目的 观察补肾助孕方对高催乳素血症（HPRL）模型大鼠生殖功能的影响，并探讨催乳素受体（PRLR）泛素化在其中的作用机制。方法 将40只雌性SD大鼠随机分为5组，分别为对照组、模型组、溴隐亭（2 mg·kg⁻¹）组、补肾助孕方低、高剂量（9、18 g·kg⁻¹，低剂量为临床等效剂量）组。大鼠颈部sc甲氧氯普胺40 mg·kg⁻¹，每天1次，连续20 d，对照组sc给予等量0.9%氯化钠溶液，制备HPRL模型。造模成功后开始给药，每天1次，连续ig给药30 d，对照组、模型组给予等体积纯水。采用苏木素-伊红（HE）染色观察大鼠下丘脑、垂体、卵巢、子宫组织的形态学变化；酶联免疫吸附（ELISA）测定大鼠血清催乳素（PRL）、促卵泡激素（FSH）、促黄体生成素（LH）、雌二醇（E₂）水平；Western blotting检测下丘脑吻素（Kisspeptin）、PRLR以及Janus激酶2/信号转导及转录激活因子5（JAK2/STAT5）信号通路相关蛋白表达；实时荧光定量PCR（qRT-PCR）检测Kisspeptin mRNA表达水平；免疫荧光共聚焦观察PRLR与Kisspeptin神经元的共定位表达；免疫共沉淀（Co-IP）检测PRLR泛素化水平，PRLR与去泛素化酶COP9信号复合体亚基5‌‌（CSN5）结合水平。结果 与对照组比较，模型组大鼠下丘脑神经元、垂体、卵巢、子宫组织出现病理性改变；大鼠血清PRL水平显著上升，FSH、LH和E₂水平显著下降（P＜0.01）；大鼠下丘脑PRLR、Kisspeptin、CSN5蛋白表达水平以及磷酸化JAK2和STAT5蛋白水平显著下降（P＜0.01）；kisspeptin mRNA表达量显著降低（P＜0.01）；PRLR泛素化水平升高；PRLR与去泛素化酶CSN5结合水平显著下降。与模型组相比，溴隐亭组和补肾助孕方低、高剂量组下丘脑区神经细胞排列较规整，胞质胞核较清晰，炎症细胞数量有所减轻；溴隐亭组和补肾助孕方高剂量组PRL水平显著下降，FSH、LH和E₂水平显著升高（P＜0.05、0.01）；PRLR、Kisspeptin、CSN5蛋白表达水平以及磷酸化JAK2和STAT5蛋白水平显著升高（P＜0.05、0.01）；PRLR泛素化水平显著下降，且PRLR与CSN5的结合水平上升。结论 补肾助孕方能够降低过高的PRL水平，使HPRL大鼠下丘脑中Kisspeptin表达增加，从而改善生殖轴功能，其机制可能与其增加PRLR与去泛素化酶CSN5结合，降低PRLR泛素化水平，提高PRLR介导JAK2/STAT5信号通路表达有关，同时也证明该方具有调节心（脑）-肾-子宫轴的作用。

Objective To investigate the effects of Bushen Zhuyun Decoction (BZD) on the reproductive function of HPRL rat model and the mechanism of PRLR ubiquitination involved in it. Methods Forty female SD rats were randomly divided into five groups, including the normal control group, the high prolactin model group, the bromocriptine group, the low-dose herbal medicine group, and the high-dose herbal medicine group. The model was established by sc metoclopramide 40 mg·kg⁻¹in the neck of rats once a day for 20 d. Equal volume of 0.9% NaCl solution was given to the control sc. After successful modeling, the drug was started to be administered once a day for 30 d consecutive ig administration, and equal volume of pure water was given to the control and model groups. The morphological changes of the hypothalamus, pituitary gland, ovary, and uterus tissues were observed by hematoxylin-eosin (HE) staining. The levels of PRL, FSH, LH, and E₂ in the blood were determined by enzyme-linked immunosorbent assay (ELISA). The expression of Kisspeptin, PRLR, and JAK2/STAT5 signaling pathway-related proteins in the hypothalamus were detected by Western blotting. The expression level of Kisspeptin mRNA was detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The co-localization expression of PRLR and Kisspeptin neurons was observed by immunofluorescence confocal microscopy. The level of PRLR ubiquitination was detected by immunoprecipitation (Co-IP), and the binding level of PRLR with deubiquitinase CSN5 was detected. Results Compared with the normal control group, Model group of rats showed pathological changes in hypothalamic neurons, pituitary gland, ovary, and uterine tissues; Significant increase in serum PRL levels in rats, FSH, LH and E₂ levels decreased significantly (P < 0.01); The expression levels of PRLR, Kisspeptin, CSN5 and phosphorylated JAK2 and STAT5 in rats (P < 0.01); kisspeptin mRNA Expression level was significantly reduced (P < 0.01); The PRLR ubiquitination level is increased; PRLR binds at a significantly less significant level to the deubiquitinating enzyme CSN5. Compared with the model group, nerve cells in the hypothalamic region were organized at low and high doses, and the number of inflammatory cells decreased; PRL decreased and FSH, LH and E₂ increased (P < 0.05, 0.01); PRLR, Kisspeptin, CSN5 protein expression levels and phosphorylated JAK2 and STAT5 levels increased (P < 0.05, 0.01); PRLR ubiquitination decreased, and PRLR binding to CSN5 levels increased. Conclusion BZD can reduce the high PRL level and increase the expression of Kisspeptin in the hypothalamus of HPRL rats, improving the reproductive axis function. The mechanism may be related to the increase of PRLR and the deubiquitinating enzyme CSN5, reduce the level of PRLR ubiquitylation, and improve the expression of PRLR to mediate the JAK2/STAT5 signaling pathway, and also prove the function of regulating the cardiac (brain) -kidney-uterine axis.

R285.5

国家自然科学基金青年基金项目（82205160）；江苏省中医药管理局面上项目（MS2021103）