血管紧张素受体-脑啡肽酶双重阻滞对雌性高血压性行为和下生殖道重塑的影响及机制

The prevalence of sexual dysfunction was higher in women with hypertension than in normotensive women in reproductive age. Our previous research indicated that irbesartan, an angiotensin Ⅱ receptor 1 blocker (ARB), could improve female sexual function in some aspects, but not the total score of female sexual function. We also found that renin angiotensin system (RAS) were out-of -balance in female hypertensive rats. We hypothesis that a new type of agent LCZ696, which is an angiotensin Ⅱ receptor-neprilysin inhibitor, would have more positive effects on female sexual function, by enhancing blood flow responses of vagina and clitoris in sexual arousal and ameliorate oxidative stress and fibrosis through inhibition of the RAS in vagina and clitoris. Female reproductive-age spontaneously hypertensive rats (SHR) randomized into treatment with LCZ696, thiazide diuretic chlortralidone, LCZ696 + chlortralidone + Mas receptor inhibitor A779 or vehicle for 12 weeks. Normotensive control Wistar Kyoto (WKY) rats receive no treatment. Sexual behavior will be evaluated using animal ethological methods. Vagina and clitorises will be processed for immunohistochemistry for detecting the index of fibrosis. Observation of clitoral and vaginal smooth muscle cell morphology will be performed by microscopy. To reveal the mechanism, we intend to using Western Blot and Real time-PCR for detection of RAS components expression, and the levels of indicators for oxidative stress including eNOS dipolymer and nNOS. We will using chemiluminescence method to measure the NO bioavailability. In addition, The gene expression of a-SMA, collagen I and III in the fibroblasts will be measured , after the cells be isolated from vagina wall of female WKY rats, and treated with Ang II and LCZ696 or A779. The expression of ERK and p-ERK protein in vagina fibroblasts will be measured to indentify the action of MAPK signal transduction pathway. Through the above methods, this study intends to provide experimental evidences for the mechanism of female sexual dysfunction, and sexual function protection effects from angiotensin Ⅱ receptor-neprilysin inhibitor in reproductive-age women with hypertension.

高血压可致育龄女性性功能障碍，我们前期研究发现雌性高血压阴道肾素血管紧张素系统（RAS）失衡，血管紧张素受体阻滞剂可改善高血压女性性功能的某些层面。推测血管紧张素受体-脑啡肽酶双重阻滞剂LCZ696通过增加阴道阴蒂血流、抑制纤维化，更有益女性性功能。本项目拟用雌性育龄自发性高血压大鼠，从行为学、生理、组织、细胞、分子多层面研究LCZ696对其性功能和下生殖道重塑的影响。评估性行为，激光多普勒测盆神经刺激下阴道血流，用免疫组化、PCR、Western Blot等方法测阴道阴蒂纤维化指标平滑肌肌动蛋白、转化生长因子β1、Col系列胶原，RAS表达ATl、AT2和MasR等，及氧化应激指标一氧化氮和其合酶活性等。并分离大鼠阴道成纤维细胞研究LCZ696能否降低胶原合成，及MAPK/ERK通路是否参与。对阐明女性高血压性功能障碍机制有意义，为育龄高血压女性性功能保护新的治疗方法提供依据。

本项目用雌性育龄自发性高血压（SHR）大鼠，从行为学、生理、组织、分子多层面研究LCZ696（沙库巴曲缬沙坦）对性行为和下生殖道结构重塑和纤维化的影响。16周龄雌性SPF级SHR大鼠随机分①LCZ696组：LCZ696 70mg/kg.day；②缬沙坦组：SHR雌鼠给予缬沙坦30mg/kg/day。③利尿剂组：氯噻酮8mg/kg.day；④SHR对照组：SHR给予生理盐水灌胃。另设立血压正常对照组： 16周龄雌性SPF级WKY大鼠，给予生理盐水灌胃。干预12周后，行心脏彩超检查，使用阴道涂片细胞学检查大鼠动情周期，比较各组动情周期时间，并于动情期采血、行性行为评估，进行手术，分离左心室肌、阴道及外阴组织，行realtime-PCR及western blot检测RAS各组分包括ATl、AT2和MasR等的表达，用免疫组化、PCR、western Blot等方法测阴道阴蒂纤维化指标α-SMA、TGF β1、Col系列胶原等表达。为阐述机制，用western Blot测定氧化应激指标NOS活性、NFκB P65/P38通路表达，及用PCR测定炎症因子IL-6和IL-1βmRNA表达。结果：雌性SHR其发情周期缩短，雌激素水平下降，阴蒂腺、下生殖道细动脉和小动脉以及尿道旁勃起组织已发生形态学改变：血管重塑及纤维化过程较WKY突出，阴蒂腺发生类似于老年化改变。其阴道eNOS和nNOS活性下降，Col I 、Col III及αSMA、TGFβ1表达增加，P38和NFκB的表达明显高于WKY组。和缬沙坦、氯噻酮相比，LCZ696组降低收缩压的幅度更显著；LCZ696组雌性SHR大鼠发情周期延长更显著，雌激素水平上升，性行为参数改善更明显。LCZ696组eNOS和nNOS活性升高更多，Col I 、Col III表达下降更明显。LCZ696组和缬沙坦组的P38和NFκB的表达均下降，但两组间没有明显差异。LCZ696组的炎症因子IL-6和IL-1β的mRNA比SHR组降低，其中IL-6mRNA水平与缬沙坦组相当，而 IL-1β mRNA水平比缬沙坦组减低。结论：雌性SHR大鼠下生殖道组织发生血管重塑和纤维化改变，这一过程与心肌组织类似。LCZ696可通过NFκB P65/P38通路、抑制氧化应激和降低炎症因子水平，改善雌性SHR大鼠下生殖道血管重塑和纤维化。