Function And Regulatory Mechanism Of Melatonin In The Progression Of Polycystic Ovary Syndrome

Polycystic ovary syndrome(PCOS)is a reproductive disorder arising from the interaction of genetic and environmental factors.It seriously affects the reproductive health of childbearing age women.Its clinical features mainly include rare ovulation or non-ovulation,hyperandrogenism and polycystic ovarian changes.Despite many advances in the understanding of the pathobiology and therapy of PCOS have been obtained over the past decades,a lot of questions remain to be answered.In addition,there is no unified standard for the treatment of this disease,and the pathogenesis of this disease is also not completely clear.Melatonin,also known as N-acetyl-5-methoxytrypamine,is an indolamine hormone mainly secreted by pineal gland.Melatonin is also generated in other organs such as gastrointestinal tract,skin,retina,bone marrow,lymphocytes,and female reproductive organs.Melatonin has been identified to excert different pharmacological properties,including antioxidant,immunomodulatory,and anti-angiogenic,etc.Moreover,melatonin is also a powerful free radical scavenger to protect oocytes and fetuses from oxidative stress damage.Although melatonin can improve menstrual cycle disorder and reduce the level of serum testosterone in PCOS patients,however,the regulatory mechanism of melatonin in the progression of PCOS is still unclear.First of all,the melatonin and its metabolite 6HMS in follicular fluid of PCOS patients and control were determined in this study.The results showed that the level of melatonin and its metabolite 6HMS in follicular fluid of PCOS patients was significantly lower than the control group,indicating that there was a close relationship between the level of melatonin in follicular fluid and the occurrence of PCOS.A PCOS mouse model was used in this study,the estrus cycle of PCOS mice was restored after injection of melatonin.In addition,it significantly reduced the levels of T,LH,E and P,reduced LH/FSH ratio,and increased FSH level in serum.The insulin sensitivity of PCOS mice was also significantly improved.In PCOS mouse model,melatonin treatment could increase the total antioxidant capacity of ovarian tissue,increase the antioxidant enzymes activities of SOD、CAT、GPX and GR,GSH content and GSH/GSSG ratio,and reduce the content of MDA,a marker of oxidative stress in PCOS mice.The effect of melatonin on mouse gut flora was analysed by 16s r DNA sequencing.The results showed that melatonin could increase the abundance of Firmicutes,Tenericutes and Patescibacteria,and reduce the abundance of Bacteroidetes,Epsilonbacteraeota and Actinobacteria in PCOS mice.At family level,melatonin could raise the abundance of Lachnospiraceae and Ruminococcaceae,and decline the abundance of Prevotellaceae,Muribaculaceae and Coccophora＿langsdorfii.The results of non-target metabonomic analysis indicated that 20 metabolites such as DL-leucine and L-tryptophan were increased significantly,however,140 metabolites such as tryptophol,phytantriol and N-arachidonoyl proline were decreased obviously in PCOS mice compared with the control group.Addition of melatonin could reverse the contents of 46 metabolites,including tryptophol,phytantriol and L-tryptophan,etc.The results of KEGG pathway enrichment analysis found that these differential metabolites were mainly involved in tryptophan metabolism,biosynthesis of amino acids,steroid hormone biosynthesis,etc.Further research found that L-tryptophan was positively correlated with Coccophora＿langsdorfii and Nitrosomonadaceae,and negatively correlated with Familly＿XIII.Tryptophan metabolite tryptophol was positively correlated with Grateloupia＿livida and Pseudoalteromonadaceae.Molecular docking and molecular dynamics simulation technology displayed that tryptophol could bind to Aryl hydrocarbon receptor(Ah R),and the RMSD complex fluctuated within±0.05.Inhibition of Ah R could prevent melatonin from improving the endocrine function,significantly up-regulate the expression of Bax and the activity of Caspase-3,down-regulate the expression of Bcl-2,and raise the apoptosis rate of ovarian Granulosa cells(GCs).In order to further study the mechanism of melatonin relieving PCOS,RNA-seq was used to screen the differential expression gene in mouse ovary.The results showed that melatonin could restore 431 differential expression genes.Fluorescence quantitative PCR and immunoblotting further verified that melatonin could up-regulate the expression of Malic enzyme 1(Me1)in the ovary of PCOS mice,but this effect could be blocked by Ah R inhibitor CH223191.Silencing of Me1attenuated the improvement of melatonin on the endocrine function and apoptosis of GCs,blocked the effect of melatonin on NADPH content and NADPH/NADP~+ratio in GCs.The activity of GR,the content of GSH and the GSH/GSSG ratio in GCs were reduced.In melatonin-treated GCs,addition of NADPH could save the decrease of GR activity and GSH content caused by Me1 deficiency.Inhibition of GR could prevent NADPH from rescuing GSH content and GSH/GSSG ratio.Further research found that melatonin could reduce ROS level in GCs,while transfection of Me1 si RNA could block the regulation of ROS by melatonin.However,this effect could be weakened by NADPH and GSH.Meanwhile,either NADPH or GSH treatment could improve the endocrine function disorder of GCs caused by Me1 deficiency to finally remarkably reduce the apoptosis rate of GCs.Mitochondria are important organelles that produce energy in cells.After treated with melatonin,the ATP level,mt DNA copy number and mitochondrial membrane potential increased significantly,however,the mitochondrial oxidative stress sensitive protein decreased significantly in GCs.Silencing of Me1 could attenuate the effect of melatonin on mitochondrial function,however,this effect could be blocked by NADPH and GSH.Further research found that melatonin could reduce the phosphorylation of Ser616 in Drp1,prevent Drp1 from being recruited to the outer membrane of mitochondria,and restrain mitochondrial network fragmentation.However,transfection of Me1 si RNA could prevent melatonin from regulating mitochondrial division and fragmentation.But adding NADPH or GSH could alleviate the antagonistic effect of Me1 deficiency on melatonin.In summary,melatonin could promote the metabolism of tryptophan to tryptophol by improving gut flora.After combining with Ah R,tryptophol could regulate the endocrine function of GCs through Me1-NADPH-GSH pathway by preventing apoptosis of GCs,maintaining the integrity of mitochondrial function,reducing mitochondrial division,so as to participate in the occurrence of PCOS.These results will provide important theoretical basis for further exploring the pathogenesis of PCOS and the clinical development of safe and effective PCOS therapeutic drugs.