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.

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.