人参皂甙Rg3通过VEGFR-2介导的PI3K/Akt/mTOR信号通路抗子宫内膜异位症血管生成机制研究

The results show that neovascularization is important characteristics of endometriosis.Among pro-angiogenic factors, vascular endothelial growth factor(VEGF)is consided to be the most critical one.Its receptor signal pathway is also cinsided to be the most promising of target for inhibiting angiogenesis.Recent studies found Ginsenoside Rg3 has the effect of inhibiting proliferation of tumor vascular endothelial cells and anti-apoptosis of tumor cells.VEGFR-2-mediated PI3K/Akt/mTOR signal transdution pathway can anti-angiogenesis of tumor cells,which play role in the tumor incidence.Therefore regulating the PI3K/Akt/mTOR signal transdution pathway related molecules may become a new treament of endometriosis.This study chooses ginsenoside Rg3,which is the only antiangiogenic agents into clinical application. Using the flouorescent quantitative RT-PCR and immunohistochemistry technology study the effect of ginsenoside Rg3 on endometriosis cells' PI3K/Akt/mTOR signal pathway related molecules ,and explore whether the effect of ginsenoside Rg3 on endometriosis is through regulating the VEGFR-2-mediated PI3K/Akt/mTOR signaling pathway to inhibit angiogenesis and promote apoptosis of endometrial cells.Thus verify the relationship between the PI3K/Akt/mTOR signaling pathway in the incidence of endometriosis,and explore the role of ginsenoside Rg3 in PI3K/Akt/mTOR signaling pathway.The study will provide experimental and theory basis for the diagnoses and theragy of the effect of ginsenoside Rg3 on endometriosis.

研究表明，新生血管生成是子宫内膜异位症的重要特征。VEGF是最强的促血管生成因子，其受体信号通路被认为是最有前途的抑制血管生成靶点。近来研究表明，人参皂甙Rg3具有抑制肿瘤血管内皮细胞增殖及抗凋亡的作用。VEGFR-2介导的PI3K/Akt/mTOR信号通路可抑制肿瘤血管新生，参与癌的发生。因此通过调节PI3K/Akt/mTOR信号通路的相关分子，可能会成为治疗EMs的新途径。本实验采用目前全世界唯一进入临床应用的肿瘤新生血管抑制剂-人参皂甙Rg3，运用荧光定量RT-PCR和免疫组化等技术，研究人参皂甙Rg3对EMs中信号通路相关分子的影响，探索人参皂甙Rg3是否通过VEGFR-2介导的PI3K/Akt/mTOR信号通路抑制血管新生，诱导异位内膜细胞的凋亡实现的。从而证实该通路与EMs发病的关系，探索人参皂甙Rg3在此通路的作用环节，为人参皂甙Rg3防治EMs提供实验和理论依据。

目的：阐明人参皂甙Rg3抑制异位内膜生长的作用机制与VEGFR-2介导的PI3K/Akt/mTOR信号通路抑制血管新生，诱导异位内膜细胞凋亡有关。.方法：本研究将造膜成功的子宫内膜异位症大鼠随机分成5组，即人参皂甙Rg3低剂量组（A组）、人参皂甙Rg3高剂量组（B组）、孕三烯酮组（C组）、模型组（D组）及去势组（E组）。分别给予模型对照组及去势组仅药物溶剂0.5%CMC-Na，孕三烯酮组0.5 mg/kg/d，人参皂甙Rg3 5mg/kg/d、10mg/kg/d，每天灌胃给药，3周后处死大鼠，测量异位内膜的体积，计算抑制率。ECLI法测定血清E2和P水平，并取下大鼠异位子宫内膜，运用免疫组化、westernblot法检测人参皂甙Rg3对VEGF、VEGFR-2、p-Akt及p-mTOR蛋白表达的影响，实时定量PCR法检测人参皂甙Rg3对VEGF、VEGFR-2、Akt及mTOR基因表达的影响。Tunel法检测各组异位细胞的凋亡率。.结果：用药后，B组、C组及E组对异位内膜体积抑制率比较均显著低于D组 (P<0.05)；组织学观察可见，异位内膜呈退化趋势，B、C、E组间大鼠血清E2值水平与D组相比有明显下降(P<0.05)。C组及E组的P水平与D相比有明显下降(P<0.05),但A、B组P水平与模型组相比无显著性差异。免疫组化显示，用药后，B、C、E组VEGF、p-Akt及p-mTOR的表达与D组相比有统计学差异（P<0.05），与westernblot结果一致。实时定量PCR检测也显示，用药后大鼠异位内膜除VEGFR-2外，VEGF、Akt及mTOR各组表达下调，B、C、E组与D组间有统计学差异(P＜0.05)。Tunel法检测各组异位细胞的凋亡率，结果显示与D组相比，B组异位内膜的荧光细胞明显增多，即凋亡细胞数增多，有统计学差异(P＜0.05)。.结论：人参皂甙Rg3能抑制子宫内膜异位症大鼠异位内膜生长，可能通过降低血清E2水平，进而降低局部雌激素含量，使异位内膜萎缩。但对P水平无明显影响，故对受孕无影响。人参皂甙Rg3还能降低VEGF、Akt及mTOR在异位内膜中的的表达，从而抑制VEGFR-2介导的PI3K/Akt/mTOR信号通路，抑制血管新生，诱导异位内膜细胞凋亡。