| Objective:To study the mechanism of action of Tang Naikang about inflammation mediating insulin resistanceMethods:Experiment was divided two parts1 Experiment in vitroTo Apply 3T3-L1 adipocytes as cellular model of insulin resistance.First,3T3-L1 pre -adipocytes were differentiated into mature adipocytes and dyed by oil red "O" to identi -fy.Then make use of dexamethasone and insulin to induce insulin resistance in 3T3-L1 adipocytes.At the same time,prepared the drug serum containing TNK,Pioglitazone and blank and established serum concentration with MTT method.Divided adipocytes into seven groups,such as normal adipocyte group,model group,blank serum group(6.4% serum),TNK-low dosage group(2.56%serum),TNK-middle dosage group(6.4%serum), TNK-high dosage group(16%serum),Pioglitazone group(6.4%group).Treated the cells in 72th and collected supernates to test the levels of tumor necrosis-alpha(TNF-α), interleukin-6(IL-6),C-reactive protein(CRP),floating fatty acid(FFA) by enzyme linked immunosorbent assay(ELISA) and colorimetric method.To detect the expression contents of InsR,p-IRS-1 Tyr,p-IRS-1 Ser307,GLUT-4 protein and PPAR-γmRNA,IKKβmRNA by western blot and Real-time PCR.Tested the acitivity of NF-κB and AP-1 protein by electrophoretic bobility shift assay(EMSA).2 Experiment in vivoTo apply spontaneous GK rats accompanied high fat forages to duplicate insulin resistance animal model of T2DM.Specific pathogen free(SPF) and male GK rats 55 were entered into the experiment according to random blood sugar value≥11.1mmol/L and divided randomly into five group,such as model group(same volum distilled water), Pioglitazone group(0.405mg/ml/100g.d),TNK-low dosage group(0.116mg/ml/100g.d), TNK-middle dosage group((0.232mg/ml/100g.d),TNK-high dosage group(0.464mg/ml/ 100g.d),and Wistar rats 10 were normal control group(same volum distilled water).The rats were administrated successivly for 8 weeks and observed separately oral glucose tolerance test(OGTT) after 4 and 8 weeks.To dectect fasting blood glucose(FBG),fasing insulin level(Fins),calculating insulin resistance index(HOMA-IR),total cholesterol(TC), triacylglycerol(TG),low density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C),floating fatty acid(FFA),tumor necrosis-alpha(TNF-α),interleukin -6(IL-6) and C-reactive protein(CRP),and observe pathological changes of pancreas after 8 weeks. Results:1 Experiment in vitroCompared with model group,TNK could decrease significantly the levels of TNF-α,IL-6,CRP and FFA in 3T3-L1 adipocytes of insulin resistance(P＜0.01).To reduce the expression contents of IKKβmRNA and the acitivity of NF-κB and AP-1 protein,at the same time,and increase the expression of PPAR-γmRNA.Increased the expression contents of InsR,p-IRS-1 Tyr,GLUT-4 protein and decreased the expresion of p-IRS-1 Ser307.2 Experiment in vivoCompared with model group,TNK could decrease blood sugar,Fins,HOMA-IR in GK rats(P＜0.05 or P＜0.01).To reduce the levels of TG,LDL-C,FFA(P＜0.01) and increase the levels of HDL-C(P＜0.01).To decrease the levels of TNF-α,CRP,IL-6 (P＜0.01).To improve pathological damages of pancreas and protect islet cells.Conclusion:TNK could decrease blood sugar,inprove insulin resistance condition and protect islet cells.Its mechanisms are as follow:(1)TNK could up-regulate the expression of PPAR-γ,inhibit the inflammatory signaling pathways,decrease the generation of inflammatory factors and reduced interfering with the insuiln signaling transduction pathway.(2)TNK can recovery the abnormal signaling proteins in insulin signaling transduction pathway and improve insulin resistance in post-receptor level. |
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