目的 觀察補(bǔ)腎助孕方對高催乳素血癥（HPRL）模型大鼠生殖功能的影響，并探討催乳素受體（PRLR）泛素化在其中的作用機(jī)制。方法 將40只雌性SD大鼠隨機(jī)分為5組，分別為對照組、模型組、溴隱亭（2 mg·kg⁻¹）組、補(bǔ)腎助孕方低、高劑量（9、18 g·kg⁻¹，低劑量為臨床等效劑量）組。大鼠頸部sc甲氧氯普胺40 mg·kg⁻¹，每天1次，連續(xù)20 d，對照組sc給予等量0.9%氯化鈉溶液，制備HPRL模型。造模成功后開始給藥，每天1次，連續(xù)ig給藥30 d，對照組、模型組給予等體積純水。采用蘇木素-伊紅（HE）染色觀察大鼠下丘腦、垂體、卵巢、子宮組織的形態(tài)學(xué)變化；酶聯(lián)免疫吸附（ELISA）測定大鼠血清催乳素（PRL）、促卵泡激素（FSH）、促黃體生成素（LH）、雌二醇（E₂）水平；Western blotting檢測下丘腦吻素（Kisspeptin）、PRLR以及Janus激酶2/信號轉(zhuǎn)導(dǎo)及轉(zhuǎn)錄激活因子5（JAK2/STAT5）信號通路相關(guān)蛋白表達(dá)；實時熒光定量PCR（qRT-PCR）檢測Kisspeptin mRNA表達(dá)水平；免疫熒光共聚焦觀察PRLR與Kisspeptin神經(jīng)元的共定位表達(dá)；免疫共沉淀（Co-IP）檢測PRLR泛素化水平，PRLR與去泛素化酶COP9信號復(fù)合體亞基5‌‌（CSN5）結(jié)合水平。結(jié)果 與對照組比較，模型組大鼠下丘腦神經(jīng)元、垂體、卵巢、子宮組織出現(xiàn)病理性改變；大鼠血清PRL水平顯著上升，F(xiàn)SH、LH和E₂水平顯著下降（P＜0.01）；大鼠下丘腦PRLR、Kisspeptin、CSN5蛋白表達(dá)水平以及磷酸化JAK2和STAT5蛋白水平顯著下降（P＜0.01）；kisspeptin mRNA表達(dá)量顯著降低（P＜0.01）；PRLR泛素化水平升高；PRLR與去泛素化酶CSN5結(jié)合水平顯著下降。與模型組相比，溴隱亭組和補(bǔ)腎助孕方低、高劑量組下丘腦區(qū)神經(jīng)細(xì)胞排列較規(guī)整，胞質(zhì)胞核較清晰，炎癥細(xì)胞數(shù)量有所減輕；溴隱亭組和補(bǔ)腎助孕方高劑量組PRL水平顯著下降，F(xiàn)SH、LH和E₂水平顯著升高（P＜0.05、0.01）；PRLR、Kisspeptin、CSN5蛋白表達(dá)水平以及磷酸化JAK2和STAT5蛋白水平顯著升高（P＜0.05、0.01）；PRLR泛素化水平顯著下降，且PRLR與CSN5的結(jié)合水平上升。結(jié)論 補(bǔ)腎助孕方能夠降低過高的PRL水平，使HPRL大鼠下丘腦中Kisspeptin表達(dá)增加，從而改善生殖軸功能，其機(jī)制可能與其增加PRLR與去泛素化酶CSN5結(jié)合，降低PRLR泛素化水平，提高PRLR介導(dǎo)JAK2/STAT5信號通路表達(dá)有關(guān)，同時也證明該方具有調(diào)節(jié)心（腦）-腎-子宮軸的作用。

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

國家自然科學(xué)基金青年基金項目（82205160）；江蘇省中醫(yī)藥管理局面上項目（MS2021103）