人参皂苷Rg3和PEG-PLGA-Rg3纳米微粒对Lewis肺癌小鼠的作用及其机制

Preliminary study for the roles and mechanisms of 20( R)-ginsenoside Rg3 and PEGPLGA-Rg3 nanoparticles in the Lewis lung cancer mice

目的:观察并比较20(R)-人参皂苷Rg3[20(R)-ginsenoside Rg3,Rg3]和聚乙二醇(polyethylene glycol,PEG)-聚乳酸羟基乙酸(poly lactic-co-glycolic acid,PLGA)-Rg3纳米微粒(Rg3-N)对Lewis肺癌荷瘤小鼠的影响,探讨它们体内抗肿瘤作用的机制。方法:建立小鼠Lewis肺癌动物模型,60只小鼠随机分为人参皂苷Rg3纳米微粒组(Rg3-N)、PEG-PLGA纳米载体组(PEG)、20(R)-人参皂苷Rg3单体组(Rg3)、生理盐水组(NS)和空白对照组(C),每组12只,灌胃给药共14 d。每隔2 d测量小鼠的体重并绘制小鼠体重变化曲线,观察各组小鼠毛色、活动和精神状态等一般情况。实验结束当天处死小鼠,计算抑瘤率和肿瘤质量与体重比值;免疫组织化学法CD31抗体标记来计算微血管密度(microvessel density,MVD),以评价Rg3和Rg3纳米缓释微粒的体内抗血管生成作用,并通过实时定量PCR、免疫组织化学和Western blot法检测基质金属蛋白酶(matrix metalloproteinase-9,MMP-9)、缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)、血管内皮生长因子(vascular endothelial growth factor,VEGF)、Ki-67等血管新生和细胞增殖相关因子水平,以探讨其体内抗肿瘤作用的分子机制。结果:NS组和PEG组小鼠的体重呈现先升后降的趋势,而其余3组小鼠的体重则呈现逐渐上升并保持稳定的趋势。相对于NS组,Rg3组和Rg3-N组小鼠的毛色更亮,精神状态更好,更加活跃,一般状况较好。PEG组、Rg3组和Rg3-N组的肿瘤质量与NS组相比差异无统计学意义,但Rg3组和Rg3-N组的肿瘤质量与体重比值和微血管密度明显下降,与NS组之间差异有统计学意义(P<0.01),Rg3组和Rg3-N组之间差异无统计学意义。与NS组相比,Rg3组和Rg3-N组的VEGF mRNA、MMP-9、HIF-1α、VEGF的蛋白表达显著下降,且Rg3-N组上述各指标相对于Rg3组有进一步降低的趋势,PEG组、Rg3组和Rg3-N组Ki-67的表达均未见明显差异。结论:人参皂苷Rg3和PEG-PLGA-Rg3纳米微粒能抑制Lewis肺癌小鼠VEGF、MMP-9和HIF-1α的表达,从而间接地发挥它们的抗肿瘤作用,并改善小鼠的一般状况;此外,PEG-PLGA纳米微粒包埋可以增强Rg3的体内抗肿瘤作用。

Objective： To comparatively observe the effects of 20（ R）-ginsenoside Rg3 and PEG-PLGARg3 nanoparticles on the Lewis lung cancer mice and to explore the mechanisms of Rg3 and PEG-PLGARg3 nanoparticle anti-cancer in vivo. Methods： Lewis lung cancer mouse model was established and 60 mice were randomly divided into 5 groups with twelve in each group： PEG-PLGA-Rg3 nanoparticles group（ Rg3-N）,PEG-PLGA group（ PEG）,Rg3 group（ Rg3）,normal control group（ C）,saline control group（ NS）,and received intragastric administration for 14 days. The weights of the mice were measured every 2 days and the weight curves were obtained. At the same time,the color pattern,activity and mental status were observed. The mice were sacrificed when the administration was over,and the effects of 20（ R）-ginsenoside Rg3 and PEG-PLGA-Rg3 nanoparticles on tumor weight,and the tumor： weight ratios were analysed. In addition,the tumor microvessel density（ MVD） was measured by immunohistochemical staining with anti-CD31 antibody to compare the effects of Rg3 and PEG-PLGA-Rg3 nanoparticles on the tumor angiogenesis in vivo. Furthermore,the levels of such angiogenesis and proliferation factors as MMP-9,HIF-1α,VEGF,Ki-67 were examined by RT-PCR,Western blot and immunohistochemistry to explore the internal molecular mechanisms of anti-tumor effects in vivo. Results： The trends of variation of the mice weights in NS group and PEG group were rising early but declining later. In contrast,the trends of the other three groups were rising early and became stable later. In comparison with NS group,the mice of Rg3 group and Rg3-N group had better general status： brighter color,more active and better spirit. Compared with NS group,the tumor weight in PEG group,Rg3 group and Rg3-N group showed no significant difference but the tumor： weight ratio and MVD in Rg3 group and Rg3-N group declined significantly（ P〈0. 01）. Besides,there was no significant difference between Rg3 group and Rg3-N group.At the same time,the level of VEGF mRNA,the protein expression of MMP-9,HIF-1α,VEGF in Rg3 group and Rg3-N group decreased compared with NS group. Furthermore,the level of each index abovementioned in Rg3-N group was lower than that in Rg3 group. The expression of Ki-67 in PEG group,Rg3 group and Rg3-N group showed no significant difference compared with NS group. Conclusion： Rg3 and PEG-PLGA-Rg3 nanoparticle may suppress the expression of VEGF,MMP-9 and HIF-1α in Lewis lung cancer mice,thereby indirectly contributing to their antitumor effects and alleviating the mice＇s general status. In addition,PEG-PLGA nanoparticles embedding can promote Rg3 antitumor effect in vivo.