| BackgroundInfertility due to premature ovarian insufficiency(POI)and age-related reduced ovarian reserve are a thorny problem worldwide,due to the increasing trend towards late marriage and childbearing.More and more cancer survivors are becoming candidates for iatrogenic POIs.Radiotherapy and chemotherapy lead to impaired ovarian function,which severely affects the reproductive health and endocrine system of young women.At present,hormone replacement therapy(HRT)is mainly used to improve symptoms in patients with POI,but long-term hormone replacement may also cause a series of estrogen-progesterone-related complications.On the other hand,when ovulation induction is performed by assisted reproductive technology in patients with low ovarian reserve,the incidence of ovarian hyporesponsiveness is increased,resulting in lower egg acquisition,higher cycle cancellation rate,and lower pregnancy rate.Therefore,it is particularly important to study drugs that can improve ovarian function and assisted reproduction.In recent years,there are many ways for traditional Chinese medicine to improve ovarian function,among which Dingkun Pill has shown certain advantages.Dingkun Pill is composed of more than 30 kinds of traditional Chinese medicine with complex chemical composition.Clinical studies have shown that Dingkun Pill can improve ovarian function and is gradually widely used in assisted reproduction,which can improve ovulation rate and pregnancy rate.Although there are many clinical studies,basic experiment and mechanism studies on this drug are still lacking.In order to study the occurrence and development of the disease and its treatment,animal models are usually used in scientific research.Mouse models of cyclophosphamide(CTX)-induced ovarian insufficiency are commonly used modeling methods at present.Chemotherapy may accelerate follicular apoptosis and follicular pool activation through a variety of molecular reactions,damaging the ovary interstitium.Studies have shown that disruption of ovarian function by CTX may lead to overactivation of primordial follicle by activating the PI3K/AKT signaling pathway.PTEN functions as reverse regulation of upstream genes of the PI3K/AKT pathway.Whether CTX affects the development of follicles by regulating the expression of PTEN,and how Dingkun Pill works,is our research direction.ObjectivesIn this experiment,CTX-induced mouse POI model was used to study the effect of different doses of Dingkun Pill on the ovarian function of mice in this model,and its possible mechanism of action was elucidated based on PTEN/PI3K/AKT/FOXO3 a signaling pathway,which provided scientific basis for Dingkun Pill in assisted reproduction medication and improvement of ovarian function,and provided a new therapeutic direction for the study of targeted treatment of POI.Methods1.Fifty female ICR mice were randomly divided into normal control(NC)group,model control(MC)group,and Dingkun Pill low,medium and high dose(DKP-L,M,H)groups.2.Intraperitoneal injection of CTX to induce POI mouse model.After successful modeling,mice in the DKP-L,M and H groups were given 0.9g/kg,1.8g/kg and 3.6g/kg Dingkun Pill suspension,respectively,for continuous gavage for 21 days.Mice in the NC and MC groups were given the same amount of normal saline.3.Observe the changes of body weight,estrous cycle and gonadal index of mice in each group.4.Serum FSH,LH,E2 and AMH levels are measured by ELISA.5.Hematoxylin-eosin(HE)staining observed the changes of ovarian pathological morphology and follicle count in each group of mice.6.qRT-PCR detects PTEN and FOXO3 a gene expression levels in ovarian tissue.7.Western-blot and immunohistochemistry(IHC)detected the expression of PTEN/PI3K/AKT/FOXO3 a signaling pathway-related proteins,respectively.Results1.Dingkun Pill could increase the body weight and ovarian index of chemotherapy-induced POI mice,and the ovarian index of mice in the low-dose group was statistically significant(P(27)0.05).After treatment with Dingkun Pill,the estrus cycle of mice improved to a certain extent.2.The serum FSH and LH levels of POI model mice rose,and the levels of E2 and AMH reduced.FSH levels decreased in the medium-dose group(P(27)0.05),LH levels in the medium-and high-dose group(P(27)0.01),and E2 levels in the high-dose group(P(27)0.05).There were no significant differences in AMH levels between all dose groups.3.The growth follicles of POI model mice at all levels were significantly reduced,and the atresia follicles were significantly increased.After treatment with Dingkun Pill,the number of primordial follicles and growing follicles in the low-and medium-dose groups increased significantly.The number of growing follicles decreased in the high-dose group,but the number of corpus luteum increased significantly(P(27)0.001),and atresia-locked follicles decreased in all three dose groups.4.qRT-PCR results showed that the expression levels of PTEN and FOXO3 a m RNA in POI model mice were significantly reduced.The expression of ovarian PTEN m RNA was significantly upregulated(P(27)0.0001)after treatment in the low-and medium-dose groups of Dingkun Pill,and there was no significant change in FOXO3 a m RNA.5.The results of Western-blot and IHC showed that the expression of PTEN protein in POI model mice was significantly downregulated,and the phosphorylation levels of PI3 K,AKT and FOXO3 proteins were significantly increased.The medium-dose group Dingkun Pill upregulated the expression of PTEN(P(27)0.05)in the ovarian tissues of POI mice,inhibited the levels of p-PI3 K and p-AKT protein,thereby reducing the expression of the key protein p-FOXO3a(P(27)0.01).However,high-dose Dingundan may not be as effective as low-and medium-dose groups.Conclusions1.Dingkun Pill can improve the serum sex hormone level of CTX-induced POI mice,increase the number of primordial follicles and growth follicles,promote follicle growth and induce ovulation,and have a better effect in medium and low dose groups.2.Dingkun Pill may improve ovarian function by upregulating the expression of PTEN in the ovarian tissues of POI mice,thereby inhibiting the excessive phosphorylation of downstream molecules PI3 K,AKT,and FOXO3 a proteins,and preventing excessive activation of follicles. |
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