基于VEGF与血管形成在卵泡发育中的作用研究四物汤改善卵巢早衰的机制

Premature ovarian insufficiency is one of the most important as well as urgent problem which must be solved first in infertility treatment. In the light of the discussion about the relationship between the fecundity and the wax and wane of pulse in the Huangdi Neijing, as well as the fact that Siwu Decoction, as the basic recipe of regulating menstruation, has good effects in clinical application, combined with modern medical theory that ovarian microenvironment, angiogenesis and related growth factors play significant role in follicle formation and development, a hypothesis is suggested as below: Siwu Decoction may improve microcirculation locally, promote angiogenesis in ovary, optimize the ovarian microenvironment, induce the proliferation and differentiation of granulosa cells, and contribute to the follicular recruitment, dominance and ovulation, so as to achieve the purpose of regulating menstruation cycle and improving fertility in the end. In this study, the mice model of premature ovarian failure (POF) induced by cyclophosphamide serves as the research object. The methods of morphological analysis, molecular biology, immunohistochemistry are applied to detect the number of different stages of follicles, the proliferation of vascular endothelial cell, the vascular distribution and microvessel density, the expression and location of related factors as well as their receptors such as VEGF, GDF-9, BMPs and so on, and the apoptosis of granulosa cells along with the levels of pro- and anti-apoptotic proteins in ovary. In addition, the effects of the mediated serum of Siwu Decoction on cell proliferation, apoptosis and activation of the PI3K/AKT and MEK/ERK signaling pathways in ovarian vascular endothelial cells, early antral follicles or granulosa cells which injured by 4-hydroperoxycyclophosphamide are studied in vitro. This study is to investigate the effects of Siwu Decoction on angiogenesis, follicular development and the survival and proliferation of ovarian granulosa cells, to lay the foundation for clinical application of Siwu Decoction in regulating menstruation and infertility treatment, and to provide experimental basis for combining traditional Chinese medicine with modern reproductive medicine in the therapy of infertility.

卵巢储备功能不足是治疗不孕症一开始就必须面对的棘手问题。基于《内经》中生育功能与“脉盛脉衰”关系的论述，及四物汤作为调经打底方的临床事实，结合现代医学卵巢微环境、血管形成及其相关生长因子在卵泡发育中的重要意义，提出假说：四物汤可能改善微循环，促进血管形成，优化卵巢微环境，诱导颗粒细胞的增殖与分化，有助于卵泡的募集和优选，从而改善卵巢功能。将以环磷酰胺诱导卵巢早衰模型小鼠为研究对象，应用形态学定量、分子生物学、免疫组化等技术，在体部分观察卵巢中血管分布及密度、各级卵泡计数以及内皮细胞的增殖，卵巢内VEGF、HIF、GDF-9、BMPs 及其受体的表达，颗粒细胞凋亡及相关蛋白等指标，体外实验观察四物汤含药血清对卵巢来源的内皮细胞、窦卵泡和颗粒细胞的PI3K/AKT信号通路及凋亡、增殖的影响，验证假说，为四物汤作为调经、助孕的打底方，以及传统中医更好地与现代生殖医学相结合治疗不孕症提供实验依据。

由于社会、经济因素的变化，女性生育年龄不断推迟，由此带来的与生育相关的临床问题，如卵巢储备功能不足、卵巢早衰（Primary Ovary Failure，POF）、不孕症等显著增加。国家“二胎”、“三胎”政策的开放以来，高龄女性加入再育行列。 女性生育能力与年龄密切相关，37岁以后生育力显著下降，出现所谓的“折棍现象”，因此改善高龄不孕症患者的卵巢功能是解决不孕症和响应国家政策需要解决的首要问题。四物汤（Si Wu Tang, SWT）被称为“妇科第一方”，广泛用于月经病和不孕症的治疗，然而SWT治疗POF的潜在作用机制尚不明确。本研究采用高效液相色谱法（HPLC）对SWT主要成分进行了鉴定，使用C57/BL6小鼠单次腹腔注射环磷酰胺100 mg/kg建立POF模型，SWT或DHEA灌胃治疗，连续28天。观察小鼠的卵巢功能、激素水平、卵泡发育、血管生成、抗氧化能力的变化，并对生育能力进行了统计分析。体外实验部分使用4-过氧氢环磷酰胺处理人卵巢颗粒细胞系KGN，观察SWT对颗粒细胞增殖、凋亡的影响。结果表明SWT可以显著改善POF小鼠的卵巢功能，提高AMH和雌激素水平；调控卵巢局部卵泡发育因子，并激活PI3K/AKT/FOXO1信号通路，减少颗粒细胞凋亡，以促进卵泡发育；SWT治疗也显著提高了POF小鼠卵巢微血管的形成，激活了STAT3/HIF-1α/VEGF信号通路；此外SWT通过激活Nrf2/HO-1通路提高了POF小鼠的抗氧化能力，并带来了更好地生育结局。血管生成对卵泡发育和排卵，及黄体功能维持十分重要。我们的研究结果证实了SWT通过促进VEGF与血管形成而改善卵巢功能，同时可以调节卵巢局部卵泡发育因子、抑制颗粒细胞凋亡，发挥保护POF小鼠生育能力的作用，这一发现，不仅为临床应用SWT治疗POF提供了科学依据，也对POF的论治提供了新的思路。