Study On The Improvement Effect And Mechanisms Of Celastrol On Lipid And Glucose Disorders Mices Induced By High Fat Diet

Metabolic sydrom has become one of problems which has been a threat to human beings,and glucose and lipid disorders is the impartant parts of it.NLRP3 inflammatory pathway play an important role in the process of metabolic sydrom.Celastrol(CEL),which extracted from traditional Chinese medicine,with anti-oxidation,anti-cancer neovascularization,anti-rheumatoid effect,get the attentions because of a better effect of improving obesity and insulin resistance.However,whether CEL could ameliorate the high fat-induced glucose and lipid disorders through the NLRP3 inflammatory pathway is not yet clear.Therefore,this study established a high fat-induced glucose and lipid disorders mouse model to verify the effect of CEL on the model mice by useing of Western blot detection and nuclear magnetic resonance spectroscopy(1H-NMR)to explore the effect of CEL on the NLRP3 inflammatory pathways and finally provide a theoretical basis for the study of CEL pharmacological mechanism and further clinical application.Objectives1.To explore the effect of CEL on glucose and lipid disorders that induced by high fat diet.and possible mechanisms.2.To revealed the changes of metabolomics in mices that has glucose and lipid disorders by 1H-NMR.Materials and Method1.Grouping of Animals and Collection of SamplesIn this study,C57BL/6J mice were randomly divided into 4 groups: control group(LFD),normal diet with Celastrol treated group(LFD+CEL),and model group(HFD)and high fat diet with Celastrol treated group(HFD+CEL).Group of HFD and HFD+CEL were fed up with high fat diet for 10 weeks.Mice in LFD+CEL group and HFD+CEL group were injected intraperitoneally with Celastrol(100?g/kg/qd)for 6weeks.The mice in the HFD and LFD group were injected intraperitoneally with the same amount of vehicle.After 6 weeks,glucose tolerance test and insulin tolerance test were conducted.After the end of these experiments,the mice were executed and the tissues and blood were collected.The blood samples were centrifuged at-4°C and tissues were conserved at-80 °C.2.Determination of Biochemical ParametersThe levels of triglyceride(TG),total cholesterol(TC),high density lipoprotein(HDL-C),low density lipoprotein(LDL-C),fasting blood glucose(FBS)and insulin(INS)were measured with the kit according to instructions rigorously.3.Glucose Tolerance Test and Insulin Tolerance TestThe mice were fasted for 6 hours before the experiment,then injected with glucose saline solution(2g/kg).Blood samples were taken from the tail vein at 0,15,30,60 and 120 min,and ACCU-CHEK per forma were used to determine the level of blood glucose levels.4.Western BlotThe procedure of SDS-PAGE gel electrophoresis,transferring,blocking and antibody incubation were carried out orderly and rigorously.The main proteins expression of NLRP3-mediated inflammatory pathway(NLRP3,pro-Caspase-1,Caspase-1p20 and IL-1?)were detected by western blot assay.5.Process and Test of1H-NMR Plasma SamplesSamples(200?l),D2O(200?l)and distilled water(100?l)were mixed and centrifuged,and then the supernatant(450?l)were transferred into 5-mm nuclear tube.AVANCE III NMR spectrometer equipped with a 5.0-mm wideband observation(BBO)probe was used under 600.13 MHz.The NMR spectrum was recorded using the water-presaturated standard one-dimensional Carr-Purcell-Meiboom-Gill(CPMG)pulse sequence.6.1H-NMR Mapping and Data Pro-processingUse Topspin3.0 to get the resulting data Fourier transform,manual baseline correction and phase processing.AMIX software to deal with the image: 3.03 ppm creatinine as a standard calibration chemical shift,0.04 ppm as the smallest integral unit will be 0.5-9.5ppm divided into 225 integral sections,remove the water peak(4.84-5.8ppm).Followed by normalization,and export the ASCII format data to Microsoft Excel for Pareto-scaling(Par)calculations.7.Identification of Potential BiomarkersThe potential biomarkers were screened according to the VIP and P <0.05 and compared and identificatid by standard compounds(www.hmdb.ca;www.bml-nmr.org)and the Chenomx NMR software suite(Vers.7.6,Chenomx,Edomonton,Cana,da)Biochemical reactions were identified by the Kyoto Gene,Genome Encyclopedia(KEGG)and Human Metabolites Database(HMDB).8.StatisticsSPSS21.0 software was used for statistical analysis.The measurement data were expressed as `X±S.Two sets of independent samples were used for T test.One-way ANOVA was used for comparison between groups.When P<0.05,the difference was statistically significant.Results1.Effects of Celastrol on lipid and glucose disorders mice induced by high fat diet.Compared with LFD group,the body weight,Groin fat ? Epididymal fat ?perirenal fat tissue weight and adipose tissue and body weight ratio,HOME-IR,FBS,INS,TC,TG and LDL-C levels in HFD group were increased 23%?67%?62%?59%?59%?51%?47%?65%?28%?46%?15%?20% and 29% respectively(P<0.01 or P<0.05);Liver,heart and kidney tissue and body weight,HDL-C levels were decreased 14% ? 38% ? 13% and 20%(P<0.01 or P<0.05)respectively;Insulin tolerance and glucose tolerance test showed that glucose homeostasis and insulin resistance were observed in HFD mice;The content of TG in the liver of the mice increased by 99.5%,and there were Steatosis and inflammatory infiltration obviously;The adipocytes became larger and the boundary was unclear.Compared with HFD group,the body weight,adipose tissue weight,adipose tissue and body weight ratio,HOME-IR,FBS,INS,TC,TG,LDL-C levels were significantly decreased23% ?67%?62%?59%?59%?51%?47%?65%?28%?46%?15%?20% and 29% respectively(P<0.01 or P<0.05);Liver,heart and kidney tissue and body weight,HDL-C levels were increased 17%?16%?25% and 19% respectively(P<0.01 or P<0.05).Insulin tolerance and glucose tolerance test showed that the glucose imbalance and insulin resistance in HFD + CEL group were significantly improved(P<0.01 or P<0.05).TG in the liver of the mice was reduced by 43.7%;there was no obvious steatosis and inflammatory infiltration in the liver and the volume of adipocytes decreased and the boundary was clear;There was no significant difference between LFD and LFD +CEL group in above indexes(P>0.05).2.Effects of Celastrol on NLRP3 inflammasome in lipid and glucose disorders mices induced by high fat diet.Compared with LFD,the results showed that the expression of NLRP3 protein,Caspase-1 p20 protein and IL-1? protein in HFD group were increased by 230%,133% and 310% respectively(P<0.01 or P<0.05).Compared with HFD,the expression of NLRP3,Caspase-1p20 and Il-1? protein in HFD + CEL mice liver decreased by 57%,73% and 78% respectively(P<0.01 or P<0.05).In LFD and LFD+ CEL groups,the expression of above three proteins were no difference.At the same time,the expression of pro-caspase-1 protein was not different between four groups.3.Effect of CEL on Metabolomics in lipid and glucose disorders mices induced by high fat diet.The major differences in metabolites of the four groups were analyzed by PCA and OPLS-DA.Compared with LFD group,in HFD group,LDL / VLDL,FBS,3-hydroxybutyrate and acetoacetate were increased by 20%,45%,10% and 10%respectively,while,pyruvate,lemon Acid,lactate,creatine levels were reduced by20%,20%,20%and 15% respectively;Meanwhile,lactate,alanine,valine,leucine,isoleucine,arginine levels all had different degrees of reduction.Compared with HFD group,LDL/VLDL,FBS,3-hydroxybutyrate and acetoacetate were decreased by 40%,27%,20% and 20% respectively in LFD+CEL mice plasma;Addition to the different increase of valine?leucine?isoleucine?arginine?creatine,Alanine and lactic acid levels were all increased by 30%,30% and 35%;But the citrate and Pyruvate had no effect.However,celastrol had a certain effect on the metabolism of normal mice.According to the analysis of biomarkers,it can be deduced that the regulation of celastrol could be related to the following metabolic pathways: lipid metabolism,gluconeogenesis and glycolysis,tricarboxylic acid(TCA)cycle,creatine metabolism,ketone body synthesis,amino acid metabolism,metabolism of steroid organisms and beta-oxidation of fatty acids.Conclution1.Celastrol can significantly ameliorate disorders of glucose and lipid metabolism induced by high fat diet,Celastrol may improve lipid and glucose disorders induced by high fat diet via inhibiting NLRP3 inflammatory.2.CEL may play an important role in regulating glucose and lipid metabolism by regulating the metabolic pathways of gluconeogenesis,glycolysis,lipid metabolism,TCA cycle,creatine metabolism,amino acid metabolism and fatty acid beta-oxidation.