| Objective:To construct a PCOS rat model by intraperitoneal injection of insulin(INS)combined with human chorionic gonadotropin(HCG)and high-fat and high-glucose diet.The biochemical changes of rat urine were studied by dynamic metabolomics and nuclear magnetic resonance.According to the significant changes in the metabolism of metabolites in the urine of PCOS rats,the correlation between the syndromes and the syndrome types of TCM was analyzed.Methods:Twenty healthy female SD rats were randomly divided into control group and model group(10 rats in each group).The model group was fed with high-fat diet and glucose water(50g.L-1).Protamine zinc recombinant human insulin injection(NPH)was injected subcutaneously into the abdomen every night from day 1 to 10 at a dose of 0.5IU to 5.0IU(i.e.0.5IU on the first day and 1.0IU on the second day,with an increase of 0.5IU per day,and so on).NPH 6.0IU was injected subcutaneously every night from day 11 to22,and HCG 3.0IU(dissolved in 0.2 m L)was given at the same time.The normal group was given normal feed and drinking water and injected with the same liquid as the model group every day.After 22 days,the body weight,ovarian pathological morphology and serum insulin,testosterone and glucose,TNF-α and IRS-1,PI3 K,Akt,IκB and NF-κB protein expression levels were observed to confirm the feasibility of PCOS rat construction.Then,1H-NMR method was used to study the biochemical changes of urine in the PCOS model group,and whether the metabolites and metabolic pathways that changed with the control group were found to be metabolized with arginine and proline,glycolysis/gluconeogenesis,days Metabolism of valine and glutamate,metabolism of phenylalanine,synthesis of phenylalanine,tyrosine and tryptophan,TCA cycle,lysine metabolism,taurine metabolism,tryptophan metabolism,inositol phosphate Metabolism-related,based on changes in metabolites and metabolic pathways,explores the relationship between phlegm and blood stasis in TCM is discussed.Results:1.Rats in the model group gained weight compared with the control group(P<0.05),The ovaries showed polycystic changes,serum testosterone and insulin levels increased significantly compared with the control group,and glucose levels increased significantly compared with the control group(P<0.05);TNF-alpha levels of inflammatory factors increased compared with the control group(P<0.05);The expression of Akt and I-kappa B protein was lower than that of the normal group(P<0.05),and the expression of NF-kappa B(p65)protein was significantly higher than that of the control group(P<0.05).2.Analysis of urine and control group of model group rats,found that there are significant changes in 26 metabolites,namely formic acid,phenylalanine,histidine,tryptophan,urea,tyrosine,threonine,methionine,creatinine,creatine,taurine,lactate,lysine,citrate,acetate,ethanol,trimethylamine,dimethylamine,isoleucine,glucose,succinic acid Salt,glutamine/glutamate,phenylacetylglycine,glutamic acid and inositol.3.On the 10 th day of modeling,metabolic pathway disorders mainly occurred in 10 metabolic pathways,namely,arginine and proline metabolism,glycolysis/gluconeogenesis,aspartic acid and glutamic acid metabolism,phenylalanine metabolism,phenylalanine,arginine,tyrosine and tryptophan synthesis,TCA cycle,lysine metabolism,taurine metabolism,tryptophan metabolism,inositol phosphoric acid metabolism.It is mainly related to the metabolism of arginine and proline.4.On day 22,metabolic disorders may occur in TCA cycle,arginine and proline metabolism,lysine metabolism,phenylalanine metabolism,glycolysis/gluconeogenesis,taurine and taurine metabolism,and inositol phosphoric acid metabolism.Conclusion: 1.The weight of rats in model group was higher than that in control group,and the ovaries showed polycystic changes.Serum levels of T,INS,GLU and TNF-alpha increased significantly.The expression of IRS-1,PI3K,Akt and I-kappa B protein was lower than that of the normal group(P<0.05),and the expression of NF-kappa B(p65)protein was significantly higher than that of the control group(P<0.05).2.This study found that the increase of TNF-alpha indicated that there was inflammatory reaction in PCOS.The decrease of IKK protein indicated that IKK was activated and promoted the activation of NF-kappa B pathway.The decrease of IRS-1,PI3K and Akt protein indicated that the activation of IKK promoted the serine phosphorylation of IRS,which led to the inhibition of insulin signaling pathway and inhibited the downstream transmission of insulin signal,leading to IR.3.This experiment simulated the pathogenesis of phlegm-blood stasis junction,and used 1H-NMR to study the mechanism of phlegm-blood stasis junction PCOS for the first time.In the process of phlegm and blood stasis,the disorder of glycolipid metabolism,the increase of inflammatory factors and the change of Hemorheology are often accompanied.In the PCOS model group,inflammatory factors increased significantly.From the analysis of urine,26 metabolites were found to change,12 endogenous metabolites with significant differences were found to be potential biomarkers in the pathological process of PCOS at different stages,and 10 metabolic pathways were found to be involved in the development of PCOS at different stages,which were mainly related to the metabolism of arginine and proline.One of the main causes of phlegm and blood stasis in POCS is excessive accumulation of insulin,which causes abnormal insulin signaling pathway.4.PCOS process is accompanied by chronic low-grade inflammation and impaired vascular endothelial cell function.The role of inflammatory mediators is related to arginine and proline metabolism and NO pathway.Arginine and proline metabolic disorders are the major metabolic pathways in the pathogenesis of PCOS,which are significantly correlated with insulin signaling pathway and NF-kappa B pathway.These pathways are mainly activated by inflammatory factors through IKK pathway,which induces phosphorylation and degradation of I-kappa B and dissociates from NF-kappa B.This indicates that there are abnormalities of insulin signaling pathway and NF-kappa B pathway in the pathogenesis of phlegm-blood stasis junction and PCOS.The two pathways have a cross-action and are mainly mediated by inflammatory factors. |
PDF Full Text Request