目的 探讨人参固本口服液联合荆防颗粒对少弱精子症小鼠的生殖保护作用，并运用蛋白质组学探究其作用机制。方法 将小鼠随机分为对照组，模型组，人参固本口服液低、中、高剂量（0.1、0.2、0.4 mL/只）组，荆防颗粒（4 g/kg）组和人参固本口服液（0.4 mL/只）联合荆防颗粒（4 g/kg）组，每组10只。采用奥硝唑诱导建立少弱精子症小鼠模型，连续造模同时给药56 d后麻醉处死。采用全自动精子分析仪检测精子数量及精子活动指标；采用苏木素-伊红（hematoxylin-eosin，HE）染色观察小鼠睾丸、附睾组织病理变化；ELISA检测小鼠血清中性激素水平；微量法检测小鼠睾丸组织中谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）、超氧化物歧化酶（superoxide dismutase，SOD）活性；应用蛋白质组学技术分析小鼠睾丸组织中蛋白种类和表达情况，筛选差异表达蛋白及其相关蛋白信号通路；采用Western blotting检测差异表达蛋白表达。结果 与模型组比较，各给药组可明显提高精子数量和精子活动指标（P＜0.05、0.01、0.001），显著增强睾丸组织中GSH-Px活性（P＜0.01、0.001）。其中，人参固本口服液联合荆防颗粒组可显著增加睾丸和附睾指数（P＜0.001），显著增加精子数量（P＜0.01），显著改善睾丸、附睾组织病理状态，显著回调血清中促卵泡生成素（follicle-stimulating hormone，FSH）、黄体生成素（luteinizing hormone，LH）、雌二醇（estradiol，E₂）、睾酮（testosterone，T）水平（P＜0.01、0.001）。蛋白质组学结果表明，人参固本口服液联合荆防颗粒治疗少弱精子症主要与氨基酸代谢、碳代谢、脂肪酸代谢、过氧化物酶体增殖物激活受体（peroxisome proliferator-activated receptor，PPAR）信号通路、磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）/蛋白激酶B（protein kinase B，Akt）信号通路有关。Western blotting结果显示，人参固本口服液联合荆防颗粒组可显著提高睾丸组织中p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR蛋白表达水平（P＜0.05、0.01）。结论 人参固本口服液联合荆防颗粒可协同治疗少弱精子症，能够提高精子数量及精子活动指标，改善性激素紊乱、氧化应激，减轻睾丸、附睾病理状态，其作用机制可能与激活PI3K/Akt/mTOR信号通路有关。

Objective To explore the reproductive protection effect of Renshen Guben Oral Liquid (人参固本口服液) combined with Jingfang Granules (荆防颗粒) on oligoasthenospermia mice and explore its mechanism by proteomics. Methods The mice were randomly divided into control group, model group, low, medium- and high-dose (0.1, 0.2, 0.4 mL/mice) groups of Renshen Guben Oral Liquid, Jingfang Granules (4 g/kg) group, Renshen Guben Oral Liquid (0.4 mL/mice) combined with Jingfang Granules (4 g/kg) group, with 10 mice in each group. The mouse model of oligoasthenospermia was induced by ornidazole, and the mice were sacrificed by anesthesia after 56 d of continuous modeling and administration. The number of sperm and its activity index were detected by fully automatic sperm analyzer. The pathological changes of testis and epididymis were observed by hematoxylin-eosin (HE) staining. Serum sex hormone levels in mice were detected by ELISA. The activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in mouse testis were detected by micro method. Proteomics technology was used to analyze the types and expression of proteins in mouse testis, screen differentially expressed proteins and their related protein signaling pathways. The expressions of differentially expressed proteins were detected by Western blotting. Results Compared with model group, each administration group could significantly increase the number of sperm and sperm activity indexes (P < 0.05, 0.01, 0.001), significantly enhance the activity of GSH-Px in testicular tissue (P < 0.01, 0.001). Among them, Renshen Guben Oral Liquid combined with Jingfang Granules group could significantly increase the testis and epididymis indexes (P < 0.001), significantly increase the number of sperm (P < 0.01), significantly improve the pathological state of testis and epididymis, significantly callback the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E₂) and testosterone (T) in serum (P < 0.01, 0.001). Proteomics results showed that the therapeutic effect of Renshen Guben Oral Liquid combined with Jingfang Granules on oligoasthenospermia was mainly related to amino acid metabolism, carbon metabolism, fatty acid metabolism, peroxisome proliferator-activated receptor (PPAR) signaling pathway and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Western blotting results showed that Renshen Guben Oral Liquid combined with Jingfang Granules could significantly increase the expression levels of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/ mTOR proteins in testicular tissue (P < 0.05, 0.01). Conclusion Renshen Guben Oral Liquid combined with Jingfang Granules could synergistically treat oligoasthenospermia, increase sperm count and activity indexes, improve sex hormone disorder, oxidative stress, and reduce the pathological state of testis and epididymis. The mechanism may be related to the activation of PI3K/Akt/mTOR signaling pathway.

R285.5

山东省泰山产业领军人才工程(tscx202312104)