The Effects Of Gypenosides In Confronting Age-related And Chemotherapy Induced Ovarian Damage In Double-platform

Objective:To investigate the antagonistic effects of gypenosides on age-related ovarian function decline in drosophila melanogaster.Methods:1)The target gene of gypenosides(GP)was obtained from the TCMSP platform,and it was compared with the genes related to ovarian dysfunction retrieved from Gene Cards and OMIM databases.The related bioinformatics analysis was performed based on the overlapping target genes,including Gene Ontology and KEGG pathway analysis.2)In w1118 drosophila melanogaster,total 400 drosophila were divided into control group,GP treatment group(1day-death),GP treatment group(1day-25 day),and GP treatment group(25 day-death).The mean,median and maximum lifespan of drosophila in each group were calculated to determine whether GP had an effect on the extension of lifespan in each group of male and female drosophila.At the same time,stress related experiments(high sugar diet and starvation diet)were conducted after 25 days of GP treatment to explore whether GP could prolong healthy lifespan.The number of eggs production and the hatching rate of female drosophila in each group were counted to estimate the ovarian function of each group.Results:1)Gene Ontology analysis showed that the confronting effect of gypenosides was involved in cell proliferation,oxidative stress and steroid hormone synthesis.KEGG Pathway analysis revealed that PI3K-AKT Pathway was the most significant pathway.At same time,FOXO,TNF and Insulin pathways were also found to be involved in the biological regulatory process of ovarian damage,revealing the potential role of GP in improving ovarian injury.2)In order to explore whether GP would affect the healthy condition of drosophila melanogaster,we counted the lifespan changes of both male and female drosophila in each group.Of female drosophila,it was discovered that GP could significantly extend the overall and healthy lifespan.The extension of lifespan as compared with control group,each group with the treatment of GP of females had longer maximum,mean and median lifespan.Among them,the group of 25 days to death could maximize lifespan extension effect as the maximum,mean and median lifespan were extended 4.6%,6.7%,4.7% respectively;The extension of healthy lifespan was manifested in female drosophila that GP treatment could significantly assist to resist stress stimuli(high glucose diet stimulation,starving diet stimulation)after 25days’ administration with GP,and its survival rate and lifespan were improved compared with the control group.3)Of male drosophila,GP treatment during the period from 1 day of eclosion to 25 day could prolong the overall lifespan to the greatest extent,with the maximum and mean lifespan were extended by 3% and 0.8%,respectively.The median lifespan had no prolonging effect,and GP also had little effect on the prolonging of healthy lifespan.4)The number of eggs production is the most representative index to evaluate the ovarian function of female drosophila.Compared with the control group,every group with the treatment of GP by feeding food containing 10mg/ m L of GP could increase the number of eggs production in female drosophila,and the medication time from 25 days after eclosion to death had the most obvious effect in improving the total number of eggs production in the late stage of life cycle.It was also found that the egg hatching rate of female drosophila was significantly improved after 25 days of GP treatment.Conclusion:GP treatment could improve the age-associated decline of ovarian function by increasing the number of eggs producution and the hatchability of female drosophila,and extend the overall lifespan of both male and female drosophila effectively at 1d-25 d and 25d-death respectively,as well as the healthy lifespan of female drosophila.ObjectiveTo investigate the confronting effects of gypenosides on acute ovarian dysfunction induced by chemotherapeutic drug cyclophosphamide in mice.Methods1)The protective effect of GP in ovarian dysfunction induced by cyclophosphamide(CTX)was studied in female C57 mice as mammalian platform.Mice were divided into four groups(control group,NC;Cyclophosphamide group,CTX;Cyclophosphamide + GP lowdose group,G-L;Cyclophosphamide + GP high-dose group,G-H).2)Ovarian index and vaginal secretion smear of every mice in each group before death was observed and counted.Ovarian morphology and the number of follicle counting were performed on H&E staining ovarian tissue sections.The serum concentrations of estradiol(E2)and progesterone(P)of mice were detected to evaluate the damage effect of CTX on ovarian function of C57 mice and whether GP could alleviate ovarian damage.Meanwhile,the fertility of each group was detected by counting mean number of pups and mean number of litters.3)In order to research whether GP would impact the effect of CTX on killing cancer cell,tumor weight and tumor volume were calculated in the MCF-7 tumor-bearing nude mouse(control group,NC;Cyclophosphamide group,CTX;Cyclophosphamide + gypenosides,GP).4)Ovarian function of mice in each group were observed including estrous cycle,ovarian morphology,the number of follicles counting,and the serum concentration of AMH.5)γ-H2 AX,TUNEL staining and Western Blotting were used to study the mechanism of GP in relieving ovarian injury induced by CTX.Results1)In C57 mice,we found that the both high and low dose of GP group could effectively improve the ovarian dysfunction caused by CTX with showing higher ovarian index and regular rates of estrus.By observing H&E staining section,GP group showed significant higher number of primordial follicles,functional follicles,and less number of atretic follicles.Moreover,GP could improve the level of estrogen and progesterone hormone in serum of female mice.For the fertility,GP obviously increased the mean pups and mean number of litters.In summary,the protective effect of high-dose GP was stronger than that of low-dose group.Based on the above results,high-dose GP was used uniformly in subsequent experiments.2)To investigate whether GP would interfere with the killing effect of CTX on tumor cells,further studies were conducted in MCF-7 tumor-bearing mouse.After 28 days of continuous intraperitoneal injection of GP,there was no significant difference in tumor size and tumor weight between the GP group and the CTX group,and the tumor volume and tumor weight were significantly smaller than that of the NC group.3)Compared with CTX group,the ovarian function of GP group was significantly improved,which was manifested as the increased proportion of regular estrous cycles,the enhanced number of functional follicles and the decreased number of atretic follicles.Meanwhile,the concentration of AMH in serum was significantly increased.4)Furthermore,GP could effectively alleviate the impaired ovarian function induced by CTX in tumor-bearing mice by reducing DNA damage,cell apoptosis and inhibiting primordial follicle activation.ConclusionGP could ameliorate acute ovarian injury induced by cyclophosphamide via regulating PI3K/AKT signaling in inhibiting follicle activation,and decreasing DNA damage and cell apoptosis without influencing the anti-tumor effect of CTX.