Mechanism Study Of Bushen Culuan Decoction In Treating Premature Ovarian Insufficiency By Inhibiting Granulosa Cell Apoptosis

BackgroundPremature ovarian insufficiency(POI)is a clinical syndrome defined by ovarian function loss before the age of 40 years.POI is characterized by menstrual disturbance with raised gonadotrophins(fundamental FSH>25U/L)and low estradiol.Hormone replacement therapy(HRT)is the standard treating management recommended by European Society of Human Reproduction and Embryology(ESHRE).HRT has great benefit in relieving clinical discomfort and preventing long-term complications.No matter what the cause of POI is,the mechanism involves apoptosis of ovarian granulosa cells(GCs),and then initiate abnormal follicular atresia and ovarian function damage.This shows that inhibiting GC apoptosis is an important way to protect ovarian function.Bone morphogenetic proteim-7(BMP-7),a homodimer of glycoprotein,is one member of transforming growth factor p superfamily.There were studies proved that BMP-7 had strong anti-apoptotic function of GCs,but it was unclear about the signal transferring pathway.There is lack of study about traditional Chinese medicine(TCM)treating POI or any disease in female reproductive system by regulating BMP-7,therefore,figuring out the underlining mechanism is very important to explore a new idea in TCM treating POI.Bushen Culuan Decoction(BCD)was created and refined by my supervisor in clinical work over the past three decades.It’s a TCM prescription which can nourish kidney and activate blood in TCM theory.In previous clinical study it was proved that BCD could improve serum sex hormone indexes,increase antral follicle number and relief discomfort from estradiol level decline in POI.In aim to make sure the action mechanism of BCD in treating POI,we use ovarian GC as study object to explore the action mechanism of BCD in treating POI.ObjectiveThis research contained in vivo and in intro studies.In vivo study used tripterygium wilfordii polyglycoside(TWP)gavage to establish POI mouse model,then Culuan Decoction middle dose group(BCDM group),Bushen Culuan Decoction low dose group(BCDL group)and estradiol valerate group(EV group).GCs were added by relevant medicated serums and cultured.The proliferating rate,apoptosis rate and BMP-7,caspase-3 protein expressions of GCs were tested.In vitro ovarian granulosa cells were divided into 6 groups:BCD medicated serum group(BCD group),Smad pathway inhibiting group(anti-Smad group),p38 MAPK pathway inhibiting group(anti-p38 MAPK group),NF-κB pathway inhibiting group(anti-NF-κB group),PI3K pathway inhibiting group(anti-PI3K group)and PKA pathway inhibiting group(anti-PKA group).GCs in every group were added medicated serum with or without relevant pathway inhibitors and cultured.BMP-7 and caspase-3 mRNA and protein expression level were tested after GCs collection.ResultsⅠ.POI mouse model establishment studyCompared with blank groups.TWP intervening increased irregular estrous cycle in model groups(P<0.01).Compared with BG1,the FSH and LH level were significantly higher(P<0.015 P<0.05),and the E2 level was significantly lower(P<0.05)in model group.TWP decreased the ovarian index and uterus index of the mice in model group(P<0.05).Compared with BG1,there were less primordial follicles(P<0.01),primary follicles,secondary follicles,mature follicles,corpus luteum(P<0.05)and more atretic follicles(P<0.01)in model group.Compared with BF group,MF group had smaller litter size and newborn mice survival count(P<0.05).Ⅱ.pharmacodynamic studyCompared with model group,BC group had significantly less mice with irregular estrous cycles(P<0.01).The ovarian index in BC group was significantly higher than it in model group(P<0.05).Compared with model group,the serum FSH level was significantly higher(P<0.05)and the E2 and AMH level was lower(P<0.05)in BC group.Compared with model group,there were more primordial follicles,primary follicles,secondary follicles,mature follicles,corpus luteum(P<0.05)and less atretic follicles(P<0.05)in BC group.Compared with model group,the apoptotic rate was lower in BC group(P<0.01).Compared with model group,there was significantly higher BMP-7 protein expression(P<0.05)and significantly lower caspase-3 protein expression(P<0.05)in BC group.Ⅲ.In intro studyCompared with model group,there were significantly higher proliferating rate GCs in BCDM group at 96h,120h and 144h(P<0.05).Compared with model group,GCs of BCD group had a significantly lower apoptotic rate(P<0.01).Compared with model group,there were significantly more BMP-7 protein expression(P<0.05)and less caspase-3 protein expression(P<0.05)in BCD group.Compared with BCD group,the BMP-7 mRNA expression level in anti-Smad group and in anti-NF-κB group were significantly lower(P<0.05).Compared with BCD group,the caspase-3 mRNA expression level in anti-Smad group and in anti-NF-κB group were significantly higher(P<0.05).Compared with BCD group,the BMP-7 protein expression level in anti-Smad group,anti-p38 MAPK group and anti-NF-κB group were significantly lower(P<0.05).Compared with BCD group,the caspase-3 protein expression level in anti-Smad group and in anti-NF-κB group were significantly higher(P<0.05).Conclusion1.Gastric gavage with TWP 80 mg/(kg d)for 14 days is a feasible way to establish a POI mouse model.The mouse ovarian functions didn’t recovery after 14 days from stopping TWP intervening.2.Bushen Culuan Decoction could recover mouse serum sex hormone levels,increase developing follicle counts,decrease atretic follicle count.It also could up-regulate BMP-7 protein expression and down-regulate caspase-3 protein expression to decrease granulosa cell apoptosis rate to protect ovarian function.3.Bushen Culuan Decoction could inhibit ovarian granulosa cell apoptosis and protect the cells by up-regulating BMP-7 protein expression and down-regulating caspase-3 protein expression in vitro.The possible regulating signal pathways contain Smad pathway and NF-κB pathway.