The Development Of Ischemia/Reperfusionin-induced Myocardial Injury In Mice With Diabetes Mellitus And The Role Of IL-33

Background:Diabetes Mellitus (DM) is a very common chronic metabolic disease in clinical setting. Patients with DM are secondary to insulin insufficiency or decreased sensitivty of the body system to insulin. The DM is an independent risk factor to the development of coronary heart disease (CHD). However, DM and CHD are two diseases which are commonly seen in same patient. Acute myocardial infacrtion (AMI) is one of common presentations of the CHD. In general, the prognosis of AMI in diabetic patients is worse than those patients without DM. However, the underlying cause remanis unknown.Interlukin-33(IL-33) is a cytokine with molecule weight of30Kd. It is a new member of IL-1family which includes IL-1β, IL-1α and IL-18. IL-33excerts bilogical roles through interaction with its receptor, ST-2L. Previous studies have demonstrated that IL-33has beneficial actions on various cardiovascular pathologies. For instance, by interacting with its receptor ST-2L, IL-33can1) prevent myocardial injury-induced by ischemia/reperfusion;2) anti-myocardial fibrosis in mice with ventricular over-load;3) attenuate artery arthrosclerosis in mice with Apo E deficiency;4) prevent choronic inflammation in obesed mice. The objective of the present study is to1) exlore the changes in myocardial IL-33expression in mice with DM, and the impact of IL-33expression changes on the vulnerability of the myocardium to ischemia/reperfusion injury in mice with DM;2) to study the intracellular signaling pathway involved in the myocardial protection of exogenus IL-33.Methods:Animal experiments:Mouse model of DM was induced in8week-old mice (C57/BL6) with intraperitoneal injection of stroptozotocin (STZ); Mouse model of myocardial ischemia/repefusion (I/R) was induced by ligation of LAD of coronary artery for45min followed by releasing. Myocardial IL-33expersion was assessed with immunofluoresence staining and Western blot. In addition, myocardial infarction size was evaluated with Evans blue and TTC staining; myocardial apoptosis assessed with TUNNEL staining and cell death ELISA; cardiac function was measured with a mouse pressure-volume analysis system.Cell experiments:isolated cardiomyocytes were cultured with high concertration of glucose (30mM) followed by challenging the cardiomyocytes with45min anoxia and reoxygenation (A/R) in order to mimic the in vivo conditions of DM with I/R. The cardiomyocyte oxidative stress was assessed with dihydrorhodaminel23(DHR123); cardiomyocyte apoptosis was detected with caspase-3activity and cell death ELISA kit. Intracellular PKCβⅡ and JNK phosphorylation was deternined with specific antibodies by Western blot.Results:1), DM was successfully induced in mice after3dose of STZ (50mg/kg); and mice incurred myocardial infarction after LAD ligation followed by releasing. Cardiomyocyte oxidative stress was induced by either HG or A/R as indicated by increase DHR123fluoresence reading.2), DM results in decreased myocardial levels of IL-33and DGKzeta, and activation of PKCβⅡ; I/R challenge of the DM mice resulted in futher activation of PKCβⅡ, which led to an enlarged myocardial infarction size, increased myocardial apoptosis and decreased myocardial contractility as compared with those without DM. Exogenus IL-33attanuated the enhanced myocardial I/R injury and improved myocardial function in mice with diabetes.3), A/R challenge to cardiomyocytes resulted in cardiomyocyte oxidative stress (increase DHR123fluorescence reading), activation of PKCβⅡ and JNK (increased phosphorylation of PKCβⅡ and JNK) and cardiomyocyte apoptosis (increased caspase3activity and cell death). Treatment of cardiomyocytes with exogenus IL-33increased DGKzeta expression. Exogenus IL-33prevented the A/R-induced oxidative stress, increase in phosphorylation of PKCβⅡ and JNK, and attenuaed A/R-induced cardiomyocyte apoptosis.Conclusions:Results of the present study indicate,1) increased vulnerability to I/R-induced myocardial injury in diabetic mice is attributed to decreased myocardial levels of IL-33;2) Decreased myocardial levels of IL-33leads to down-regulation of DGK-zeta expression which further activation of PKCβⅡ after I/R;3) exogenous recombinant IL-33prevents myocardial ischemia/reperfusion-induced injury via increases DGKzeta expression thereby inhibits the PKCβⅡ and JNK phosphorylation.