Effect Of Bushen-Huoxue Rule On Ovarian Granulosa Cells Apoptosis In Mice Of Premature Ovarian Failure And Controlled Ovarian Hyperstimulation Implantation Dysfunction

Section ?Effect of Bushen-Huoxue Recipe on Cyclophosphamide-induced Premature Ovarian Failure in Mice and Possible MechanismsObjectiveTo investigate the effect of Bushen Huoxue recipe(BHR)on cyclophosphamide(CTX)-induced premature ovarian failure(POF)in mice and possible mechanisms.MethodsPOF mice model was established by a single intraperitoneal injection of CTX.Forty female C57BL/6 mice were randomly divided into four groups:normal control group(NC).CTX-induced POF model group(CTX),BHR group(BHR),dehydroepiandrosterone group(DHEA).The mice in different groups were intragastrically given saline,BHR or DHEA individually 1 week before and 3 weeks after modeling.Body weight,ovarian index and the concentrations of serum 17(3-estradiol(E2)and progesterone(P4)were examined.HE staining of ovarian tissues was made,and the numbers of atretic follicles were recorded to evaluate ovarian function.TUNEL was applied to measure apoptosis of ovarian granulosa cells.Real-time PCR and Western blotting were used to detect the expression of apoptosis-related factors Bax,Bcl-2 and Caspase-3 mRNA and protein respectively in ovarian tissues of mice.ResultsCompared with group NC.body weight,ovarian index,and the concentrations of serum E2 and P4 were significantly decreased in group CTX.The numbers of atretic follicles and apoptosis of ovarian granulosa cells were greatly increased in group CTX.After treatment with BHR,body weight,ovarian index,and the concentrations of serum E2 and P4 were significantly increased.The numbers of atretic follicles and apoptosis of ovarian granulosa cells were greatly decreased.Results of Real-time PCR and Western blotting revealed that BHR down-regulated the expression of Bax and Caspase-3 and up-regulated Bcl-2.Bcl-2/Bax protein ratio showed significant difference between BHR group and COH group.ConclusionBHR can prevent and treat CTX-induced POF,and improve ovarian reserve function by decreasing the apoptosis of ovarian granulosa cells in mice,probably by down-regulating the expression of Bax and Caspase-3 and up-regulating the expression of Bcl-2.Section ?Effect of Bu-Shen-An-Tai Recipe on Ovarian granulosa cells Apoptosis in Mice with Controlled Ovarian Hyperstimulation Implantation Dysfunction and Possible MechanismsObjectiveTo investigate the effect of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation(COH)implantation dysfunction and possible mechanisms.MethodsCOH implantation dysfunction mice model was established by intraperitoneal injection of 7.5 IU pregnant mare serum gonadotrophin(PMSG),and 48 h later,7.5 IU human chorionic gonadotropin(HCG)was given.Then the female mice were made mated with male mice at 6:00 pm by 2:1 ratio.Normal females were given intraperitoneal injection of normal saline and mated at the corresponding time.Day 1 of pregnancy was recorded by examining vaginal smears at 8:00 am of the next day.Fifty pregnant KM mice were equally randomly divided into 5 groups:normal control pregnant group(NC),COH implantation dysfunction model group(COH).low dose of Bu-Shen-An-Tai recipe group(LOW),middle dose of Bu-Shen-An-Tai recipe group(MID)and high dose of Bu-Shen-An-Tai recipe group(HIGH).Then the mice in different groups were respectively intragastrically given with corresponding therapy at 9:00 am for 5 days.The concentrations of serum 17?-estradiol(E2)and progesterone(P4)were determined with radioimmunoassay(RIA).The ultrastructural changes of ovarian tissue were observed by TEM.The histopathological changes of ovarian tissues were examined by HE staining.And the numbers of atretic follicles and pregnant corpus luteum were recorded.TUNEL was applied to measure apoptosis of ovarian granulosa cells.Western blotting was used to detect the expression of apoptosis-related factors Bax,Bcl-2 and Cleaved-caspase-3 protein in ovarian tissue of mice.ResultsOvarian weight,the concentrations of E2 and P4,and the number of atretic follicles and pregnant corpus luteum were significantly increased in group COH.TEM indicated that apoptosis bodies were seen in COH group.TUNEL showed that the numbers of apoptotic granulosa cells were greatly decreased after Bu-Shen-An-Tai recipe treatment.Results of Western blotting revealed that Bu-Shen-An-Tai recipe down-regulated the expression of Bax and Cleaved-caspase-3 and up-regulated the expression of Bcl-2.ConclusionBu-Shen-An-Tai recipe could improve the micro-environment of corpus luteum during COH implantation dysfunction in mice,decrease the loss of embryos and increase the success rate of pregnancy,probably by down-regulating the expression of Bax and Cleaved-caspase-3 and up-regulating the expression of Bcl-2...