Study On Improving Ovarian Dysfunction With Human Umbilical Cord Mesenchymal Stem Cells, Umbilical Cord Plasma And Chinese Herbal Medicine Extract

With the progress of society and the development of medical technology,the average life expectancy of women increases year by year,and perimenopausal syndrome（MPS）will plague more and more women.The decline of ovarian function during perimenopause is the main cause of MPS,but the mechanism of other biological behavioral changes caused by decreased estrogen secretion remains unclear.Hormone replacement therapy（HRT）has been recommended as a first-line treatment regimen for MPS.However,due to the side effects and the risks,the rate of utilization in our country is low,it is particularly important to find other effective treatment regimen.Studies have demonstrated that humans umbilical cord mesenchymal stem cells（HUMSCs）can improve perimenopausal ovarian function in mice,but the mechanism remains unclear.human umbilical cord blood plasma（hUCBP）,as the homologous biological products of HUMSCs,has been the focus of research on its role in improving aging.The screening of effective traditional Chinese medicine（TCM）preparations is the focus of domestic MPS treatment.In this study,two different perimenopausal models with different triggers were selected.Different formulations were administered for treatment to observe their effects on improving ovarian function decline and explore the underlying mechanisms.The aim is to provide evidence for finding more effective ways to treat MPS in clinical settings.Objective:1.To investigate whether ovarian function decline in perimenopausal rats is related to the occurrence of ferroptosis.2.To investigate whether HUMSC can improve perimenopausal ovarian function by inhibiting ferroptosis.3.To investigate whether human umbilical cord plasma can improve ovarian function in perimenopausal period,and the mechanism is related to inhibiting ferroptosis.4.To explore whether Chinese herbal extracts A and B can improve ovarian function in perimenopausal period.Methods:Part I:Study on the improvement of perimenopausal ovarian function by HUMSCs.To establish an animal model of perimenopause caused by two causes.Perimenopausal model of natural aging:SD rats aged 14-15 months with estrous cycle disorder were randomly divided into model group and treatment group(.Normal 3-month-old SD rats were used as control group.The treatment group was injected with 1mL HUMSCs（1×106cells/mL）through caudal vein every day,and the control group and model group were injected with equal dose of normal saline.Vcd-induced perimenopausal mouse model:8-week-old female ICR mice were selected.Model group and treatment group were given 160mg/（kg·d）ip×20d of 4-vinylcyclohexene diepoxide（VCD）,and randomly divided into model group and treatment group.Mice aged 8 weeks were selected and given equal dose of sesame oil ip×20d as control group.The treatment group was injected with 0.2mL HUMSCs（1×106cells/mL）daily through caudal vein,and the control group and model group were injected with equal dose of normal saline.After 7,14 and 21 days,10 rats and mice in each group were sacrificed randomly.The levels of estradiol（E₂）,anti-mullerian hormone（AMH）and follicle-stimulating hormone（FSH）in serum were detected by enzyme-related immunosorbed assay（ELISA）.The ovarian tissue structure was observed by H&E staining,and the follicle and corpus luteum were counted.The levels of Fe,lipid oxide（MDA）and glutathione（GSH）in ovarian tissue were determined by biochemical method.The expressions of iron death related proteins SLC7A11,DMT1,ACSL4 and GPX4 in ovary were analyzed by Western Blot.Part Ⅱ:Study on the improvement of perimenopausal ovarian function by hUCBP.A total of 120 VCD-induced perimenopausal mice models were established and randomly divided into model group and low,medium and high treatment groups（30 mice in each group）.Thirty normal 8-week-old mice were selected as control group.Mice in the low,middle and high treatment groups were injected 175μL,240μL,300μL/hUCBP through the tail vein on consecutive days,and 10 mice in each group were randomly killed at 7,14 and 21 days after the first injection.The serum levels of E₂,AMH and FSH were detected by ELISA.The ovarian tissue structure was observed by H&E staining,and the follicle and corpus luteum were counted.The Fe,MDA and GSH levels of the ovary were detected by the kit.Part Ⅲ:Study on the improvement of perimenopausal ovarian function by crude extracts of traditional Chinese medicine.A total of 120 VCD-induced perimenopausal mice models were established and randomly divided into model group,low-dose A,medium-dose A and high-dose A groups,and low-dose B,medium-dose B and high-dose B groups（10 mice in each group）.Ten normal 8-week-old mice were selected as control group.Each mouse in low-dose A,medium-dose A and high-dose A groups was given 0.75mg/g,1.5 mg/g and 3.75 mg/g crude extract A by intragastric administration for 28 days.Each mouse in B low,B medium and B high dose treatment groups was given 0.75mg/g,1.5 mg/g and 3.75 mg/g crude extract B through continuous tube for 28 days.All mice were sacrificed one day after the last administration,and E₂,AMH,FSH,LH in serum were detected by ELISA,The tissue structure of ovary and uterus was observed by H&E staining,and follicles and corpus luteum were counted.Results:Part Ⅰ:Study on the pmprovement of perimenopausal ovarian function by HUMSCs.Natural aging perimenopausal model:Compared to the control group,the model group of rats showed extended and disrupted estrous cycles,as well as a decreased ovarian index.The number of primordial and primary follicles significantly decreased,while the number of atretic follicles significantly increased（P<0.05）.Compared to the model group,the treatment group receiving HUMSCs showed shortened and normalized estrous cycles,an increased ovarian index,a significant increase in the number of primordial and primary follicles,and a significant decrease in the number of atretic follicles（P<0.05）.Serum levels of E₂ and AMH significantly increased（P<0.05）and gradually rose over time,while serum FSH levels significantly decreased（P<0.05）and gradually declined over time.Ovarian tissue levels of Fe and MDA significantly decreased（P<0.05）and gradually declined overtime,while GSH levels significantly increased（P<0.05）and gradually rose over time.VCD-induced perimenopausal model:Compared to the control group,the model group of mice showed prolonged and disrupted estrous cycles,a decreased ovarian index,a significant decrease in the number of primordial and primary follicles,and a significant increase in the number of atretic follicles（P<0.05）.Compared to the model group,the treatment group receiving HUMSCs showed shortened and normalized estrous cycles,a significant increase in the number of primordial and primary follicles,and a significant decrease in the number of atretic follicles（P<0.05）.Serum levels of E₂ and AMH in the treatment group significantly increased（P<0.05）and gradually rose over time,while serum FSH levels significantly decreased（P<0.05）and gradually declined over time.Ovarian tissue levels of Fe and MDA in the treatment group significantly decreased（P<0.05）and gradually declined over time,while GSH levels significantly increased（P<0.05）and gradually rose over time.In the natural aging perimenopausal model group of rats,the protein expression levels of SLC7A11,DMT1,and GPX4 in the ovaries significantly decreased compared to the control group,while ACSL4 protein expression level significantly increased（P<0.05）.After receiving HUMSCs,the protein expression levels of SLC7A11,DMT1,and GPX4 in the rat ovaries significantly increased compared to the model group（P<0.05）and showed a gradual increase over time,while ACSL4 protein expression level significantly decreased（P<0.05）and showed a gradual decline over time.Part Ⅱ:Study on the improvement of perimenopausal ovarian function by hUCBP.Compared to the control group,the VCD-induced perimenopausal model group of mice showed prolonged and disrupted estrous cycles,a decreased ovarian index,a significant decrease in the number of primordial and primary follicles,and a significant increase in the number of atretic follicles（P<0.05）.Compared to the model group,the treatment group receiving hUCBP showed shortened and normalized estrous cycles;a significant increase in the number of primordial and primary follicles（P<0.05）,which showed a gradual increase with increasing treatment frequency and dosage;a significant decrease in the number of atretic follicles（P<0.05）,which showed a gradual decline with increasing treatment duration and dosage;serum levels of E₂ and AMH significantly increased（P<0.05）,which showed a gradual increase with increasing treatment duration and dosage;serum FSH levels significantly decreased（P<0.05）,which showed a gradual decline with increasing treatment duration and dosage;ovarian tissue levels of Fe and MDA in the treatment group significantly decreased（P<0.05）,which showed a gradual decrease with increasing treatment frequency,duration,and dosage;GSH levels significantly increased（P<0.05）,which showed a gradual increase with increasing treatment frequency,duration,and dosage.Part III:Study on the improvement of perimenopausal ovarian function by crude extracts of traditional Chinese medicine.Compared to the model group,mice treated with crude extract of Traditional Chinese Medicine A at low,medium,and high doses showed shortened and normalized estrous cycles.The number of follicles at various stages and corpora lutea increased,while the number of atretic follicles significantly decreased,with the lowest number observed in the medium-dose group（P<0.05）.Serum levels of E₂ and AMH significantly increased（P<0.05）,with the highest levels observed in the mediumdose group.Serum levels of FSH and LH significantly decreased（P<0.05）,with the lowest levels observed in the medium-dose group.There were no significant changes in uterine endometrial thickness.Compare to the model group,mice treated with crude extract of Traditional Chinese Medicine B at low,medium,and high doses also showed shortened and normalized estrous cycles.The number of follicles at various stages and corpora lutea increased,while the number of atretic follicles significantly decreased（P<0.05）,with a gradual decrease observed with increasing dosage.Serum levels of E₂ and AMH significantly increased（P<0.05）,with a gradual increase observed with increasing dosage.Serum levels of FSH and LH significantly decreased on average（P<0.05）,with a gradual decrease observed with increasing dosage.There were no significant changes in uterine endometrial thickness.Conclusion:1.The decline in ovarian function during the perimenopausal period caused by natural aging and VCD is associated with the occurrence of ferroptosis.2.HUMSCs may improve perimenopausal ovarian function by inhibiting ferroptosis.3.hUCBP may improve perimenopausal ovarian function by inhibiting ferroptosis.4.Chinese herbal extracts A and B can improve perimenopausal ovarian function.