Therapeutic effects and mechanism of human umbilical cord mesenchymal stem cells combined with melatonin on premature ovarian insufficiency induced by chemotherapy

doi:10.12307/2025.518

Abstract

Abstract: BACKGROUND: Current research has shown that human umbilical cord mesenchymal stem cells and melatonin can both improve ovarian function. However, the protective effect and mechanism of human umbilical cord mesenchymal stem cells combined with melatonin on chemotherapy-induced premature ovarian insufficiency have not been reported.
OBJECTIVE: To investigate the protective effect and mechanism of human umbilical cord mesenchymal stem cells combined with melatonin on chemotherapy-induced premature ovarian insufficiency.
METHODS: Forty Wistar rats with normal estrous cycle were randomly divided into control group, model group, human umbilical cord mesenchymal stem cell group, melatonin group, and human umbilical cord mesenchymal stem cell+melatonin group, with 8 rats in each group. In addition to the control group, the rat model of premature ovarian insufficiency was constructed by intraperitoneal injection of cisplatin solution. The rats in the human umbilical cord mesenchymal stem cell group and the human umbilical cord mesenchymal stem cell+melatonin group were injected with 1×106 human umbilical cord mesenchymal stem cells in the tail vein at 1, 6, and 11 days after modeling, respectively. The rats in the melatonin group and human umbilical cord mesenchymal stem cell+melatonin group were injected intraperitoneally with 10 mg/kg melatonin daily. The changes in body mass and estrous cycle of rats were monitored during treatment. After 14 days of treatment, ELISA was used to detect serum levels of estradiol, follicle stimulating hormone, luteinizing hormone, and anti Mullerian hormone. Ovarian index was calculated. Hematoxylin-eosin staining was performed to observe ovarian histological morphology. Ultrastructure of ovarian granulosa cells was observed by transmission electron microscopy. Western blot assay was conducted to detect the expression of PI3K/AKT/mTOR signaling pathway proteins and LC3 and P62 autophagy proteins in ovarian tissues.
RESULTS AND CONCLUSION: (1) Compared with the control group, the body mass of rats in the model group decreased gradually, the estrous cycle was disturbed, and the ovarian index decreased significantly (P < 0.01). The levels of estradiol and anti-Mullerian hormone were decreased (P < 0.01), while the levels of follicle stimulating hormone and luteinizing hormone were increased (P < 0.01). The histological morphology and granulosa cell ultrastructure of ovary were seriously damaged. The protein expressions of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, and P62 were significantly decreased (P < 0.01), while the protein expressions of LC3II/I were significantly increased (P < 0.001). (2) Compared with the model group, the body mass of rats in all treatment groups recovered gradually, the estrous cycle of some rats returned to normal, and the ovarian index was significantly increased (P < 0.01); the levels of estradiol and anti-Mullerian hormone were increased (P < 0.05), while the levels of follicle stimulating hormone and luteinizing hormone were decreased (P < 0.05). The histological morphology and ultrastructure of ovary were significantly improved. The protein expressions of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, and P62 were significantly increased (P < 0.001), while the protein expression of LC3II/I was significantly decreased (P < 0.01). Human umbilical cord mesenchymal stem cell+melatonin group showed more significant improvement in the above indexes. The results suggest that human umbilical cord mesenchymal stem cells combined with melatonin may inhibit ovarian granulosa cell autophagy by upregulating PI3K/AKT/mTOR signaling pathway to treat premature ovarian insufficiency.

Key words: premature ovarian insufficiency, human umbilical cord mesenchymal stem cell, melatonin, estrogen, autophagy, PI3K, AKT, mTOR

CLC Number:

Wei Luxiao, Huang Bingxue, Du Jing, Shi Shuanxia, Wang Jitian, Wang Ling. Therapeutic effects and mechanism of human umbilical cord mesenchymal stem cells combined with melatonin on premature ovarian insufficiency induced by chemotherapy[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(25): 5281-5288.

Figures/Tables 6

2.1 人脐带间充质干细胞的分离培养、鉴定和诱导分化结果组织贴壁法分离培养至第7天可见贴壁梭形细胞从组织周围爬出，生长状态良好，见图1A，约15 d时组织周围细胞生长达80%以上，见图1B。第3代人脐带间充质干细胞表面抗原CD44、CD73、CD90阳性表达，CD34、CD45、HLA-DR阴性表达，符合国际细胞治疗协会提出的间充质干细胞鉴定标准[13]，说明实验分离培养所得细胞为人脐带间充质干细胞，见图2。成脂诱导分化2周后，显微镜下观察可见脂滴形成，油红O染色后脂滴呈红色，见图3A；成骨诱导分化3周后，显微镜下观察可见钙结节形成，茜素红染色后钙结节呈红色，见图3B；成软骨诱导分化3周后，可见直径约1 mm的软骨球形成，石蜡切片阿利新蓝染色呈蓝色，见图3C。 2.2 大鼠一般情况及动情周期变化对照组大鼠随饲养时间的延长，体质量逐渐增加，精神、饮食、活动状态均无异常。其余组大鼠经过顺铂造模后，出现精神萎靡、进食水减少、活动减少、喜抱团、毛色发黄等情况，部分出现不同程度的腹泻，体质量逐渐下降，于造模第14天体质量达最低水平。经过治疗后，各治疗组大鼠精神好转，进食水及活动增加，毛色变白，体质量逐渐恢复，见图4。性成熟大鼠动情周期为四五天，分为动情前期、动情期、动情后期和动情间期[14]。动情前期主要特征是大量圆形或椭圆形的有核细胞；动情期主要特征是大量不规则片状无核角化细胞；动情后期是动情期向动情间期的过渡，主要特征是有核细胞、无核角化细胞和白细胞同时存在；动情间期主要特征是大量白细胞和少量无核角化细胞[15]，见图5。与对照组比较，模型组大鼠动情周期紊乱，表现为动情周期延长，停滞在动情间期或动情后期，部分大鼠阴道涂片时偶见血性分泌物。经过14 d治疗后，人脐带间充质干细胞组和褪黑素组动情周期正常大鼠占37.5%，人脐带间充质干细胞+褪黑素组动情周期正常大鼠占50%，而模型组无动情周期正常大鼠，表明人脐带间充质干细胞联合褪黑素治疗可使早发性卵巢功能不全大鼠动情周期恢复正常。 2.3 大鼠卵巢指数的变化与对照组比较，模型组卵巢指数显著下降(P < 0.01)；与模型组比较，各治疗组卵巢指数均显著增加(P < 0.01)，其中人脐带间充质干细胞+褪黑素组差异更为显著(P < 0.001)；与褪黑素组比较，人脐带间充质干细胞+褪黑素组卵巢指数增加更为明显(P < 0.05)，表明人脐带间充质干细胞联合褪黑素治疗可明显改善卵巢萎缩，增加卵巢指数，见图6。 2.4 大鼠血清性激素水平的变化与对照组比较，模型组雌二醇、抗缪勒管激素水平降低(P < 0.01)，卵泡刺激素、促黄体生成素水平升高(P < 0.01)；与模型组比较，各治疗组雌二醇、抗缪勒管激素水平均升高(P < 0.05)，卵泡刺激素、促黄体生成素水平均降低(P < 0.05)；与人脐带间充质干细胞组和褪黑素组比较，人脐带间充质干细胞+褪黑素组雌二醇、抗缪勒管激素水平升高更为明显(P < 0.05)，卵泡刺激素、促黄体生成素水平降低更为明显(P < 0.05)，表明人脐带间充质干细胞联合褪黑素治疗可明显改善大鼠卵巢的内分泌功能，见图7。"

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