| Objective:To investigate the erythrocyte membrane dysfunction and the intervention effect of Gegen Qinlian decoction（GQD）in obese pre-Type 2 Diabetes Mellitus（T2DM）rats with insulin resistance and hypoxia（IR-hypoxia）.Methods:1.Established the model of obese pre-T2DM rats with IR-hypoxiaSD rats were given high-fat diet with 60%fat,and the normal group was given control diet.Orbital blood samples were collected at the 13th week and serum samples were prepared to measure Fasting plasm-glucose（FPG）,Fasting insulin（Fins）levels and to calculate IR index;the levels of Hypoxia inducible factor-1α（HIF-1α）,Hypoxia inducible factor-2α（HIF-2α）and Erythropoietin（EPO）in serum were detected by ELISA kits;the establishment of model of obese pre-T2DM with IR-hypoxia was based on a significant increase in IR index and HIF-2α levels in rats.2.Pharmacodynamic of GQD in obese pre-T2DM rats with IR-hypoxiaAfter the successful establishment of the model,rats were randomly divided into model group,rosiglitazone group（positive drug group）,GQD low,medium and highdose groups.Normal group was given control diet,model group and each administration groups were continued to feed with high fat diet;dosages:Rosiglitazone group(5 mg·kg-1),GQD low,medium and high dose groups(crude drug concentration of 1.65,4.95,14.85 g·kg-1),administration volume:10 mL·kg-1.After 16 weeks of administration,blood gas indexes were detected by blood gas analyzer,and levels of FPG,Fins,HIF-1α,HIF-2α and EPO were detected in serum samples,and IR index was calculated to determine the pharmacodynamics of GQD.3.Lipidomics of erythrocyte membrane in obese pre-T2DM rats with IR-hypoxia and after GQD interventionAt the 13th week of high fat diet,the rats with significantly increased IR index,HIF-2α and EPO in high fat group were divided into hypoxia group,and the rats with only significantly increased IR index were divided into non-hypoxia group.Erythrocyte membrane samples were extracted from the rats fed high fat diet for 16 weeks and GQD administration for 16 weeks.Lipidomics techniques were applied to detecting and collecting data.VIP>1,and the criteria of fold change（FC）≥1.3 or ≤0.77 and P<0.05 were used to screen the differential metabolites of erythrocyte membrane in obese preT2DM rats with IR-hypoxia and after GQD intervention.In addition,fragment matching was performed in HMDB database,and metabolite pathway analysis was performed in combination with MetaboAnalyst 5.0 online database,to explore the metabolic mechanism of erythrocyte dysfunction in pre-T2DM rats and the mechanism of action after GQD intervention.4.Metabolomics of erythrocyte contents and serum in obese pre-T2DM rats with IRhypoxia after GQD interventionSamples of erythrocyte contents and serum were collected from each group at 16 weeks of GQD administration.Metabolomics techniques were applied to detecting and collecting data,identifying potential biomarkers,performing metabolic pathways,and elucidating the relevant metabolic mechanisms.Results:1.Establishment of the model of obese pre-T2DM rats with IR-hypoxiaAfter 13 weeks of high fat diet,IR index of rats in high fat group was significantly increased（P<0.01）,indicating that all rats produced IR at this time,but only 50%of the rats had significant increase of HIF-2α levels in serum（P<0.01）,resulting in hypoxia phenomenon.The model of obese pre-T2DM rats with IR-hypoxia was successfully established,with a modeling rate of 50%.2.Pharmacodynamic results of GQD intervention in obese pre-T2DM rats with IRhypoxiaWhen GQD was administered for 16 weeks,the IR index,HIF-2α and EPO levels were significantly increased in the model group compared with the normal group（P<0.01）;compared with model group,IR index of rats was significantly decreased in all GQD dose groups（P<0.05,P<0.01）,the HIF-2α levels in serum of rats in low and high dose GQD groups were significantly decreased（P<0.05,P<0.01）,and EPO levels in serum of rats in the GQD medium and high dose groups were significantly decreased（P<0.05）.Correlation analysis showed that IR correlated more strongly with HIF-2αand EPO and less strongly with HIF-1α.The results of blood gas analysis showed that there was no statistical difference in all the indexes except for a significant increase in plasma lactate concentration（cLac）and a significant decrease in partial pressure of carbon dioxide（pCO2）in the model group（P<0.05）.3.Erythrocyte membrane lipidomic results in obese pre-T2DM rats with IR-hypoxiaCompared with normal group,IR index,HIF-2α and EPO contents of rats in hypoxia group were significantly increased（P<0.05,P<0.01）,and IR index of rats in non-hypoxia group was increased（P<0.01）,but HIF-2α and EPO had no significant changes（P>0.05）after feeding high fat diet for 13 weeks;at the 16th week of high fat diet,blood gas analysis results showed no significant changes in various indexes of the either group.Lipidomics analysis of erythrocyte membranes of rats in hypoxia group revealed significant changes in the content of 57 metabolites,mainly phospholipids,glycerol esters and cholesterol esters;statistical analysis of the metabolites identified by classification revealed that the contents of total phospholipids,total saturated phospholipids and total cholesterol esters in erythrocyte membrane of rats in hypoxia group and non-hypoxia group were significantly increased（P<0.01）,which probably affected the function of erythrocyte.4.Erythrocyte membrane lipidomics results of GQD intervention in obese pre-T2DM rats with IR-hypoxiaAfter 16 weeks of GQD administration,101 different metabolites were identified in the model group.Classification statistics showed that there was no significant difference in total phospholipid content of erythrocyte membranes（P>0.05）,a significant increase in total saturated phospholipids and total cholesterol esters（P<0.01）,and a significant decrease in total unsaturated phospholipids（P<0.01）in the model group;GQD could significantly back-regulate 15 of them.Statistical analysis showed that the contents of total phospholipid and total saturated phospholipid were significantly increased（P<0.01）,and total unsaturated phospholipid contents were obviously decreased（P<0.01）in the model group.the GQD low dose group could significantly back-regulate total phospholipid,total saturated phospholipid,and total unsaturated phospholipid contents（P<0.05,P<0.01）,and total unsaturated phospholipid contents were significantly increased in the GQD medium and high dose groups（P<0.05,P<0.01）.GQD regulates erythrocyte function and improves IR and hypoxia in the body,probably by altering phospholipids,sphingolipids and glycerides.5.Metabolomics results of erythrocyte contents in obese pre-T2DM rats with IRhypoxia after GQD interventionAfter 16 weeks of GQD administration,a total of 47 metabolites were significantly altered in the model rats;GQD could modulate 22 metabolites and involve 11 metabolic pathways,which could affect lipid metabolism such as phospholipids and glycerides and glucose metabolism,and regulating erythrocyte function.6.Serum metabolomics results of GQD intervention in obese pre-T2DM rats with IRhypoxiaSerum metabolomics results showed that GQD could regulate 20 metabolites in serum,involving 7 metabolic pathways,mainly regulating the body’s lipid metabolism and glutathione metabolism,and improving IR and hypoxia.Conclusion:1.Obese pre-T2DM with IR-hypoxia model rats induced by high fat diet were not hypoxic in the circulatory system and tissues show chronic hypoxia with a 50%modeling rate.2.Tissue hypoxia in obese pre-T2DM with IR-hypoxia model rats induced by high fat diet was associated with altered erythrocyte membrane lipids,with abnormal erythrocyte function leading to tissue hypoxia.3.Efficacy of clearing heat and drying dampness in GQD probably could change the fluidity of erythrocyte membrane,regulate the function of erythrocyte,and thus improve the hypoxia phenomenon in obese pre-T2DM rats with IR-hypoxia.4.Glycerophospholipid metabolism had an important role in erythrocyte dysfunction. |
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