Effects Of Metformin And Exenatide Monotherapy/Combination In PCOS-IR Rats

Objective: Polycystic ovary syndrome is a common reproductive endocrine disease,which is one of the most common causes of infertility in women of reproductive age.The common clinical symptoms of PCOS include hyperandrogenemia,ovulation disorders,and insulin resistance.Metformin and GLP-1 receptor agonists have been found to be effective in reducing androgen levels,but the mechanism is unclear.In recent years,an increasing number of studies continue to demonstrate that PCOS is a chronic inflammatory disease.The chronic inflammation of the whole body may lead to chronic inflammation of the uterus,which leads to a decrease in endometrial receptivity and pregnancy rate and an increase in miscarriage rate.Clinically,it has also been found that after ovulation induction therapy,the pregnancy rate of patients is not increased.Metformin and Exenatide are two different insulin sensitizers,which are currently clinically used in the treatment of PCOS.Both drugs can reduce the inflammatory state of PCOS,promote ovulation and increase pregnancy rate,but the specific mechanism is unclear.We established a PCOS-IR rat model to study the therapeutic effects and possible mechanisms of metformin and exenatide monotherapy/combined intervention on the ovarian androgen synthesis-related enzymes and uterine inflammation in PCOS-IR rats,which may provides a new idea for clinical guidance of drug use and improvement of pregnancy rate.Materials and Methods: 30 female SPF Sprague Dawley(SD)rats,6 weeks old,weighing 170～190g,were randomly divided into two groups: control(n=6,normal diet feeding),PCOS-IR(n=24,60% high-fat diet feeding).The PCOS-IR rat models were established by gavaged letrozole(dissolved in 1% CMC)at 1 mg/(kg·d),the control rats was given an equal volume of solvent by gavage every day for 4 weeks.Four weeks later,PCOS-IR model rats were randomly divided into four groups: PCOS-IR(n=6),metformin(MET,n=6),exenatide(EX,n=6),and combined(COM,n=6).The control rats were given solvent gavage + normal saline subcutaneous injection on the back of the neck + normal diet for 3 weeks.The PCOS-IR rats were given daily subcutaneous injection of normal saline on the back of the neck;the MET rats were given 300 mg/kg of metformin(dissolved in normal saline)by gavage;the EX rats were given exenatide10μg/kg(dissolved in saline)subcutaneously in the back of the neck;the COM rats were gavaged with metformin(300mg/kg·d)+ exenatide(10μg/kg·d)subcutaneously in the back of the neck,all the treatments lasted 3 weeks.At the same time,the PCOS-IR,MET,EX,and COM rats continued to be gavaged with letrzole and 60% high-fat diet for 3weeks.Then,the fasting blood glucose,insulin,HOMA-IR and testosterone levels of all rats were determined.One ovary was collected for HE staining,the other ovary was placed at-80°C and the rat uterine tissues were placed at-80°C for use.The protein levels of 17α hydroxylase(CYP17A1),aromatase(CYP19A1),3βhydroxydehydrogenase-2(HSD3B2)were determined by Western blot in ovary tissue;the protein levels of silence information factor-1(SIRT1),NF-κB(p65),TNF-α were determined by Western blot in uterine tissue.Results:(1)Compared with control rats,the body weights,serum T、FINS,and FBG were significantly increased(P<0.05),the HOMA-IR also increased in PCOS-IR rats.Further,there were estrus cycle disorders and multiple cystic dilated follicles under ovarian microscope.(2)After drug intervention,the body weight of the rats in three treatment groups were significantly decreased(P < 0.05),the COM group was significantly lower than the MET group(P < 0.05),and there was no significant difference between the EX group and the COM group(P>0.05).(3)The estrus cycle of the rats recovered in three treatment groups,and clear oocytes were seen in the ovaries under microscope.(4)After treatment,the serum T levels,FINS levels,and HOMA-IR levels of three treatment groups were significantly decreased(P<0.05),but there was no statistical difference between the three groups(P>0.05);Compared with the PCOS-IR rats,the serum FBG of the rats in the EX group and the COM group was significantly lower(P < 0.05),but the rats in the MET group showed no statistical difference compared with the PCOS-IR group(P>0.05).(5)Compared with the control group,the protein expression of CYP17A1 of the ovarian tissue in the PCOS-IR rats was increased(P < 0.05),and the expression of CYP19A1 and HSD3B2 protein did not change significantly(P >0.05);After treatment,the protein expression of CYP17A1、 CYP19A1 and HSD3B2 did not change significantly.(6)Compared with the control group,the expression of SIRT1 protein of the uterine tissue in the PCOS-IR group was decreased(P<0.05),and the expression of NF-κB(p65)and TNF-α protein were increased(P<0.05);After treatment,compared with rats in the PCOS-IR group,the expression of SIRT1 in the three treatment groups were increased(P<0.05),and the expression of NF-κB(p65)and TNF-α were decreased(P<0.05),but the difference was not statistically significant among the three groups(P>0.05).Conclusion: Letrozole combined with a 60% high fat diet can induce a PCOS-IR rat model successfully.In PCOS-IR rats,there is hyperandrogenemia.Metformin and Exenatide can reduce serum T levels,but they may not affect the enzymes related to androgen synthesis directly.The specific mechanism needs to be further explored.Metformin and Exenatide alone/combination can reduce the chronic inflammation of the uterus,but the combined use has not shown a better effect.Metformin and Exenatide may improve the chronic inflammation of the uterus through the SIRT1-NF-κB pathway.