tumor size was measured every three days, and the tumor volume and tumor inhibition rate were calculated. When the tumor volume reached 100 mm³, the mice in the Ctrl group were administered with sterile water by gavage daily, while those in the Treat group received the cocktail via gavage until euthanized under anesthesia. After 27 days of treatment, the body weights of mice in both groups were measured, and tumors were excised and weighed, with the tumor weight/body weight ratio calculated. The content of Ki-67 protein in the tumors was determined by immunohistochemical (IHC) staining, and the lipid accumulation within tumor cells was assessed by Oil Red O staining. [Results] The cocktail of IPA, SB, and VA lowered the levels of TC and TG in hepatocellular carcinoma HepG2 cells and exerted an inhibitory effect on the proliferation of HepG2 cells. Both CCK-8 and colony formation assays indicated that the cocktail inhibited the proliferation of HepG2 cells in a dose-dependent manner. The oral administration of the cocktail inhibited the growth of hepatocellular carcinoma cells, as evidenced by smaller and lighter tumors and lower tumor weight/body weight ratios in the Treat group than in the Ctrl group (Ctrl: 723 mm³, 0.47 g, 22.23%; Treat: 526 mm³, 0.32 g, 16.65%). IHC and Oil Red O staining further demonstrated reductions in Ki-67 expression and lipid accumulation in the mice administered with the cocktail via gavage. [Conclusion] The cocktail of IPA, SB, and VA can inhibit the proliferation and suppress the lipid synthesis of hepatocellular carcinoma cells.