| Objective:High-fat diet indu To investigate the effects of high-fat diet-induced insulin resistance(IR)in pre-Type 2 diabetes mellitus(T2DM)rats with hypoxia on fecal metabolism group and the intervention of Ge Gen Scutellaria Tang(GQD),and further explore the metabolic mechanism of GQD to improve IR with hypoxia in pre-T2 DM.Methods:1.Establishment of IR with hypoxia modelEighty-four SPF male SD rats were divided into 12 normal and 72 high-fat groups according to the random number method,and the normal group was given control chow and the high-fat group was fed with high-fat chow.Fasting insulin(Fins),fasting blood glucose(FPG),steady-state assessment of IR index(HOMA-IR),serum hypoxia-inducible factor-1α(HIF-1α),hypoxia-inducible factor-2α(HIF-2α)and other indicators were monitored,and oral glucose tolerance test(OGTT)was performed to measure blood glucose values before(0 min)and after(15,30,60,120min)rats.The data were also analyzed to determine the establishment of a model of IR with hypoxia in rats with pre-obese T2 DM.2.Fecal metabolomics in the successful IR with hypoxia modelWhen the model was successfully established,six rats in the normal group and six rats in the high-fat group were selected as metabolomics samples,and the lipophilic substances were extracted by methyl tert-butyl ether(MTBE)/methanol(MEOH)extraction,and the lipophilic substances in the samples were detected,screened and identified by using metabolomics technology,and matched with the substances in the bioinformatics database.To profile the different metabolites in the feces of obese T2 DM pre-IR with hypoxia rats,screen and identify potential biomarkers from the level of lipophilic substances,and search for possible metabolic pathways in the obese T2 DM pre-IR with hypoxia model.3.Pharmacological effects of GQD in IR with hypoxia model ratsThe remaining 66 SPF-grade male SD rats were randomly divided into 6 rats in the normal group and 60 rats in the high-fat group and fed with normal control diet and high-fat diet,respectively.30 rats with successful modeling(50% success rate)were divided into model group,GQD high,medium and low dose group and rosiglitazone group according to the random number method,with 6 rats in each group.The normal group and the model group were gavaged with saline,the rosiglitazone group was gavaged at 5 mg/kg,and the GQD high,medium and low doses were gavaged at 14.85,4.95 and 1.65 g/kg of raw drug,and the volume of administration was 10 ml/kg for 16 weeks.The changes of Fins,FPG,HOMA-IR,and HIF-1α and HIF-2α were observed in each group of rats.Hematoxylin eosin(HE)staining was used to observe the morphological changes of colonic mucosa,and immunohistochemistry(IHC)technique was used to observe the changes of HIF-2α,interleukin 10(IL-10)and tumor necrosis factor α(TNF-α)in the intestinal mucosa of each group.4.Study on the metabolic mechanism of GQD intervention in IR with hypoxia model ratsStool samples were selected from normal group,model group,GQD high,medium and low dose group and rosiglitazone group,and the samples of each group were correlated by metabolomics technology,matched and identified with the substance information in HMDB database to elucidate the metabolic mechanism of GQD to improve IR and hypoxia,and to find the possible metabolic pathways.Results:1.Starting from the 2nd week of high-fat diet feeding,the body weight and lee’s index of rats in the high-fat group were significantly higher compared with the normal group.There was a statistically significant difference(P<0.01),while a significant and statistically significant difference(P<0.01)in body length was observed between the normal and high-fat groups starting from week 3.The results of OGTT from time-glucose relationship and AUC before the 13 th week of modeling showed that the glucose tolerance of rats in the high-fat group was significantly decreased compared with the normal group and was statistically significant(P<0.01).The results of detecting serum HIF-2α and HIF-1α showed that,compared with the normal group,the expression of HIF-1α in the high-fat group rats had a tendency to increase but was not statistically significant,while the expression of HIF-2α increased with significant difference and was statistically significant(P<0.01).2.After metabolomics technology,2126 metabolites were detected in positive mode and 949 metabolites were detected in negative mode,and all metabolites were screened for significantly different metabolites by unidimensional and multidimensional statistical analysis between normal group and high-fat group in positive and negative mode,and substance matching and identification of significantly different metabolites were performed by HMDB and Metabo Analyst database.The final screening of 2 groups of potential biomarkers(Cer(d18:1/16:0),Galactosylceramide(d18:1/18:1(9Z)),Phosphatidylcholine,2beta-Hydroxytestosterone,Leukotriene A4,Prostaglandin H2,Thromboxane A2,9,10-Epoxyoctadecenoic acid),etc.A total of 27 metabolic pathways were enriched by KEGG pathway enrichment analysis,involving metabolic pathways such as sphingolipid metabolism,arachidonic acid metabolism,linoleic acid metabolism,glycerophospholipid metabolism,tryptophan metabolism,etc.,indicating that This indicates that lipid disorders were observed in the model of IR with hypoxia caused by high-fat diet.3.After 16 weeks of GQD intervention,Fins,FPG,HOMA-IR and HIF-2αwere significantly increased in the model group compared with the normal group(P<0.05),and compared with the model group,there was a significant decrease in the high and low dose groups of GQD and rosiglitazone group(P<0.05),but there was no significant difference in the medium dose group of GQD.In comparison with the normal group,the model group showed disorganized colonic mucosal tissue glands,incomplete structure,thickened intestinal wall and inflammatory cell infiltration,and the colonic mucosal tissue was repaired to different degrees after different dose interventions of GQD.The expression of HIF-2α and TNF-α was increased and the expression of IL-10 was decreased in the model group.4.1728 metabolites were detected in positive mode and 2120 metabolites were detected in negative mode by metabolomics technology,and the data were analyzed in positive and negative mode to screen for significantly different metabolites in each group respectively.The HMDB and Metabo Analyst databases were used for substance matching and identification of significantly different metabolites to find potential biomarkers,and finally Arachidonic acid,PC(22:1(13Z)/P-18:1(11Z)),PC(24:1(15Z)/P-18:1(11Z)),PC(18:1(11Z)/24:0),Lyso PC(24:0/0:0),CE(5:0),Calcidiol and other 33 potential biomarkers were enriched by KEGG pathway enrichment analysis to a total of 9 metabolic pathways,mainly affecting lipid synthesis and metabolism related sphingolipid metabolism,arachidonic acid metabolism,steroid biosynthesis and other pathways,indicating that GQD ameliorates IR with hypoxia metabolic disorders in the rat model mainly through the back regulation of lipid synthesis and metabolism.Conclusion:1.This experiment showed that high-fat diets could induce IR and hypoxia in pre-T2 DM.And the analysis of rat fecal samples by metabolomics techniques revealed that hypoxia in model rats may be related to lipid-related metabolic pathways such as sphingolipid metabolism,arachidonic acid metabolism and linoleic acid metabolism.2.The results of this experiment showed that GQD could improve IR and hypoxia in the model rats and improve the inflammatory response caused by intestinal hypoxia.3.The results of this experiment showed that GQD could improve the IR and hypoxia phenomenon in pre-T2 DM through regulating the metabolic pathways of arachidonic acid metabolism,glycerophospholipid metabolism and steroid biosynthesis. |
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