| Polycystic ovary syndrome(PCOS)is the most common endocrine and metabolic disorders in women of childbearing age.The incidence of PCOS in women of childbearing age is about 6%-10%.The clinical manifestations of PCOS are diverse and heterogeneous.Its main clinical features are chronic anovulatory,oligomenorrhea or amenorrhea,multiple cystic follicles in the ovary under ultrasound examination,hyperandrogenism,infertility.In addition to reproductive abnormalities,PCOS patients can often be observed obesity,insulin resistance with compensatory hyperinsulinemia,dyslipidemia,and increased risk of type 2 diabetes and cardiovascular diseases.In domestic animals,follicular cyst is a common reproductive disorder,which directly causes the reproductive capacity of the female herd to decrease.The cause of the disease is complicated,the pathogenesis is not clear,and effective prevention and control methods are lacking.Therefore,it has become one of the important bottlenecks in the livestock breeding industry.With the discovery of adipose-derived mesenchymal stem cells(AMSC),there are more and more researches in regenerative medicine.A large number of preclinical and clinical studies have found that AMSCs can be used to treat some clinically intractable diseases.AMSC can secrete a variety of metabolic and immune-related cytokines and participate in metabolic regulation.At the same time,AMSCs have been studied more and more in female related diseases.AMSC can improve oocyte quality,promote granulosa cell proliferation and play an important role in restoring ovarian function.Recent studies have found that the paracrine of ADSC also plays an important role in tissue and organ repair.Exosomes secreted by AMSC(AMSC-EXO)can play an effective role in the repair of various tissues and organs.However,there is no report on the role of AMSC and AMSC-EXO in PCOS.In this study,PCOS rat model was constructed,and AMSC and AMSC-EXO were used to treat PCOS rats.The therapeutic effect was evaluated from the metabolic level,fertility level and tissue level.At the same time,in order to further reveal the molecular mechanism of AMSC-EXO in the treatment of PCOS,we used miRNA abundance sequencing and RNA-seq technology to give analysis on miRNA and m RNA,and used rat liver cell line(BRL-3A)to verify in vitro.The main research contents and results are as follows:1.Construction of PCOS rat model.In this study,dehydroepiandrosterone(DHEA)was used to construct PCOS rat model.Control group: 21-day-old female rats were injected sesame oil subcutaneously(0.1mL / rat)for 23 days.PCOS group: 21-day-old female rats were injected subcutaneously with DHEA(6mg / 100g)dissolved in sesame oil(0.1mL /rat)for 23 days.Results: After subcutaneous injection of DHEA,there was no significant difference in body weight between the control group and the PCOS model group.Glucose tolerance and insulin resistance were observed in the PCOS model group-The estrous cycle was disordered,the interestrus was increased,and the midestrus was decreased.The fatty degeneration of liver tissue,the number of cystic follicles in ovarian tissue and the number of corpus luteum were significantly increased in the PCOS model group.These data indicated that DHEA was successfully used to construct PCOS rat model,which could be used for subsequent experiments.2.Effect of rat adipose-derived mesenchymal stem cells on PCOS ratsWe explored whether adipose-derived mesenchymal stem cells can treat DHEA-induced PCOS rat model After successfully constructed the model.The experimental animals were divided into three groups,among which the PCOS rats were two groups: PCOS group and adipose-derived mesenchymal stem cells treatment group,12 rats per group.After three weeks of treatment with rat adipose-derived mesenchymal stem cells,it was found that compared with the PCOS model group,the estrous cycle,glucose tolerance,insulin tolerance,and litter size were significantly improved.In addition,there were less fat accumulation and fat steatosis shown on the ovarian section,abdominal fat section and liver section.ELISA test showed that the testosterone(T)level in the AMSCs treatment group had a downward(p<0.05),and up-regulated the adiponectin level(p=0.085).3.Effect of rat adipose-derived mesenchymal stem cell exosomes on PCOS ratsIn order to further explore the therapeutic mechanism of rat adipose mesenchymal stem cells in the treatment of PCOS rats,in this experiment,the exosomes of AMSC(AMSC-EXO)were extracted.The experimental animals were divided into three groups: control group,PCOS group and the AMSCs-EXO treatment group.After 3 weeks of AMSC-EXO treatment,the estrous cycle,glucose tolerance and litter size of PCOS rats were significantly improved.In addition,there were less fat accumulation and fat steatosis shown on the ovarian section,abdominal fat section and liver section.ELISA test showed that the T level in the AMSC-EXO treatment group was significantly down-regulated(p<0.05),and up-regulated the adiponectin level(p=0.066).At the same time,It is observed that the main distribution of AMSC-EXO in SD rats was liver tissue.4.Effect of rat adipose-derived mesenchymal stem cell exosomes on rat hepatocytes(BRL-3A)In order to further explore the therapeutic mechanism of rat adipose-derived mesenchymal stem cell exosomes in the treatment of PCOS rats,we sequenced the miRNA abundance of AMSC-EXO and found that miR-21-5p had the highest abundance.At the same time,We extracted the total RNA from the liver of the control group,the PCOS group and the AMSC-EXO treatment group after we observed that the main distribution position of AMSC-EXO in SD rats was the liver tissue.After RNA-seq,we found the number of 68 differential genes,and found the Btg2 gene targeted by miR-21-5p through gene joint analysis.In order to verify the relationship between miR-21-5p and Btg2 genes,rat liver cells(BRL-3A)were used for molecular mechanism studies.The results showed that AMSC-EXO can enhance the glucose uptake ability of BRL-3A,and the phosphorylation level of IRS/AKT is significantly increased(p<0.05).Exogenous overexpression of miR-21-5p can target the Btg2 gene to enhance the glucose uptake capacity of BRL-3A,and the phosphorylation level of AKT is significantly increased.Endogenous removal of miR-21-5p in AMSC-EXO(p<0.05),targeting Btg2 gene attenuated BRL-3A’s ability to take up glucose(p<0.05),and AKT phosphorylation level was significantly reduced(p<0.05).In summary,this study found that DHEA could successfully construct PCOS rat model.Rat adipose-derived mesenchymal stem cells and their exosomes can improve the infertility,estrous cycle disorder,relieve fatty degeneration in liver tissue and polycystic ovary tissue in PCOS rat model.However,compared with AMSC,it is observed that AMSC-EXO did not improve the insulin resistance in PCOS rats.Our further studies showed that AMSC-EXO promoted glucose uptake by hepatocytes through miR-21-5p targeting the Btg2 gene.This study provides a theoretical basis for the use of rat adipose stem cells and their secreted exosomes to treat PCOS,and provides a new strategy for the treatment of PCOS. |
