Expression Of Ovarian Inflammation Related Genes In PCOS Mice Based On Transcriptome Sequencing And The Effects And Mechanisms Of Metformin On Chronic Inflammation In PCOS Mice

Backgroundpolycystic ovary syndrome(PCOS)is a reproductive and metabolic disorder that affects women of reproductive age.Chronic low-grade inflammation has been regarded as a critical contributor to the pathophysiology of PCOS.In women with PCOS,inflammation leads to hyperandrogenism,obesity,and insulin resistance.Inflammatory biomarkers play an important role in the progression of chronic inflammation and the incidence rate of this chronic inflammation is about 95% of women with PCOS,yet its pathogenesis is not fully understood.Women with PCOS have a number of ovarian complications.These complications include inflammation,decreased folliculogenesis,anovulation,and poor oocyte quality,which all have an effect on their reproductive health.It is important to start with inflammation genes,which are controlled by these processes,because these inflammatory genes play an important role in how this disorder is treated.Materials and methodsTo achieve this,we used a deep sequencing approach to compare the inflammatory m RNA expression profiles of ovarian tissues between a DHEA plus HFD-induced PCOS mouse model and control mice.Additionally,the chemokine C-X-C motif ligand 13(CXCL13)and its receptor type 5(CXCR5)are well-known to play important roles in ovarian cancer and inflammatory responses.The pathophysiology of these inflammatory chemokines in PCOS,however,remains unknown.Furthermore,metformin plays an important role in inflammatory genes and its work as an anti-inflammatory drug.Metformin is also administered to PCOS patients,but its mechanism of action is not fully known.Thus,we also aimed to determine the expression of CXCL13 and CXCR5 in PCOS mouse ovaries and to evaluate the therapeutic effect of metformin on them.The PCOS mouse model was induced using dehydroepiandrosterone(DHEA)and a high-fat diet(HFD).For the RNA-seq study,six samples were divided into two groups(control and PCOS),with three biological replicates in each group.This was followed by hierarchical clustering,Gene Ontology(GO),and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses.The relative expression levels of nine inflammatory genes were validated via quantitative reverse transcriptionpolymerase chain reaction(q RT-PCR).For the inflammatory chemokine study,the mice(25/group)were randomly divided into four groups: control,PCOS,PCOS plus metformin,and PCOS plus vehicle.Immunohistochemistry(IHC)was used to assess the expression and localization of CXCL13 and CXCR5 in ovarian tissues.q RT-PCR and western blotting were used to determine the levels of CXCL13 and CXCR5 m RNA levels and protein expression in ovarian tissues.Additionally,western blotting was also performed to determine the level of extracellular signaling-related kinase(ERK)protein expression.ResultsA total of 436 genes were differentially expressed between the control and PCOS mice.Out of these,137 genes were up-regulated while 299 genes were down-regulated.Gene ontology analysis indicated that differentially expressed m RNAs were associated with T cell-mediated cytotoxicity and homocysteine metabolic processes.Pathway analysis further showed that these abnormally expressed m RNAs were associated with signaling pathways,such as NF-kappa-B signaling,tyrosine metabolism,and phenylalanine metabolism,all of which are associated with chronic inflammation and PCOS.Additionally,the inflammatory chemokines CXCL13 and CXCR5 were found to be overexpressed in the serum and ovarian tissues of dehydroepiandrosterone(DHEA)plus high-fat diet(HFD)-induced PCOS mice.Metformin reduced ovarian CXCL13 and CXCR5 expression in PCOS mice.This indicated that metformin treatment could be used as a CXCL13 inhibitor in PCOS.Moreover,expression of CXCL13 and CXCR5 were found to be associated with enhanced activation of the phosphorylated ERK1/2 pathway.ConclusionsThe differentially expressed genes are potentially involved in the inflammation that is evident in PCOS,and so they can serve as therapeutic options for the disease.Nevertheless,prospective studies are needed to test this hypothesis.Additionally,the current study also established an association between the CXCL13/CXCR5 and PCOS,which could serve as a solid basis for further studies regarding the pathophysiology and management of this disease.Furthermore,CXCL13 and CXCR5 are upregulated in PCOS mice ovaries;and their downregulation following metformin administration point to their potential involvement in the pathogenesis of PCOS.