甘草酸二铵联合人类胎盘间充质干细胞对大鼠卵巢早衰的改善作用

Mechanistic evaluation of diammonium glycyrrhizinate combined with human placental mesenchymal stem cells in experimental rat models of premature ovarian failure

摘要:

目的 探究甘草酸二铵联合人类胎盘间充质干细胞（hPMSCs）对卵巢早衰（POF）的保护作用及机制。方法 将40只动情周期正常的SD雌性大鼠随机分为Control组（空白对照）、POF组（卵巢早衰模型）、hPMSCs组（hPMSCs治疗）及DG + hPMSCs组（甘草酸二铵联合hPMSCs治疗），每组10只。连续7 d腹腔注射环磷酰胺（CTX）诱导复制POF大鼠模型；hPMSCs组和DG+hPMSCs组分别在模型复制第15、22天经大鼠尾静脉注入hPMSCs悬浮液1 mL；DG + hPMSCs组在模型复制第10天起按150 mg/（kg·d）剂量连续腹腔注射甘草酸二铵21 d。采用酶联免疫吸附试验检测大鼠血清性激素、抗氧化酶及炎症因子水平，采用苏木素-伊红（HE）染色对大鼠卵巢组织进行病理学观察，采用Western blotting检测大鼠卵巢功能及炎症相关蛋白的表达。结果 CTX诱导后大鼠动情周期紊乱，POF大鼠模型复制成功。与POF组相比，hPMSCs组及DG + hPMSCs组大鼠血清性激素雌二醇、抗缪勒氏管激素水平均升高（P <0.05），促卵泡生成素、黄体生成素水平均降低（P <0.05）；血清抗氧化因子超氧化物歧化酶、谷胱甘肽过氧化物酶水平均升高（P <0.05）；血清促炎因子白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）及肿瘤坏死因子α（TNF-α）含量均降低（P <0.05）；卵巢组织病理学明显改善；卵巢功能蛋白CCAAT增强子结合蛋白β、细胞外调节蛋白激酶表达均升高（P <0.05），IL-1β、IL-6及TNF-α蛋白相对表达量均降低（P <0.05）；DG + hPMSCs组的作用优于hPMSCs组。结论 甘草酸二铵具有抗氧化和抗炎症作用，联合hPMSCs能改善损伤卵巢组织内的微环境，并增强hPMSCs移植治疗POF的效果。

Abstract:

Objective To investigate the protective effects and underlying mechanisms of diammonium glycyrrhizinate (DG) combined with human placental mesenchymal stem cells (hPMSCs) on premature ovarian failure (POF).Methods Forty female Sprague-Dawley (SD) rats with normal estrous cycles were randomly divided into four groups (n = 10 per group): blank control (Control), POF model (POF), hPMSCs treatment (hPMSCs), and DG combined with hPMSCs treatment (DG + hPMSCs). The POF model was established by intraperitoneal injection of cyclophosphamide (CTX) for 7 consecutive days. On days 15 and 22 of modeling, rats in the hPMSCs and DG + hPMSCs groups received 1 mL of hPMSCs suspension via tail vein injection. Additionally, the DG + hPMSCs group was administered DG (150 mg/kg/day) via intraperitoneal injection from day 10 for 21 consecutive days. Serum levels of sex hormones, antioxidant enzymes, and inflammatory cytokines were measured by ELISA. Ovarian histopathology was evaluated using hematoxylin-eosin (HE) staining, while protein expression related to ovarian function and inflammation was assessed by Western blot (WB).Results Following CTX induction, the estrous cycle was disrupted, confirming successful POF modeling. Compared with the POF group, both the hPMSCs and DG + hPMSCs groups exhibited increased serum levels of estradiol (E₂) and anti-Müllerian hormone (AMH) (P < 0.05), along with decreased follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (P < 0.05). Antioxidant markers, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), were elevated (P < 0.05), while pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) were reduced (P < 0.05). Ovarian histopathology showed significant improvement, with upregulated expression of CCAAT/enhancer-binding protein β (C/EBP-β) and extracellular signal-regulated kinase (ERK), and downregulated IL-1β, IL-6, and TNF-α protein levels (P < 0.05). Notably, the therapeutic effects in the DG + hPMSCs group surpassed those in the hPMSCs group.Conclusion diammonium glycyrrhizinate exerts antioxidant and anti-inflammatory effects, synergizing with hPMSCs to ameliorate the ovarian microenvironment and enhance the therapeutic efficacy of hPMSC transplantation in POF.