人参皂苷Rg3联合索拉非尼对裸鼠肝癌移植瘤生长和血管生成的调控作用

Effect of ginsenoside Rg3 combined with sorafenib in inhibiting tumor growth and neovascularization in nude mice with in situ transplanted human hepatocellular carcinoma

目的:研究人参皂苷Rg3联合索拉非尼对裸鼠肝癌移植瘤生长及血管生成的影响及其机制.方法:构建裸鼠人肝癌移植瘤模型LCI-D20,将造模的26只裸鼠随机分成人参皂苷组(R组):人参皂苷Rg3 5 mg/kg,腹腔注射,1次/d;索拉非尼组(S组):索拉非尼30 mg/kg,灌胃,1次/d;联合组:人参皂苷Rg3 5 mg/kg+索拉非尼30 mg/kg;对照组:生理盐水腹腔注射,1次/d.治疗2 wk剥离瘤体称质量,计算抑瘤率;免疫组织化学法检测移植瘤组织微血管密度(microvascular density,MVD);ELISA法、Western blot法检测血管内皮生长因子(vascular endothelial growth factor,VEGF)、低氧诱导因子-1?(hypoxia inducible factor-1?,HIF-1?)和VEGFR-2的表达.结果:人参皂苷Rg3组的抑瘤率为20.6%,索拉非尼组的抑瘤率为34.74%,联合组的抑瘤率为48.64%,根据Weeb系数计算方法,人参皂苷Rg3与索拉非尼联合用药后肿瘤的生长率为51.36%,低于预期值51.81%,两者表现为协同作用.免疫组织化学法测定显示,3组的MVD均较对照组明显降低(P<0.01),但3组间差异不明显(P>0.05).ELLISA法结果显示:R组、S组及联合组血清VEGF水平均低于对照组(P<0.01、P<0.05及P<0.01),联合组较S组水平更低(P<0.05);R组及联合组血清HIF-1?水平明显低于对照组(P<0.01),S组仅有降低的趋势(P>0.05);联合组与S组间有明显差异(P<0.05);3组血清VEGFR-2水平与对照组比较差异不明显(P>0.05).Western blot法结果显示:3组VEGF、HIF-1?和VEGFR-2蛋白的表达均低于生理盐水组(P<0.05),但联合组与R、S组之间均无明显差异(P>0.05).结论:人参皂苷Rg3联合索拉非尼对裸鼠肝移植瘤生长有明显的抑制作用,两者联合具有协同增效作用;其机制可能与调控血管生成相关因子HIF-1?、VEGF、VEGFR-2的表达密切相关.

AIM：To observe the effect ot ginsenoside Rg3 combined with sorafenib in inhibiting tumor growth and neovascularization in nude mice with in situ transplanted human hepatocellular carcinoma xenografts and to explore the possible mechanism.METHODS：Twenty-six nude mice with highly metastatic human hepatocellular carcinoma transplanted in situ（LCI-D20）were randomly divided into an R group treated with ginsenoside Rg3（5 mg/kg,qd）,an S group treated with sorafenib（30 mg/kg,qd）,a combination group treated with both ginsenoside Rg3（5 mg/kg,qd） and sorafenib（30 mg/kg,qd）,and a control group treated with saline.After 2 wk of treatment,all mice were killed to collect orbital blood samples.The tumors were peeled off and weighed to calculate the tumor inhibition rate.Immunohistochemical method was used to detect the micro-vessel density（MVD） in the tumors.The expression of vascular endothelial growth factor（VEGF）,hypoxia inducible factor-la（HIF-la）,and VEGF receptor 2（VEGFR-2） in tumors was detected by ELISA and Western blot.RESULTS：The tumor inhibition rates of the R group,S group and combination group were 20.60%,34.74%and 48.64%,respectively.According to the Weeb coefficient algorithm,the combination group showed a synergistic effect in inhibiting the tumor growth in nude mice.The MVD of each treatment group was significantly lower than that of the control group（P〈0.01）,although there were no significant differences between the combination group and the R or S group（P〈0.05）.ELISA results showed that serum VEGF levels were significantly lower in the R,S and combination groups than in the control group（P〈0.01,P〈0.05 and P〈0.01）,and in the combination group than in the S group（P〈0.05）,but there was no significant difference between the combination group and R group（P〈0.05）.Compared with the control group,the level of HIF-la was significantly lower in all treatment groups（P〈0.05） except the S group（P〈0.05）,and the decrease was more significant in the combination group than in the S group（P〈0.05）.The level of VEGFR-2had no significant difference in the four groups（P〈0.05）.Western blot showed decreased expression of VEGF,HIF-la and VEGFR-2 in the three treatment groups（P〈0.05）,although there were no significant differences between the combination group and R or S group（P〈0.05）.CONCLUSION：Ginsenoside Rg3 combined with sorafenib shows a synergistic effect in inhibiting tumor growth in nude mice,via mechanisms possibly associated with regulating the expression of angiogenesis factors VEGF,HIF-la,and VEGFR-2.