NLRP3Inflammasome Involved In Insulin Induced Inflammation In Vascular Smooth Muscle Cell

Backgroud:Atherosclerosis is one of important complications of type2diabetes mellitus(T2DM),leading to major deaths of the latter one. Both diseases are divided into metabolic syndrome,which owns a character of being chronic inflammation. During this inflammation,Interleukin-1β(IL-1β) exists vast in both diseases connecting them tightly.Untill recent years, the mechanism of maturation of IL-1β is found to depend on ahigh weight molecule called inflammasomes, and NOD-like receptor family protein3(NLRP3) inflammasome is the most well studied one. To activate NLRP3inflammasome,a priming step and a activation step are needed. To T2DM patients, matured IL-1β canimpair the sensitivity of insulin, causing more severe insulin resistance finally leading tocadiovascular diseases. To patients suffering atherosclerosis, it can directly aggravate theprocess of atherogenesis. Thus IL-1β could be considered as a link between these twodiseases.Besides that, insulin is another connection between them due to its proatherosclerosiseffects as reported. This effects attributes to increasing proliferation and migration ofvascular smooth muscle cells(VSMCs), which are mediated by mitogen activated proteinkinase(MAPK) and phosphatidyl inositol-3kinase(PI3K). There is also report to delineate apro-inflammation status of VSMCs that the cells are able to secrete cytokines afterinflammatory stumilation. We have already been aware that changing of cell phenotype isalways companied with proliferation, and thus it is not hard to imagine insulin inducedprolieration would be associated with inflammation. This hypothesis could offer a newperspective of insulin treating therapy.Objectives:To identify the impact of insulin on expression of NLRP3, Caspase-1and IL-1β, and explore the preliminary possible mechanism. Then give a picture of how insulin effectatherosclerosis from inflammation view.Study contents:1. Effect of insulin on expression of NLRP3, Caspase-1and IL-1β in VSMCs.2. Impact of insulin on secretion of IL-1β and IL-6in VSMCs.3. Possible mechanisms of how insulin influence NLRP3inflammasome.4. Effect of insulin on vascular injury in leptin receptor deficient(lepr-/-) mice.Methods:1. Culture of vascular smooth muscle cells.2. RT-QPCR detection of expression of NLRP3, Caspase-1and IL-1β on mRNAlevel in insulin treated VSMCs.3. Western blot detection of expression of NLRP3, Caspase-1and IL-1β on cytosolprotein level in insulin treated VSMCs.4. ELISA detection of IL-1β and IL-6in supernates of cultured VSMCs.5. Animal breeding.6. Stripping of aortas in mice and oil-red staining.Results:1. Insulin increase the expression of NLRP3, Caspase-1and IL-1βon both mRNAlevel and cytosol protein level in treated VSMCs.2. The secretion of IL-1βand IL-6of VSMCs increases over a time line after insulintreated, reaching top at9H.3. Silibinin and ac-yvad-cmk are able to prevent the increasing effect of insulin onexpression of NLRP3and IL-1β.4. Insulin treated lepr-/-mice exhibit more aorta injury by oil-red staning.Conclusions:1. Rat vascular smooth muscle cells are able to produce IL-1β.2. Insulin is able to increase expression of IL-1β in treated VSMCs via NLRP3mediated pathway.3. Effect of insulin on NLRP3can be prevented by silibinin and ac-yvad-cmk. 4. Insulin is able to aggravate vessel injury of lepr-/-mice.5. All results suggest a proatherosclerosis effect of insulin.