雷公藤甲素调节PI3K/Akt信号通路抑制结肠癌增殖并诱导凋亡的实验研究

Experimental study of triptolide regulating PI3K/Akt signaling pathway to inhibit proliferation and induce apoptosis of colon cancer

目的探讨雷公藤甲素抑制结肠癌增殖增殖、诱导凋亡的机制。方法稳定培养结肠癌HT29细胞,分别加入含25、50、100、200、400、600 mg/L雷公藤甲素的培养基进行干预,采用CCK-8法、流式细胞术、细胞集落形成实验检测雷公藤甲素对HT29细胞增殖和凋亡的影响。构建裸鼠结肠癌移植瘤模型,将30只裸鼠随机分为对照组、雷公藤甲素100 mg/kg组和400 mg/kg组。对照组给予0.2 ml生理盐水灌胃,雷公藤甲素组灌胃剂量分别为100 mg/kg和400 mg/kg,1次/d,共14 d。比较各组裸鼠抑瘤率、检测移植瘤组织中细胞凋亡情况。结果雷公藤甲素体外能抑制HT29细胞的增殖,且抑制作用呈浓度和时间依赖性。HT29细胞对照组、100 mg/L组、200 mg/L组、400 mg/L组的细胞凋亡率分别为(7.85±1.44)%、(13.86±2.08)%、(20.45±2.83)%、(28.52±2.41)%,差异均有统计学意义(P<0.05)。随着雷公藤甲素干预浓度升高,HT29细胞中Bcl-2蛋白表达量逐渐降低,而caspase-3和Bax蛋白表达量逐渐升高(P<0.05)。与对照组裸鼠比较,雷公藤甲素不同浓度给药组小鼠的肿瘤生长均受到明显抑制。100 mg/kg组抑瘤率为31.18%,400 mg/kg组为48.77%。雷公藤甲素干预后Ki67蛋白表达强度明显低于对照组(P<0.05)。Western blotting结果显示,雷公藤甲素作用48 h后,不同剂量组HT29细胞中p-PI3K蛋白和p-Akt蛋白的表达水平逐渐降低(P<0.05)。结论雷公藤甲素在体内外均能抑制人结肠癌HT29细胞的增殖同时诱导其凋亡,并通过抑制PI3K/Akt信号通路活性抑制结肠癌裸鼠的肿瘤生长。

Objective To investigate the mechanism of triptolide in inhibiting proliferation and inducing apoptosis of colon cancer cells.Methods Colon cancer HT29 cells were cultured stably.25,50,100,200,400 and 600 mg/L of triptolide were added for intervention.CCK-8 assay,flow cytometry and cell colony formation assay were used to detect the effects of triptolide on proliferation and apoptosis of HT29 cells.Thirty nude mice were randomly divided into control group,triptolide 100 mg/kg group and triptolide 400 mg/kg group.The control group was given 0.2 ml normal saline intragastric administration,and the triptolide group was given 100 mg/kg and 400 mg/kg intragastric administration,once a day,for 14 days.The tumor inhibition rate of nude mice in each group was compared and the cell apoptosis in the transplanted tumor tissue was detected.Results Triptolide inhibited the proliferation of HT29 cells in a concentration-and time-dependent manner in vitro.The apoptosis rate of HT29 cells in control group,100 mg/L group,200 mg/L group and 400 mg/L group were(7.85±1.44)%,(13.86±2.08)%,(20.45±2.83)%,(28.52±2.41)%,respectively,and the differences were statistically significant(P<0.05).With the increase of triptolide concentration,the protein expression of Bcl-2 in HT29 cells was gradually decreased,while the protein expression of Caspase-3 and Bax was gradually increased(P<0.05).Compared with the control group,the tumor growth of mice treated with different concentrations of triptolide was significantly inhibited.The anti-tumor rate of 100 mg/kg group was 31.18%,and that of 400 mg/kg group was 48.77%.The expression intensity of Ki-67 protein after triptolide intervention was significantly lower than that of control group(P<0.05).Western blotting results showed that after treatment with triptolide for 48 h,the expression levels of p-PI3K and p-Akt protein in HT29 cells in different dosage groups were decreased gradually(P<0.05).Conclusion Triptolide can inhibit proliferation and induce apoptosis of human colon cancer HT29 cells in vitro and in vivo,and inhibit tumor growth of colon cancer nude mice by inhibiting PI3K/Akt signaling pathway activity.