| Objective: To make induction and differentiation of themouse3T3-L1pre-adipocytes，to induce3T3-L1adipocytes into theinsulin resistance model by free fatty acid （PA was used in theexperiment）. Then ecdysterone （ECR） were used to intervene in theproliferation of3T3-L1adipocytes. To explore the influence on insulinresistance of the cells and the possible molecular mechanisms in vitro,that was whether it could alleviate the insulin-resistant by inhibiting theproteins`expression of IKK β and NF-κ B in the inflammationsignaling pathway of NF-κ B;whether it could alleviate adiposeinflammation by decreasing the gene`s expression of TLR4. Methods: Tocultivate the mouse3T3-L1pre-adipocytes in the high glucose DMEMand change the high glucose DMEM every2days, when the cells mixedtogether to75%or so, to cultivate them into3T3-L1adipocytesaccording to priority by DMEM（H） with10μg/ml insulin,0.5mmol/L3-isobutyl-1-methylxanthine （IBMX）,1μmol/L dexamethasone for2days,then to cultivate them into high glucose DMEM with10μg/ml insulin for6～8days, to change the high glucose DMEM with10μg/ml insulinevery2days; at last to go on cultivating them into high glucose DMEM,it spent12～14days altogether for inducting and differentiating themouse3T3-L1pre-adipocytes into3T3-L1adipocytes. To cultivate 3T3-L1adipocytes in the high glucose DMEM with2g/L BSA for12h,then in the DMEM with0.5mmol/L PA and10g/L FAF BSA for24h toset up the insulin resistance model. At the end, to mix the ecdysterone indifferent concentration or the pioglitazone in the concentration of1×10-5mol/L in each DMEM to cultivate together for24h.To test theeffect of ecdysterone on the growth of3T3-L1adipocytes.To determinethe glucose consumption of cultivating solution in each group. Todetermine the proteins expression of IKKβand NF-κB by Western blot,to determine the m RNA expression of TLR4receptor by RT-PCR. Atthe same time,there were6groups in the experiments:（1）blank contrastgroup:3T3-L1adipocytes;（2） model group:fat cell,that was the insulinresistance model;（3）ECR groups:3groups ECR with differentconcentration (ECR1×10-7, ECR1×10-6and ECR1×10-5mol/L);（4）pioglitazone group:the concentration was1×10-5mol/L. Results:the ODvalue decreased obviously （P<0.01）,when the ECR`s concentration wasgreater than1×10-4mol/L, which inhibited the growth of3T3-L1adipocytes; Between the concentration1×10-7mol/L～1×10-5mol/L,thecells growth rate was92.70%～100%, good growth; Between theconcentration of ECR1×10-7mol/L～1×10-5mol/L, the glucoseconsumption of cells were increased; the proteins expression of IKKβand NF-κB and m RNA expression of TLR4receptor in insulinresistance cells were increased obviously, after the intervention of ECR(ECR1×10-7, ECR1×10-6and ECR1×10-5mol/L), the expression ofthem in different group decreased in different degree. Conclusions:（1）Free fatty acids could make the cells being in inflammationstate,activate TLR4signaling pathway,result in insulin resistance;（2）when the concentration of ECR was above1×10-4mol/L，which inhibitedobviously the growth of the adipocytes, so the concentration of ECRchosen in the experiment was1×10-7mol/Lã€1×10-6mol/Lã€1×10-5mol/L;（3）ECR could improve the glucose consumption of the insulin-resistant cellsand depressed the proteins expression of IKKβ and NF-κB in theTLR4signaling pathway of the fat cells and reduced the expression ofTLR4m RNA in the fat cell membrane which showed ECR could relieveinsulin resistance induced by FFA in3T3-L1adipocytes and its possiblemolecular mechanism might be associated with inhibiting the TLR4signaling pathway. |
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