Based On PI3K/Akt/mTOR Autophagy Pathway To Explore The Mechanism Of Delaying D-gal-induced Premature Ovarian Failure By Ginsenoside Rg1

Objective:Youth women have long-term hormone disorder in low estrogen and high follicle stimulating hormone,called Premature ovarian failure（POF）,which leads to the syndrome of organ atrophy and persistent amenorrhea.Estrogen and progesterone replacement therapy to establish an artificial cycle,which can quickly improve the symptoms of low estrogen and endocrine environment is the main method for the clinical intervene of POF at present,however,the previous clinical symptoms appeared immediately after stopping the drug.Estrogen use for too long will produce breast cancer and endometrial cancer sequelae,the use of hormone replacement therapy has a very high risk.Therefore,exploring a drug that can negatively regulate premature ovarian failure and positively regulate the body has become a new idea for the research of targeted drugs for POF.Ginseng,with extensive pharmacological effects,has unique application value in anti-aging.As one of the tetracyclic triterpenoid saponins in ginseng tuber,ginsenoside Rg1 can effectively resist aging,antioxidative damage and improve immunity.at the same time,it is also a kind of drug with high effectiveness,less risk and no toxic side effects.Research display that ginsenoside Rg1 resists aging of ovary mainly by inhibiting the expression of P16 gene,improving the viability of neatly arranged granulosa cells around oocytes and reducing follicular degeneration,thus effectively promoting the formation of follicles at all levels and enhancing ovarian function.In this study,ginsenoside Rg1 was used to induce BALB/c mouse POF model induced by D-galactose.The effect of ginsenoside Rg1 on POF was discussed through the regulation mechanism of ginsenoside Rg1 regulating PI3K/Akt/m TOR autophagy pathway.It provides a new target for clinical POF diagnosis and treatment,and a new idea for POF targeted drug research.Methods:Fifty-four female BALB/c mice aged from 6 to 8 weeks and weighing（21.6?1.7）g were randomly divided into three groups,18 mice in each group,which were named PBS group,D-gal group and Rg1 group respectively.In the D-gal group,200 mg/kg/d D-galactose was injected subcutaneously in the neck and back for 42 days without interruption.In PBS group,the neck and back were injected with the same amount of phosphate buffer solution（PBS）for 42 days.On the basis of D-gal treatment,Rg1 group received intraperitoneal injection of Rg1 20 mg/kg/d,for 28 days on the 15th day,while D-gal group and PBS group were also given intraperitoneal injection of the same amount of PBS,for 28 days on the 15th day.The estrous cycle was recorded by vaginal exfoliated cell smear to detect the effect of ginsenoside Rg1 on estrous cycle in mice.Record the weight and ovarian weight of mice.To detect the effects of ginsenoside Rg1 on body weight and ovarian weight coefficient of mice.The contents of serum estrogen（E₂）,catalase（CAT）,luteinizing hormone（LH）,superoxide dismutase（SOD）and follicle stimulating hormone（FSH）in mice were determined by ELISA way,and the effects of ginsenoside Rg1 on serum hormones and antioxidant indexes in mice were detected.Ovarian HE staining and SA-β-Gal staining were used to detect the morphological changes of ovarian aging.The expression of autophagy factor LC3-II,PI3K,p-Akt,TSC2,p-m TOR,p-S6k and aging regulatory factors SIRT1 and P16^INK4awere detected by Western blot.The expression of SIRT1、S6k、PI3K、TSC2、m TOR、P16^INK4aand Akt was detected by fluorescent quantitative PCR（q PCR）.Results:The results of body weight showed that the growth rate of body weight in D-gal group was significantly slower than that in PBS group and the body weight growth rate in Rg1 group was higher than that in D-gal group.The results of ovarian weight coefficient showed that compared with PBS group,the ovarian mass coefficient of D-gal group decreased.Ovarian mass coefficient in Rg1 group was higher than that in D-gal group.ELISA experimental results show that compared with the PBS group,the serum E₂,LH,SOD,CAT were significantly reduced and FSH was significantly increased of the D-gal group.the serum E₂,LH,SOD,CAT of the Rg1 group was higher than that of the D-gal group and the serum FSH of the Rg1 group was lower than that of the D-gal group.The results of SA-β-Gal staining showed that compared with the PBS group,the positive rate of SA-β-Gal staining of the D-gal group was significantly higher and the positive rate of SA-β-Gal staining of the Rg1 group was lower than that of the D-gal group.HE staining of ovary showed that the morphology and structure of follicles in PBS group were normal.Compared with PBS group,the number of follicles at all levels decreased in D-gal group except atresia.Compared with D-gal group,the number of atretic follicles in Rg1 group decreased,while the number of primordial,primary and secondary follicles increased.Western blotting showed that the protein expressions of P16^INK4a,PI3K,p-Akt,p-m TOR and p-S6k were increased,while the protein expressions of LC3-II,SIRT1 and TSC2 were decreased in D-gal group.The expressions of P16^INK4a,PI3K,p-Akt,p-m TOR and p-S6k in Rg1 group were lower than those in D-gal group,while the expressions of LC3-II,SIRT1 and TSC2 were higher than those in D-gal group.The q PCR results showed that compared with PBS group,the m RNA levels of P16^INK4a,PI3K,Akt,m TOR and S6k in D-gal group were significantly increased,while the m RNA levels of SIRT1 and TSC2 were significantly decreased.The m RNA expressions of P16^INK4a,PI3K,Akt,m TOR and S6k in Rg1 group were lower than those in D-gal group,while the m RNA expression of SIRT1 and TSC2was higher than that in D-gal group.Conclusions:1.Rg1 can delay premature ovarian failure in a mouse model of premature ovarian failure induced by D-gal2.Rg1 can delay premature ovarian failure in a mouse model of premature ovarian failure induced by D-gal by regulating the PI3K/Akt/m TOR autophagy pathway...