黄芪甲苷对乌拉坦诱导肺癌小鼠的防治作用及其机制

Preventive and therapeutic effects of astragaloside IV on urethane induced lung cancer in mice and its mechanism

目的探讨黄芪甲苷(AS-Ⅳ)对乌拉坦诱导的肺癌小鼠的防治作用及其机制。方法将100只美国癌症研究所小鼠随机分为对照组、模型组、低剂量AS-Ⅳ组、中剂量AS-Ⅳ组和高剂量AS-Ⅳ组,每组20只。模型组、低剂量AS-Ⅳ组、中剂量AS-Ⅳ组和高剂量AS-Ⅳ组小鼠腹腔注射乌拉坦600mg·kg-1,每周1次,持续10周,建立肺癌模型;对照组小鼠腹腔注射等量生理盐水。造模当天,低剂量AS-Ⅳ组、中剂量AS-Ⅳ组和高剂量AS-Ⅳ组小鼠分别给予AS-Ⅳ12.5、25.0、50.0mg·kg-1灌胃,对照组和模型组小鼠给予等量蒸馏水灌胃,每日1次,连续19周。记录各组小鼠的体质量、自主活动次数,每2周1次。各组小鼠乙醚麻醉后脱臼处死,取肺组织,计算肺癌发生率和肺肿瘤数;苏木精-伊红染色观察各组小鼠肺组织病理学变化。采用酶联免疫吸附法检测各组小鼠血清中癌胚抗原(CEA)、癌抗原125(CA125)水平,实时荧光定量聚合酶链式反应法检测各组小鼠肺组织中Wnt-1、β-连环蛋白(β-catenin)、C-myc、细胞周期蛋白D1(CyclinD1)mRNA表达水平,Westernblot法检测各组小鼠肺组织中Wnt-1、β-catenin、C-myc、CyclinD1蛋白表达水平。结果造模后第1、3周,5组小鼠体质量和自主活动次数比较差异无统计学意义(P>0.05)。造模后第5~19周,模型组小鼠体质量显著低于对照组(P<0.05),低剂量AS-Ⅳ组、中剂量AS-Ⅳ组和高剂量AS-Ⅳ组小鼠体质量分别从第5、7、11周开始高于模型组(P<0.05)。造模后第5~19周,模型组小鼠自主活动次数显著少于对照组(P<0.05),中剂量AS-Ⅳ组和高剂量AS-Ⅳ组小鼠自主活动次数从第11周开始多于模型组(P<0.05),低剂量组小鼠自主活动次数从第13周开始高于模型组(P<0.05)。低剂量AS-Ⅳ组、中剂量AS-Ⅳ组和高剂量AS-Ⅳ组小鼠肺癌发生率和肺肿瘤数低于模型组(P<0.05),中剂量AS-Ⅳ组、高剂量AS-Ⅳ组小鼠肺癌发生率和肺肿瘤数低于低剂量AS-Ⅳ组(P<0.05),高剂量AS-Ⅳ组小鼠肺癌发生率和肺肿瘤数低于中剂量AS-Ⅳ组(P<0.05)。对照组小鼠肺组织形态正常,无增生和炎症细胞浸润;模型组小鼠肺组织被大量肿瘤细胞浸润,出现明显瘤区,失去原有肺组织形态;低剂量AS-Ⅳ组、中剂量AS-Ⅳ组和高剂量AS-Ⅳ组小鼠肺组织结构基本清晰,较少出现细胞核密集增生和炎症细胞浸润。模型组、低剂量AS-Ⅳ组、中剂量AS-Ⅳ组和高剂量AS-Ⅳ组小鼠血清中CEA和CA125水平高于对照组(P<0.05),低剂量AS-Ⅳ组、中剂量AS-Ⅳ组和高剂量AS-Ⅳ组小鼠血清中CEA和CA125水平低于模型组(P<0.05),中剂量AS-Ⅳ组、高剂量AS-Ⅳ组小鼠血清中CEA和CA125水平低于低剂量AS-Ⅳ组(P<0.05),高剂量AS-Ⅳ组小鼠血清中CEA和CA125水平低于中剂量AS-Ⅳ组(P<0.05)。模型组、低剂量AS-Ⅳ组、中剂量AS-Ⅳ组和高剂量AS-Ⅳ组小鼠肺组织中Wnt-1、β-catenin、C-myc和CyclinD1mRNA相对表达量高于对照组(P<0.05),低剂量AS-Ⅳ组、中剂量AS-Ⅳ组和高剂量AS-Ⅳ组小鼠肺组织中Wnt-1、β-catenin、C-myc和CyclinD1mRNA相对表达量低于模型组(P<0.05),中剂量AS-Ⅳ组、高剂量AS-Ⅳ组小鼠肺组织中Wnt-1、β-catenin、C-myc和CyclinD1mRNA相对表达量低于低剂量AS-Ⅳ组(P<0.05),高剂量AS-Ⅳ组小鼠肺组织中Wnt-1、β-catenin、C-myc和CyclinD1mRNA相对表达量低于中剂量AS-Ⅳ组(P<0.05)。模型组、低剂量AS-Ⅳ组、中剂量AS-Ⅳ组小鼠肺组织中Wnt-1、β-catenin、C-myc和Cyclin D1蛋白相对表达量高于对照组(P<0.05),低剂量AS-Ⅳ组、中剂量AS-Ⅳ组和高剂量AS-Ⅳ组小鼠肺组织中Wnt-1、β-catenin、C-myc和CyclinD1蛋白相对表达量低于模型组(P<0.05),中剂量AS-Ⅳ组、高剂量AS-Ⅳ组小鼠肺组织中Wnt-1、β-catenin、C-myc和CyclinD1蛋白相对表达量低于低剂量AS-Ⅳ组(P<0.05),高剂量AS-Ⅳ组小鼠肺组织中Wnt-1、β-catenin、C-myc和CyclinD1蛋白相对表达量低于中剂量AS-Ⅳ组(P<0.05),高剂量AS-Ⅳ组小鼠肺组织中C-myc蛋白相对表达量高于对照组(P<0.05),高剂量AS-Ⅳ组与对照组小鼠肺组织中Wnt-1、β-catenin和CyclinD1蛋白表达量比较差异无统计学意义(P>0.05)。结论AS-Ⅳ可显著抑制乌拉坦诱导的小鼠肺癌发生,其作用机制可能与减少Wnt-1、β-catenin、C-myc、CyclinD1蛋白表达及抑制Wnt/β-catenin信号通路活化有关。

Objective To explore the preventive and therapeutic effects of astragaloside Ⅳ(AS-Ⅳ)on urethaneinduced lung cancer mice and its mechanism.Methods One hundred Institute of Cancer Research mice were randomly divided into the control group,model group,low-dose AS-Ⅳ group,medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group,with 20 mice in each group.The mice in the model group,low-dose AS-Ⅳ group,medium-dose AS-Ⅳ group and high-dose AS-IV group were made the tung cancer model by intraperitoneal injection of urethane 600 mg·kg^-1 once a week for 10 weeks,and the mice in the control group were intraperitoneally injected with the same amount of normal saline.From the day of modeling,the mice in the low-dose AS-Ⅳ group,medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group were given AS-Ⅳ 12.5,25.0,50.0 mg·kg^-1 by gavage,while the mice in the control group and model group were given the same amount of distilled water once a day for 19 weeks.The body weight and the number of autonomous activities of mice in each group were recorded every two weeks.The incidence of lung cancer and the number of lung tumors were calculated.The mice in each group were killed by dislocation after diethyl ether anesthesia,and then the lung tissues of the mice were obtained,and the pathological changes of lung tissues were observed by using hematoxylin-eosin staining.The levels of serum carcinoembryonic antigen(CEA)and cancer antigen 125(CA125)of mice in each group were detected by enzyme linked immunosorbent assay.The expressions of Wnt-1,β-catenin,C-myc and cyclin D1 mRNA were detected by real-time fluorescent quantitative polymerase chain reaction,the expressions of Wnt-1,β-catenin,C-myc and cyclin D1 protein were detected by Western blot.Results At the 1 stand the3 rdweek after modeling,there was no significant difference in the body weight and the number of autonomous activities of mice among the five groups(P>0.05).From the 5 thto the 19 thweek after modeling,the body weight of mice in the model group was significantly less than that in the control group(P<0.05),while the body weight of mice in the low-dose AS-Ⅳ group,medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group was more than that in the model group from the 5 thweek,the 7 thweek and the 11 thweek,respectively(P<0.05).From the 5 thto the 19 thweek after modeling,the number of autonomous activities of mice in the model group was significantly less than that in the control group(P<0.05);the number of autonomous activities of mice in the medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group was more than that in the model group since the 11 thweek(P<0.05);the number of autonomous activities of mice in the low-dose AS-Ⅳ group was higher than that in the model group from the 13 thweek(P<0.05).The incidence of lung cancer and the number of lung tumors in the low-dose AS-Ⅳ group,medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group were significantly lower than those in the model group(P<0.05),the incidence of lung cancer and the number of lung tumors in the medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group were significantly lower than those in the low-dose AS-Ⅳ group(P<0.05),the incidence of lung cancer and the number of lung tumors in the high-dose AS-Ⅳ group were significantly lower than those in the medium-dose AS-Ⅳgroup(P<0.05).The lung tissues morphology of mice in the control group were normal,without hyperplasia and inflammatory cells infiltration.The lung tissues of mice in the model group were infiltrated by a large number of tumor cells,showed obvious tumor area and loose the original lung tissue morphology.The lung tissue structure of mice in the low-dose AS-Ⅳgroup,medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group was basically clear,with less nuclear proliferation and inflammatory cells infiltration.The levels of serum CEA and CA125 in the model group,low-dose AS-Ⅳ group,medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group were significantly higher than those in the control group(P<0.05),the levels of serum CEA and CA125 in the low-dose AS-Ⅳgroup,medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group were significantly lower than those in the model group(P<0.05),the levels of serum CEA and CA125 in the medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group were significantly lower than those in the low-dose AS-Ⅳ group(P<0.05),the levels of serum CEA and CA125 in the high-dose AS-Ⅳ group were significantly lower than those in the medium-dose AS-Ⅳ group(P<0.05).The expressions of Wnt-1,β-catenin,C-myc and cyclin D1 mRNA in lung tissues of mice in the model group,low-dose AS-Ⅳ group,medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group were significantly higher than those in the control group(P<0.05),the expressions of Wnt-1,β-catenin,C-myc and cyclin D1 mRNA in lung tissues of mice in the low-dose AS-Ⅳ group,medium-dose AS-Ⅳ group and high-dose AS-Ⅳgroup were significantly lower than those in the model group(P<0.05),the expressions of Wnt-1,β-catenin,C-myc and cyclin D1 mRNA in lung tissues of mice in the medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group were significantly lower than those in the low-dose AS-Ⅳ group(P<0.05),the expressions of Wnt-1,β-catenin,C-myc and cyclin D1 mRNA in lung tissues of mice in the high-dose AS-Ⅳ group were significantly lower than those in the medium-dose AS-Ⅳ group(P<0.05).The expressions of Wnt-1,β-catenin,C-myc and cyclin D1 protein in lung tissues of mice in the model group,low-dose AS-Ⅳ group and medium-dose AS-Ⅳ group were significantly higher than those in the control group(P<0.05),the expressions of Wnt-1,β-catenin,C-myc and cyclin D1 protein in lung tissues of mice in the low-dose AS-Ⅳ group,medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group were significantly lower than those in the model group(P<0.05),the expressions of Wnt-1,β-catenin,C-myc and cyclin D1 protein in lung tissues of mice in the medium-dose AS-Ⅳ group and high-dose AS-Ⅳ group were significantly lower than those in the low-dose AS-Ⅳ group(P<0.05),the expressions of Wnt-1,β-catenin,C-myc and cyclin D1 protein in lung tissues of mice in the high-dose AS-Ⅳ group were significantly lower than those in the medium-dose AS-Ⅳ group(P<0.05);the expression of C-myc protein in lung tissues of mice in the high-dose AS-Ⅳ group was significantly lower than that in the control group(P<0.05),there was no significant difference in the expression of Wnt-1,β-catenin and cyclin D1 protein in lung tissues of mice between the high-dose AS-Ⅳ group and the control group(P>0.05).Conclusion AS-Ⅳ can significantly inhibit urethane induced lung cancer in mice.Its mechanism may be related to reducing the expressions of Wnt-1,β-catenin,C-myc,Cyclin D1,and inhibiting the activation of Wnt/β-catenin signaling pathway.