Effects Of Obesity And Type 2 Diabetes Mellitus On NALP3 Inflammatory Complex Of 3T3 - L1 Cells And Its Mechanism

Objective:obesity and type2diabetes mellitus(T2DM), which are threatening human health, are often observed along with each other. However, the pathogenesis of obesity and T2DM is still unclear. As a kind of metabolic inflammatory disease, the pathogenesis of obesity and T2DM must related to inflammation. Recently, we discovered that NALP3inflammasome regulates interleukin-1β(IL-1β), which plays an important role in obesity and T2DM.In this research, we selected glibenclamide, repaglinide, voglibose, metformin, sitagliptin, rosiglitazone and orlistat which are obesity and T2DM therapeutic drugs, and observed these drugs’influence on NALP3inflammasome of3T3-L1cell. Tn this way, we were able to validate the relationship between NALP3inflammasome and these two diseases, and screen drugs which affect the NALP3inflammasome, besides that, we might know the mechanism preliminarily through the following methods:firstly, we investigated the change of upstream factors including pannexin-1, cathepsinB, TXNIP and NF-κB, which are key factors of stimulating NALP3inflammasome, and the change of IL-1β,IL-18, IL-6and TNF-a which are inflammatory mediums of obesity and T2DM. And because this research aimed at3T3-L1cell, we wondered whether the drugs promoted/inhibited the differentiation of3T3-L1cell as well as the expression of NALP3inflammasome. We answered this question by literatures survey and an experiment about sitagliptin.Method:we grew3T3-L1cells and stimulated differentiation when the density of cells>90%. At the same time, drugs were added as designed:metformin4mmol/L(mM)、glibenclamide200umol/L (μM)、repaglinide60μM、voglibose300μM、orlistat15μM、sitagliptin20nM,、rosiglitazone40μM.10days later, the cells were differentiated successfully, we compared the expression of NALP3inflammasome, Pannexin-1, cathepsinB, TXNIP and NF-κB though real-time PCR and the level of IL-1β、IL-18、IL-6、TNF-α though ELISA. On the other hand, we observed the effect of sitagliptin(5nM, lOnM,25nM,50nM,100nM) on3T3-L1cells’ differentiation by oil red O staining and on factors representing differentiation which include PPARy, LPL, C/EBPa and FABP4.Results:1.4mM metformin,200μM glibenclamide,60μM repaglinide,300μM voglibose,15μM orlistat,20nM sitagliptin,40μM rosiglitazone inhibited the expression of NALP3inflammason which is composed by NALP3, ASC, caspase-1;2.4mM metformin,200μM glibenclamide,60μM repaglinide,300μM voglibose,inhibited the expression of pannexin-1, cathepsinB, TXNIP and NF-κB,15μM orlistat only inhibited the expression of pannexin-1, cathepsinB and NF-κBmRNA, and had no effects on TXNIP,20nM sitagliptin only inhibited the expression of TXNIP, cathepsinB and NF-κB, and had no effects on pannexin-1,40μM rosiglitazone inhibited the expression of pannexin-1, TXNIP and NF-κB, and increased the expression of cathepsinB;3.4mM metformin,200μM glibenclamide,60μM repaglinide,300μM voglibose,15μM orlistat,20nM sitagliptin,40μM rosiglitazone decreased the level of IL-1β、IL-18、IL-6、TNF-a;4. Sitagliptin (5nM、10nN、25nM、50nN、100nM) inhibited the differentiation of3T3-L1cells and10nM sitagliptin suppressed the expression of factors representing differentiation which include PPARy, LPL, C/EBPa and FABP4;5. As reported, rosiglitazone and glibenclamide promote3T3-L1cell differentiate, so there is no consistency of drug’s effect on NALP3inflammasome and drug’s effect on3T3-L1cell differentiation.Conclusion:our research selected the drugs which can suppress NALP3inflammasome and the inflammative factors IL-1β、IL-18、IL-6、TNF-α,these drugs include glibenclamide, repaglinide, voglibose, metformin, sitagliptin, rosiglitazone and orlistat. And we observed that this impact might be realized by regulating pannexin-1、 cathepsinB、TXNIP and NF-κB, but was not consistent with drug’s impact on3T3-L1cell differentiation.