Study On The Effect Of Celastrol And Its Analogues To Insulin Resistance

Objects:In vitro and cellular model antioxidant experiments,we study the antioxidant activity of five pentacyclic triterpene acids,including celastrol,ursolic acid(UA),oleanolic acid(OA),corosolic acid(CA)and malonic acid(MA).The cellular and mouse IR models were made to study the effects of the tested drugs on IR,and the molecular mechanisms were preliminarily explored in the present study.Methods:The antioxidant activity of the tested drugs was evaluated by DPPH free radical scavenging assay.AAPH was used to construct the oxidative stress damage model of HAEC cells to evaluate the protective effect of the tested drugs on the intracellular oxidative stress injury;IR model in HAEC cells,induced by high concentration of insulin and obese IR model in C57BL/6 mice induced by high fat diet,were used to evaluate the effects of the tested drugs on IR;Total RNA and the total protein were extracted from HAEC cells and liver,blood vessel and fat from mice.RT-qPCR and Western Blot were used to detect the mRNA transcription and protein expression levels of related genes in IR signaling pathway.Results:1.In vitro antioxidant experiment showed that at 0.4,0.6,0.8,1 mg/mL concentration gradient,DPPH radical scavenging rate of celastrol was above 90%,other tested drugs'were above 60%;the antioxidant capacity of the tested drugs were associated with active double bond,hydroxyl and carboxyl groups in the moleculars.2.It was confirmed that the optimal concentration of AAPH was 5 mmol/L.except OA and MA,other tested drugs had significant inhibitory effects intracellular ROS,suggesting that the tested drugs had significant protective effects on oxidative stress.Celastrol showed significant protective effects in 0.125,0.25,0.5 ?g/mL series dose(P<0.05).3.It was found that the optimal concentration of insulin was 10-6 mol/L,and all of the tested drugs could significantly increase the glucose consumption of IR model cells and improve the cellular IR in HAECs.We determined the optimal concentrations of tested drugs for the next mechamism study:0.5 ?g/mL for celastrol,10 ?g/mL for UA and OA,2?g/mL for CA and MA;After treating IR cells with tested drugs at these concentrations,we extracted the total cellular RNA and protein for the mechanism assay.4.The possible mechanisms of tested drugs improving HAECs IR:celastrol and its analogues can increase transcriptional levels of InsR,IRS1,PI3-K,GLUT4 and PDE3B,increase the protein expression of AKT and PPAR-?.5.The C57BL/6 mouse IR models were induced by high-fat diet,celastrol dosage was 2 mg/kg/day,the total triterpene acids from loquat leaf(UA,OA,CA and MA)at doses of 200 and 400 mg/kg/day,with i.g.administration.Tested drugs showed significant improvement on IR in mice;through the analysis of serum biochemical indexes,weight detection,organ's HE staining and Lee's index determination,the results show that the tested drugs could improve hepatic injury,lipid metabolism,oxidative stress and leptin resistance,which were resulted by obesity,to improve the mice's IR,and celastrol also had significant anti-obesity effect(P<0.01).Conclusion:oxidative stress is an important factor which induces IR.Celastrol and its analogues have the ability to scavenge free radicals,and show good antioxidant capacity in vitro and in HAEC cells.This indicating that celastrol and its analogues have potential activity in improving IR.The cell and animal experiments proved that the tested drug does improve IR in some degree.The molecular experiment indicated that the tested drugs work by regulating the transcription of the mRNA and the expression levels of the proteins in insulin signaling pathway.