| Part1 Literatures researchObjectiveTo elaborate Diminished Ovarian Reserve(DOR)on traditional Chinese and western medical research progress in detail by reviewing the existing literatures of DOR in the last decade.It can provide evidences for making animal experiment researches.MethodsBy consulting the domestic and foreign literatures in CNKI and PUBMED nearly 10 years,it has elaborated detailed disease name,etiology,diagnostic standard,therapeutic method of DOR,the research progress of DOR pathogenesis,dormancy and active regulatory factors of primordial follicles and the intracellular signaling pathways involved;it has also elaborated detailedly traditional Chinese medicine(TCM)name of DOR?etiology and pathogenesis?treatment based on syndrome differentiation from the perspective of TCM.ResultsRecently the diagnostic standard and therapeutic plan have not reach a consensus yet.The etiology involves heredity,autoimmunity,metabolic disorders and iatrogenic factors.TCM holds that the basic pathogenesis of DOR is kidney deficiency,combining liver stagnation and/or spleen deficiency and/or blood stasis.Kidney deficiency and liver stagnation syndrome(KDALSS)is the most common syndrome of DOR.Therefore,therapeutic principle of DOR is tonifying the kidney,smoothing the liver and strengthening the spleen,promoting the blood circulation.The intracellular signaling pathways involved in dormancy and active regulatory factors of primordial follicles include Mammals target protein of rapamycin signal pathway(TSC/mTOR)and Phosphatidylinositol 3-kinase signal pathway(PI3K),which the action mechanism in DOR remains controversial.ConclusionsDOR is a kind of disease which has complex etiology and pathogenesis,highly characteristic clinical manifestations,and increased incidence.The pathogenesis of DOR is related to the regulation of the dormancy,activation and atresia of primordial follicles.The treatment of DOR is quite difficult,so it's very important to work out therapeutic plans with good effects and less adverse effect.The therapy of TCM on DOR have unique advantages and characteristics,and it is worthy of exploring and researching.Yijing decoction is a famous prescription of Fuqing in Qing period for treating menstruation delay and amenorrhea,clinical practice shows it is definitely useful for DOR,however the mechanism is yet to be investigated.Part2 Effect of Jiajian Yijing Decoction on Tsc/mTOR signal pathway in rats with diminished ovarian reserveObjective(1)To discuss the mechanism of Tsc/mTORCl signal pathway in oocyte of DOR rats,and the interaction with PI3K signal pathway.(2)In comparison with Progynova(P)and Rapamycin(RPM),to study the effects of Jiajian Yijing Decoction(JD)on improving ovarian function in DOR SD rats which had treated by CTX,and the effects on Tsc/mTORCl signal pathway in DOR rats,and elaborate the curative effects and possible mechanism of JD,help provide experimental evidences for widely using JD in clinical study.MethodsThe animal experiment had adopted prospective and randomized methods.100 SPF adult female SD rats had been randomly divided into blank control group(NG),model control group(MG),Progynova group(PG),Rapamycin group(RPM),JD group(JG)(Except rats in NG had been induced by the same volume of saline,the other rats had been induced by 75mg/kg CTX).NG,MG,PG,RPM,JG had been administrated respectively by gavage of the same volume of saline,the same volume of saline,0.09mg.kg-1 Progynova,0.9g.kg-1 RPM,11.79g.kg-1 JD for 4 weeks continuously.10 rats at diestrus in every group had been broken the neck to death in the 4th,6th week after administration.The general situation,vaginal exfoliative cell,the serum concentrations of FSH,LH,E2 and AMH,ovary index,ovarian tissue pathologic structure,follicle count,the expression of PS6K1,rpS6,PDK1,Tscl and mTORCl in ovarian tissue of rats all had been observed in different groups and different weeks.Results(1)The general situation of model:The MG rats had loose hair?activity decreases?dispiritedness?stooping?loose stool?limb weakness,6 weeks later the MG rats recovered.After 4 weeks administration,the appearance signs,activities,mental state,feeding,drinking,hair,breaths,mouth,eyes,noses,ears,stools and urine color of every treatment group rats had returned to normal and had no significant difference to NG.(2)The impact on estrus cycle(EC):After molding,the MG rats regular ECs that had 4?6d had disappeared,it extended to 8?17d and the anestrous extended to 5?7d from 1.5?3d.The ECs in MG had regained after molding for 6w.The ECs in PG?JG had regained after molding for 4w.The ECs in RPM had regained after molding for 5w.(3)The impact on ovarian index(01):Comparing with NG,the 01 in MG significantly reduced in the 4th,6th week after modeling(P<0.01).Comparing with NG and MG,the 4th week 01 in PG?JG began to go up apparently and returned to normal,until the 6th week(P<0.01);the 4th week 01 in RPM began to go up visibly until the 6th week,it returned to normal(P<0.05).The OI in JG and PG werec apparently higher than the RPM OI in the 4th,6th week(P<0.05 or P<0.01).The OI in PG and JG had no significant differences in the 4th,6th week(P>0.05).(4)The impact on ovarian pathological structure(OPS):After molding,ovaries in MG had atrophied medulla,cortex hyperplasia,fibrosis,vessel hypertrophy,reduced primitive and mature follicles count,increased primary,secondary and atretic follicles count and corpus luteum regression.The OPS in MG still had a little cortex hyperplasia and fibrosis,primitive and mature follicles count increased,primary,secondary and atretic follicles count decreased,corpus luteum count increased after molding in the 6th week.The OPS in JG had almost regained after molding for 4w.The OPS in PG had regained after molding for 6w.The OPS in RPM had mostly regained after molding for 6w.(5)The impact on all levels of follicle count(OI):Comparing with NG,the number of primitive and mature follicles in MG in the 4th,6th week decreased significantly(P<0.01).Comparing with NG and MG,the number of primitive and mature follicles in every treatment group increased apparently in the 4th week,until the 6th week(P<0.05 or P<0.01);the number of primitive follicles in JG returned to normal in the 4th week(P>0.05);the number of primitive follicles in PG,RPM returned to normal in the 6th week(P>0.05).The number of primitive follicles in JG was obviously more than in PG and RPM in the 4th,6th weeks after modeling(P<0.05 or P<0.01).Mature follicles in JG and PG was apparently higher than in RPM in the 4th,6th week after modeling(P<0.05 or P<0.01),but had no significant differences between them in the 4th,6th week(P>0.05).Comparing with NG,the number of the primary,secondary,atretic follicles in MG in the 4th,6th week increased significantly(P<0.01).The number of the primary,secondary and atretic follicles in MG significantly decreased in the 6th week than in the 4th week(P<0.05 or P<0.01).Comparing with NG and MG,the number of the primary,secondary,atretic follicles in every treatment group decreased significantly in the 4th week,until the 6th week(P<0.05 or P<0.01);the number of primary follicles in JG returned to normal in the 4th week(P>0.05);the number of secondary,atretic follicles in JG returned to normal in the 6th week(P>0.05);the number of the primary,secondary,atretic follicles in PG returned to normal in the 6th week(P>0.05).The number of the primary,secondary,atretic follicles in Jd was much less than in RPM in 4th,6th week after modeling(P<0.01).The number of the primary,secondary follicles in JG was much less than in PG in 4th,6th week after modeling(P<0.05).Comparing with PG,the number of atretic follicles in JG had no significant differences during every week(P>0.05).(6)The impact on serum concentrations of FSH?LH?E2:Comparing with NG,serum concentrations of FSH?LH in MG in the 4th,6th week increased significantly(P<0.01);Serum concentration of FSH in MG significantly decreased in the 6th week than in the 4th week(P<0.01).Comparing with NG and MG,the serum concentrations of FSH and LH in every treatment group went down apparently in the 4th week,until the 6th week(P<0.05 or P<0.01);the serum concentrations of FSH in JG and PG return to normal in the 6th week(P>0.05);the serum concentrations of LH in JG and PG return to normal in the 4th week(P>0.05),and in the 6th week in RPM(P>0.05).Comparing with PG,the serum concentrations of FSH and LH in JG had no significant difference in every week(P>0.05).Comparing with RPM,the serum concentrations of FSH in JG were much lower than in every week(P<0.01),the serum concentration of LH in JG was much lower than in the 4th week(P<0.01).Comparing with NG,serum concentrations of E2 in MG in the 4th,6th week decreased significantly(P<0.01);serum concentration of E2 in MG significantly increased in the 6th week than in the 4th week(P<0.01).Comparing with NG and MG,the serum concentrations of E2 in every treatment group went up apparently in the 4th week,until the 6th week(P<0.05 or P<0.01);the serum concentrations of E2 in PG returned to normal in the 4th week(P>0.05),and in the 6th week in JG(P>0.05).Comparing with RPM,the serum concentrations of E2 in JG and PG were much higher than in every week(P<0.01).The serum concentration of E2 in JG was much lower than in PG in the 4th week(P<0.01),but had no significant differences in the 6th week(P>0.05).(7)The impact on serum concentrations of AMH:Comparing with NG,the serum concentrations of AMH in MG in the 4nd,6th week decreased significantly(P<0.01).Comparing with NG and MG,the serum concentrations of AMH-in every treatment group went up apparently in the 4th week,until the 6th week(P<0.05 or P<0.01),and began to return to normal in the 6th week in JG and PG(PP>0.05).The serum concentrations of AMH in PG.JG were obviously higher than in RPM in the 4"d week(P<0.01).(8)The impact on PS6K1,rpS6 protein and gene expression in ovarian tissue:Comparing with NG,the PS6K1 and rpS6 expression value of ovarian tissue in MG in the 4th,6th week increased significantly(P<0.05 or P<0.01);increased in every treatment groups in the 4th,6th week(P<0.05 or P<0.01),but had a downtrend in the 6th week.Comparing with MG,the PS6K1,rpS6 expression value of ovarian tissue in every treatment groups decreased in the 4th,6th week(P<0.05 or P<0.01).The PS6K1 expression value of ovarian tissue in JG and PG were obviously lower than in RPM in the 6th week(P<0.05 or P<0.01);the PS6K1 expression value of ovarian tissue in JG was obviously lower than in PG in the 6th week(P<0.01).The rpS6 expression value of ovarian tissue in JG was obviously lower than in PG and RPM in the 6th week(P<0.01).All the Western-blotting(WB)tested results were similar as using Q-PCR.(9)The impact on PDK1,Tscl and mTORCl protein and gene expression in ovarian tissue:Comparing with NG,the PDK1 and mTORCl expression value of ovarian tissue in MG in the 4th,6th week increased significantly(P<0.05 or P<0.01);increased in every treatment groups in the 4th,6th week(P<0.05 or P<0.01),but had a downtrend in the 6th week.Comparing with MG,the PDK1 expression value of ovarian tissue in PG decrease significantly in the 4th,6th week(P<0.05 or P<0.01);decrease significantly in JG and RPM in the 6th week(P<0.05 or P<0.01);the mTORCl expression value of ovarian tissue in every treatment groups decrease significantly in the 4th,6th week(P<0.05 or P<0.01).The PDK1 expression value of ovarian tissue in JG was significantly lower than in RPM and PG in the 6th week(P<0.05 or P<0.01).The mTORC1 expression value of ovarian tissue in JG and PG were significantly higher than in RPM in the 4th,6th week(P<0.05 or P<0.01);was significantly lower in JG than in PG in the 4th and 6th week(P<0.05 or P<0.01).Comparing with NG,the Tscl expression value of ovarian tissue ovarian significantly decreased in 4th,6th week in MG(P<0.05 or P<0.01);decreased in JG and PG in the 4th,6th week(P<0.05 or P<0.01),but had a uptrend in the 6th week,decreased in RPM in the 4th,6th week(P<0.05 or P<0.01).ovarian Tscl expression in all treat groups were obviously higher than in MG(P<0.05 or P<0.01).The Tscl expression value of ovarian tissue ovarian in JG and PG was significantly higher than in RPM and MG in 4th,6th week(P<0.05 or P<0.01).The Tscl expression value of ovarian tissue ovarian in RPM had no significant differences than in MG in every week(P>0.05).All the values were similar in WB test,Q-PCR and immuno histochemistry(IHC)testing.Conclusions(1)JD,RPM and P could obviously improve ovarian function of DOR rats,decreased FSH and LH concentrations,increased E2 concentrations,AMH and OI,increased primitive and mature follicles count,cut down primary,secondary and atretic follicles count.As to improving ovarian function,the efficacies of JG has no significant differences but superior in some respects than P,were more quickly,great than RPM.(2)After CTX modeling,the primitive and mature follicles count decreased,the primary?secondary and atretic follicles count increased,which indicated model success,the presence of primordial follicles is over activated and follicular atresia increased;the expression of downstream molecules in signal pathway of DOR rats' ovarian tissue,such as PS6K1 and rpS6 were up regulated,the expression of key molecules,such as PDK1,and mTORC1,were up regulated,Tsc1 was down regulated,which indicated the overactive state of primordial follicles pool and follicular atresia increased were realized through Tsc/mTORC1 pathway and PI3K pathway.(3)Both JG and P significantly improved the expression of the key molecules like PDK1 and mTORC1,up regulated Tsc1;down regulated the expression of the downstream molecules like PS6K1,rpS6;JG and P had the same effects but did better than RPM.All the facts indicated that JG and P could decrease follicles over activated by inhibiting the work of Tsc/mTORC1 pathway and PI3K pathway.(4)RPM significantly improved the expression of the key molecules such as PDK1 and mTORCl in DOR rats ovarian pathways,the expression of the downstream molecules like PS6K1,rpS6,but had no significant effect on Tsc1.It showed the most powerful effects on reducing the expression of mTORC1,which reminded that PI3K pathway and Tsc/mTORC1 pathway were not the same signal pathway.They may both collaborated in regulating and controlling the stationary and activation of primordial follicles,maybe the Tsc/mTORC1 pathway played a more direct role in keeping primordial follicles in a dormant state.(5)RPM,as an inhibitor of mTORC1,could protect ovarian function by reducing follicles over activated through suppressing Tsc/mTORC1 pathway.This study can provide experimental evidence for the clinical application of rapamycin in the prevention and treatment of DOR. |
PDF Full Text Request