The Research On The Abnormal Expression Of SCF/c-kit In Ovarian Granulosa Cells Induced By Hyperandrogen And Mechanism Of Ziyin Decoction Intervention

Objective:SCF regulates proliferation and apoptosis of ovarian cells,participates in recruitment and growth of follicles by binding with its receptor c-kit and is abnormally expressed in blood and follicular fluid in PCOS patients,which was reported to cause different results with much controversy and might be related to highly heterogeneous of PCOS phenotypes.The Zinyin-Decoction of traditional Chinese medicine is an effective empirical formula for clinical treatment of PCOS.This research,taking the ovarian local regulatory factor SCF/c-kit as the breakthrough point,was aimed to study the abnormal expression of SCF/c-kit in antral follicles'granulosa cells of hyperandrogen-induced PCOS,to discuss the impacts of Zinyin-Decoction(ZYD)medicated serum on SCF/c-kit expression in granulosa cells via the PI3K/AKT signal pathway,and to further explain the mechanism of ZYD in improving ovarian dysfunction induced by hyperandrogen.Methods:Experiment 1:Injection with DHEA was used to created models of female rats with PCOS.A total of25 female rats(23d)were randomly divided into five groups,including the vehicle control,2-wk DHEA,4-wk DHEA,6-wk DHEA,and 8-wk DHEA,injected with vehicle or DHEA daily for 2,4,6,and 8 weeks respectively and mated at 80d.ELIS A was adopted to test the concentrations of serum hormone FSH,LH,T and E2.Vaginal opening time and estrous cycle changes were observed.Data,including mating rate,pregnancy rate,number of offspring and post-weaning survival amounts,was collected.The PCOS rat model,induced by hyperandrogen,was constructed and optimized to explain the impacts of adrenache dysfunction on gonadarche.Experiment 2:The above mentioned method was used to construct PCOS models.IHC was adopted to observe position of SCF in PCOS ovarian cells and sub-cells.ZYD of 10%,ZYD of 20%,FSH and FSH+ZYD of 10%were respectively administered in vitro culture of granulosa cells for 24h,48h and 72h.We detected supernate concentrations of SCF and E2 by ELISA.Western Blot and real time RT-PCR were used to observe the differences of SCF protein and mRNA expression in different groups and determine SCF,c-kit,p-AKT protein and mRNA expressions by blocking PI3K with LY294002.Results:Experiment 1:(1)The T level was elevated after 2 weeks of DHEA injection,fell to normal after drug withdrawal in the 2-wk and 4-wk DHEA groups,and remained at a high level in the 6-wk group.(2)The E2 level was markedly increased in at 2 and 4 weeks but showed a falling trend at 6 and 8 weeks.(3)The vaginal opening time was significantly advanced in the 2-wk DHEA group as compared with the controls([26.60±0.89]d vs[35.20±2.49]d,P<0.01).(4)The estrous cycle exhibited no significant change until the 4th week(86.67%vs 20.00%,P<0.05)and returned to normal with drug withdrawal in the 2-wk and 4-wk groups,but not in 6-wk and 8-wk groups.(5)Compared with the oil controls,the rats in the 6-wk and 8-wk groups showed a dramatically lower number of offspring(10.2± 1.48 vs 6.40±1.14 and 6.60±1.52,P<0.01)as well as a lower survival rate of the offspring(9.40±1.67 vs 5.60±0.55 and 5.40±1.67,P<0.01).However,no statistically significant difference were found in the rates of mating and pregnancy among the five groups.Experiment 2:(1)In DHEA-induced PCOS-like antral follicle granule cells,secretions of SCF and E2 were decreased(P<0.05),and expressions of SCF,c-kit proteins and mRNA were significantly lowered(P<0.05).(2)Intervention with 10%ZYD,2 0%ZYD and FSH significantly increased 24-72h supernate SCF and E2 concentrations(P<0.05)and elevated expressions of SCF,c-kit proteins and mRNA(P<0.05).(3)The intervention with FSH+10%ZYD better facilitated the expression of SCF mRNA than simple FSH intervention(P<0.05),but didn't increase the expression of SCF.(4)ZYD promoted SCF/c-kit expression in granulosa cells by regulating and controlling the PI3K/AKT signal pathway.Conclusion:(1)Extending the time of DHEA injection to six weeks optimized and constructed the PCOS rat models.Prepubertal exposure to hyperandrogen may advance adrenarche and gonadarche and affect the initiation of the sexual gland function in female rats.(2)Hyperandrogen induced decreased SCF secretion of antral follicle granulosa cells and the Ziyin-Decoction regulated and controlled the expression of SCF/c-kit via the PI3K/AKT signal pathway and,therefore,improved adverse impacts of hyperandrogen on granulosa cells and promoted the development of PCOS antral follicles.