葫芦巴碱对荷胆囊癌小鼠抑制作用及其机制

Inhibitory effect of trigonelline on gallbladder cancer bearing mice and its molecular mechanism

目的探讨葫芦巴碱对荷胆囊癌小鼠抑制作用及其机制。方法胆囊癌细胞系SGC-996随机分为对照组、50μmol/L组、150μmol/L组,分别采用0、50、150μmol/L葫芦巴碱处理细胞24 h,采用细胞计数试剂盒(CCK-8)分析3组细胞活力;采用Transwell检测细胞迁移,采用荧光定量聚合酶链反应(PCR)和蛋白质印迹法(Western blot)检测基质金属蛋白酶(MMP)-2和MMP-9 mRNA和蛋白质表达水平。采用胆囊癌细胞系SGC-996建立异体移植瘤模型随机分为模型组和葫芦巴碱组,葫芦巴碱组小鼠经腹腔给予葫芦巴碱50 mg/kg,模型组小鼠注射等体积生理盐水。连续治疗30 d,计算肿瘤体积和重量。计量数据比较采用t检验。结果对照组胆管癌细胞吸光度值(1.94±0.21)明显高于50μmol/L葫芦巴碱组(1.64±0.17),差异有统计学意义(t=3.091,P<0.05)。50μmol/L葫芦巴碱组细胞吸光度值(1.64±017)明显高于150μmol/L葫芦巴碱组胆管癌细胞(1.32±0.11),差异有统计学意义(t=2.784,P<0.05)。对照组胆管癌细胞迁移数量[(138.44±12.09)个]明显高于50μmol/L葫芦巴碱组细胞[(109.37±9.33)个],差异有统计学意义(t=6.018,P<0.05)。50μmol/L葫芦巴碱组细胞迁移数量[(109.37±9.33)个]明显高于150μmol/L葫芦巴碱组胆管癌细胞[(79.36±7.81)个],差异有统计学意义(t=5.091,P<0.05)。对照组胆管癌细胞MMP-2和MMP-9 mRNA表达水平(1.09±0.12、1.16±0.14)明显高于50μmol/L葫芦巴碱组细胞(0.75±0.18、0.80±0.12),差异有统计学意义(t=4.115、4.001,P<0.05)。50μmol/L葫芦巴碱组细胞MMP-2和MMP-9 mRNA表达水平(0.75±0.18、0.80±0.12)明显高于150μmol/L葫芦巴碱组(0.50±0.11、0.49±0.10),差异有统计学意义(t=3.819、3.618,P<0.05)。模型组治疗30 d后小鼠肿瘤体积[(1542.49±254.88)cm3]明显高于葫芦巴碱组[(1127.38±142.29)cm3],差异有统计学意义(t=6.099,P<0.05)。模型组治疗30 d后小鼠肿瘤质量[(5.29±0.41)g]明显高于葫芦巴碱组[(2.71±0.34)g],差异有统计学意义(t=4.971,P<0.05)。模型组治疗30 d后小鼠肿瘤组织MMP-3和MMP-9蛋白表达水平(2.39±0.14、1.52±0.16)明显高于葫芦巴碱组小鼠肿瘤组织(1.03±0.10、0.79±0.14),差异有统计学意义(t=3.901、3.428,P<0.05)。结论葫芦巴碱可显著下调MMP-2和MMP-9,抑制胆管癌细胞增殖和迁移,达到抗肿瘤作用。

Objective To investigate the inhibitory effect of trigonelline on gallbladder cancer bearing mice and and its molecular mechanism.Methods Gallbladder cancer cell line SGC-996 was randomly divided into control group,50μmol/L groups and 150μmol/L group,respectively.The cells were treated with trigonelline for 24 hours,and the cell viability of the three groups was analyzed by cell counting kit-8(CCK-8);The cell migration were analyzed by Transwell and the mRNA and protein expression levels of matrix metalloproteinase(MMP)-2 and MMP-9 were detected by fluorescence quantitative polymerase chain reaction(PCR)and Western blotting.Thirty nude mice were randomly divided into model group and trigonelline group.The allogeneic tumor model was established by gallbladder cancer cell line SGC-996.The mice in trigonelline group were given trigonelline 50 mg/kg intraperitoneally,and the mice in model group were injected with equal volume of normal saline.The tumor volume and weight were calculated after continuous treatment for 1 month.The measurement data were compared by t-test.Results The light absorption value of cholangiocarcinoma cells in the control group(1.94±0.21)was significantly higher than 50μmol/L trigonelline group(1.64±0.17,t=3.091,P<0.05).The absorbance value of cells in 50μmol/L trigonelline groupwas significantly higher than 150μmol/L trigonelline group(1.32±0.11,t=2.784,P<0.05).The migration number of cholangiocarcinoma cells in the control group[(138.44±12.09)cells]was significantly higher than 50μmol/L trigonelline group[(109.37±9.33)cells,t=6.018,P<0.05].The number of cell migration in 50μmol/L trigonelline group was significantly higher than 150μmol/L trigonelline group(79.36±7.81,t=5.091,P<0.05).The mRNA expression levels of MMP-2 and MMP-9 in cholangiocarcinoma cells of the control group(1.09±0.12,1.16±0.14)was significantly higher than 50μmol/L trigonelline group(0.75±0.18,0.80±0.12),t=4.115,4.001,P<0.05).The expression levels of MMP-2 and MMP-9 mRNA in 50μmol/L trigonelline group was significantly higher than 150μmol/L trigonelline group(0.50±0.11,0.49±0.10,t=3.819,3.618,P<0.05).After 30 days of treatment,the tumor volume in the model group[(1542.49±254.88)cm3]was significantly higher than that in the trigonelline group[(1127.38±142.29)cm3,t=6.099,P<0.05].After 30 days of treatment,the tumor mass of mice in the model group[(5.29±0.41)g]was significantly higher than that in the trigonelline group[(2.71±0.34)g,t=4.971,P<0.05].The expression levels of MMP-2 and MMP-9 in tumor tissues of model group after 30 days of treatment(2.39±0.14,1.52±0.16)was significantly higher than that in trigonelline group(1.03±0.10,0.79±0.14,t=3.901,3.428,P<0.05).Conclusion Trigonelline have antitumor effect by down-regulating MMP-2 and MMP-9 and inhibiting the proliferation and migration of cholangiocarcinoma cells.