The Mechanism Of Bushen Culuan Decoction In The Protection Of Diminished Ovarian Reserve Via Modulating PI3K And Nrf2 Pathway

BackgroudWith the delay of childbearing ages,the global fertility rate is decreasing and the infertility rate is increasing year by year.According to an investigation report on the current infertility situation in China,the incidence of infertility among the reproductive population in China is as high as 15-20%,with a total of more than 60 million patients.Especially after two-child policy was opened,woman in advanced maternal age became the main infertility patients.Among them,diminished ovarian reserve(DOR)is an important cause of infertility,if not timely intervention which can be further developed into premature ovarian failure in 1-6 years.DOR not only affects the reproductive health,but also increases the risk of fractures,osteoporosis and cardiovascular events in the long term.Modern medicine mainly adopts hormone replacement therapy(HRT)to improve clinical symptoms,and carries out pregnancy through assisted reproduction.However,due to the inability of many patients to obtain mature eggs,the pregnancy is ultimately not successful.Traditional Chinese medicine(TCM)has accumulated rich experience in the treatment of infertility.Through literatures,it is found that kidney deficiency and blood stasis is the basic pathogenesis of DOR,and tonifying kidney and promoting blood circulation is an important treatment method for DOR.Professor Ma Kun has been committed to the clinical and basic research on the treatment of ovulatory infertility for a long time.Based on years of clinical practice experience,the Bushen Culuan Decoction(BCD)was formulated based on the principle of tonifying kidney and activating blood.It has been applied to the clinical treatment of DOR for a long time.Modern pharmacological studies have shown that BCD can inhibit the apoptosis of granulosa cells(GCs)and reduce follicular atresia.For most mammals,the number of primordial follicles(PMFs)in ovaries is relatively fixed,so whatever the factors that induced DOR and ultimately can be attributed to the depletion of PMF pool in advance,and therefore cannot provide adequate number of PMF to be recruited into the growing follicle pool,ultimately unable to form mature eggs.Therefore,one of the key strategies to prevent and treat DOR is to seek effective therapeutic drugs,which can actively improve ovarian reserve and protect PMF pool,and that is exactly the embodiment of TCM’s idea of "treating pre-disease".More and more studies have shown that over-activation of PMFs is an important mechanism to induce premature depletion of ovarian reserve.In order to maintain the reproductive life,most of the PMFs in the ovary are dormant,and only a small number of them are activated each cycle.Among them,the PI3K/AKT/mTOR pathway plays a crucial role in maintaining the dormancy of PMFs.which is of great significance for maintaining the dynamic balance between activation and dormancy of PMFs.Once the excessive activation of PMFs,will be accompanied by a large number of follicular atresia,and GCs apoptosis is the central link of follicular atresia.Further,Bax/Cyt C/Caspase 3 pathways plays a key role in regulation of apoptosis,and oxidative stress is also an important factor in initiating apoptosis.The essence of apoptosis is the result of imbalance between oxidation and antioxidant systems.Among them,Nrf2/ARE pathway is the most important anti-oxidative stress system in vivo,which is significant in maintaining cell stability and inhibiting apoptosis.Therefore,in this study,we intended to explore the protecting mechanism of BCD in DOR induced by tripterygium wilfordii polyglycoside(TWP)from the PI3K/AKT/mTOR pathway.Bax/Cyt C/Caspase-3 pathway and Nrf2/ARE pathway.On this basis,the acute toxicity of BCD was studied in order to evaluate the safety and provide a basis for clinical application.ObjectiveThis study adopted TWP to establish DOR mice model.Through selecting the optimal concentration of BCD to observe protecting function in the ovarian reserve.Furthermore,the mechanism of BCD inhibiting the PI3K/AKT/mTOR pathway to reverse the overactivation of PMFs induced by TWP was investigated from the molecular perspective.as well as BCD through activating of Nrf2/ARE pathway to inhibit oxidative stress caused by TWP and to alleviate activation of ovarian Bax/Cyt C/Caspase-3 pathway,which will reduce GCs apoptosis and prevent excessive atresia of the growing follicles,thus protecting the function of ovarian reserve.At the same time,the acute toxicity of BCD was studied,so as to provide evidence for the safety of clinical medication.MethodsPart One:The acute toxicity experiment of BCDIn the pre-experiment,the mice were randomly divided into BCD group and control group.The highest concentration and the maximum volume that the mice could bear were given to the BCD group.They were given BCD solution three times a day,with an interval of about 4 hours each time.The control group was given isosomatic saline,and the LD50 value was measured for 7 days.According to the results of the pre-experiment,40 mice were randomly divided into BCD group and control group.The administration method is the same as the pre-experiment and continuous observation for 14 days.The reaction symptoms of the mice were closely monitored shape,death,weight change,feed consumption,as well as the change of main organs’ morphology and organ index.Part Two:Concentration screening experiment of BCDFemale Balb/c mice were randomly divided into blank,model,BCD low dose,BCD medium dose,BCD high dose and estradiol valerate group.In addition to the empty group,40mg/kg TWP suspension was administered daily by gavage.Meanwhile,the BCD low-dose,medium-dose and high-dose groups were given with concentrations of 0.13g/mL,0.27g/mL and 0.53g/mL BCD suspension respectively.The estradiol valerate was given 0.015mg/mL estradiol valerate suspension by gavage of 0.2mL.The blank group and the model group were given the same amount of normal saline for 30 days.The general conditions and changes of estrous cycle in each group were observed,the gonadal index was calculated,the levels of serum sex hormones were measured,and the ovarian tissue morphology and follicle count were observed by HE staining.Part Three:BCD modulates the PI3K/AKT/mTOR pathway to protect PMFs poolFemale Balb/c mice were randomly divided into blank,model,BCD and HRT groups.In addition to the empty group,40mg/kg TWP suspension was administered daily by gavage.Meanwhile,the group was given with 0.27g/mL BCD suspension.The HRT group was given estradiol valerate combined with medroxyprogesterone acetate.The blank group and the model group were given the same amount of normal saline for 30 days.The general conditions and changes of estrous cycle of mice were observed,the indexes of organs were measured,the levels of serum sex hormones were detected,and the morphological changes of uterus and ovary as well as the follicle count were observed by HE staining.In order to further clarify the mechanism of BCD on protecting PMF,the levels of PI3K/AKT/mTOR pathway key proteins,PI3K,P-PI3K,AKT,P-AKT,mTOR and P-mTOR in ovarian tissues,were detected by Western blot,and the levels of PI3K.AKT and mTOR mRNA were detected by RT-PCRPart Four:BCD regulates the Bax/Cyt C/Caspase-3 pathway to protect growing follicleOn the basis of Part three,it was found that TWP not only induced over activation of PMF,but also induced over atresia of follicle.In order to further explore whether TWP induced DOR through over activation of PMFs,resulting in increased growing follicle atresia,or directly inducing PMF atresia.TUNEL fluorescence staining was carried out in mouse ovaries,and the expression of Cyt C and Caspase-3 was observed by immunohistochemistry.In order to further clarify the mechanism of BCD in inhibiting the follicular atresia induced by TWP,the levels of related apoptosis proteins,Cyt C,Caspase-3,Bcl-2 and Bax,were detected by Western blot,and the levels of Bcl-2 and Bax mRNA were detected by RT-PCR.Part Five:BCD regulates Nrf2/ARE pathway to inhibit ovarian oxidative damageOn the basis of the above experiments,it is found that TWP can induce over-activation of PMF and over-apoptosis of GCs,which would cause abnormal atresia of follicle in DOR.In order to further explore the mechanism of TWP inducing apoptosis of GCs and the protective effect of BCD,the levels of 8-OHdG,MDA,CAT,GSH-Px and SOD in ovaries were measured,and Western blot and RT-PCR were used to detect the expression levels of Bach 1,Nrf2,Keap 1,HO-1 in Nrf2/ARE pathway in ovaries.ResultsPart One:The results showed that LD50 of BCD could not be detected,so the maximum tolerance test was used in the official acute toxicity test.The results showed that all animals survived until the end of the experiment.The mice in each group were in good condition at other time points,except that the mice in the BCD group had static lodging,decreased activity,D0 stool color black and decreased D1 intake within 2 hours after gavage.During the experiment,there was no difference in feed consumption between the two groups,and the weight of the two groups increased steadily.There was no abnormal phenomenon in the gross anatomy of each organ in the two groups,and there was no difference in the main organ index.Part Two:During the experiment,the mice in each group had no obvious abnormality in eating,activity and defecation.The hair of the mice in BCD group was dense and glossy.Except for the blank group,the weight in each group decreased first,and then gradually increased.Until the end of the experiment,there was no difference in the weight of the mice in each group.The middle and high dose groups of BCD can effectively improve the estrous cycle,increase the uterus and ovary index(P<0.05),decrease the serum FSH and LH levels(P<0.01),increase the count of PMF,antral follicle and corpus luteum(P<0.01),and decrease the count of atretic follicle(P<0.01).Part Three:The general situation,body weight and estrous cycle of mice in each group were consistent with the results of Part Two.After BCD treatment,the ovary index increased(P<0.01),the serum levels of AMH and INHB increased(P<0.01),the serum FSH and FSH/LH decreased(P<0.01,P<0.05).The number of PMF,antral follicle and corpus luteum in the ovary was increased by BCD(P<0.01,P<0.05,P<0.01),the number of secondary and atretic follicles was decreased(P<0.01),and the ratio of early-growing follicles to PMFs was decreased(P<0.01).Further study showed that BCD acn reduce the ratios of P-PI3K/PI3K,P-AKT/AKT,P-mTOR/mTOR in ovarian tissue(P<0.01),and reduce the expression of AKT and mTOR mRNA(P<0.01),and the expression of PI3K mRNA showed a downward trend.Part Four:TUNEL fluorescence staining mainly appeared in the GCs around the growing follicles,but no obvious fluorescence was found in the PMFs.After the intervention of BCD,the fluorescence area and quantity of the ovary decreased significantly.Cyt c and Caspase-3 proteins were also mainly expressed in the GCs around the growing follicles.The BCD could reduce the area ratio of positive expression of Cyt C and Caspase-3 in ovaries(P<0.01).Western blot showed that BCD could reduce the expression of Cyt C,Caspase-3 and Bax(P<0.05),increase the expression of Bcl-2 and Bcl-2/Bax(P<0.05,P<0.01),meanwhile,BCD could down-regulate the expression of Bax mRNA,and up-regulate the expression of Bcl-2 mRNA and Bcl-2/Bax(P<0.01).Part Five:BCD can reduce the contents of 8-OHdG and MDA in ovaries(P<0.05,P<0.01),and increase the activities of CAT,GSH-Px and SOD(P<0.01).Western blot showed that BCD could decline the expression of nucleoprotein Bach 1 and Keap 1(P<0.01,P<0.05),up regulate the expression of nucleoprotein Nrf2 and HO-1(P<0.05),and at the same time BCD could decrease the nuclear translocation rate of Bach 1 and raise the nuclear translocation rate of Nrf2(P<0.01).Conclusion1.The maximum tolerable dose of BCD is 660.00g crude drug/kg/d,which is 240 times of the clinical dosage.The clinical dosage of this recipe is safe and non-toxic.2.The middle and high dose of BCD can effectively protect the ovarian reserve function of DOR mice induced by TWP,and the effect of the two doses is equivalent.Therefore,the middle dose is selected in the follow-up experiment.3.BCD can inhibit the PI3K/AKT/mTOR pathway,reverse the excessive activation of PMF induced by TWP,and reduce the abnormal atresia of follicle,which play a role in protecting the ovarian reserve function.4.BCD can inhibit Bax/Cyt C/Caspases-3 pathway,up regulate Bcl-2 expression,reduce excessive apoptosis of GCs of growing follicles,and prevent atresia of follicles in DOR mice induced by TWP.5.BCD can regulate Nrf2/ARE pathway,inhibit Keap-1 over expression,promote Nrf2 nuclear translocation,activate downstream antioxidant enzyme activity,alleviate the oxidative stress induced by TWP in DOR mouse ovary,and reduce the apoptosis of GCs.