Interleukin-33 Protects Against Myocardial Apoptosis Induced By High Glucose

Objective Diabetes mellitus is an important risk factor of cardiovascular disease. There has been a continued increase in the incidence of cardiovascular disease because of the rising number of diabetes patients. Diabetic cardiomyopathy is an important complication of diabetes, which can cause a significant rise of mortality in diabetic patients. The mechanisms of diabetic cardiomyopathy are very complicated. Many factors have been reported to be responsible for it, such as myocardial metabolic disorders, mitochondrial dysfunction, oxidative stress, impaired regulation of intracellular calcium, endoplasmic reticulum stress, etc. Interleukin-33 (IL-33), a new member of IL-1 family, has been shown protective effects on many cardiovascular diseases. It can inhibit myocardial apoptosis, myocardial hypertrophy and myocardial fibrosis. It also can block the development of Diabetes. It is widely accepted that the diabetic heart is associated with cardiomyocyte hypertrophy, myocardial interstitial fibrosis and increased apoptosis. We designed this experiment to investigate the protective effects of IL-33 against high glucose (HG)-induced apoptosis in H9c2 cardiac cells and explore the potential mechanisms.Methods Cells were divided into several groups as follow:Control group, Mannitol control group, High glucose group, IL-33 treated group, N-acetyl-L-cysteine (NAC) treates group, Inhibitor of c-Jun N-terminal kinase (JNK) treated group and Inhibitor of p38 Mitogen-activated protein kinase (MAPK) treated group.H9c2 cardiac cells were treated with 30 mmol/L glucose to establish the cell injury model induced by HG. In order to control the osmotic effects of HG, the normal culture media containing 5.6 mmol/L glucose was supplemented with 24.4 mmol/L mannitol. To explore the effect of IL-33, cells were treated with various concentrations of IL-33 (0.2-5 ng/mL) for 1 h prior to exposure to HG. To investigate the functional mechanism of IL-33, cells were pretreated with NAC (1000 μmol/L), SP600125 (a selective inhibitor of JNK,10 μmol/L) or SB203580 (a selective inhibitor of p38 MAPK,3 μmol/L) for 1 h prior to exposure to HG.The first part of the experiment was to observe viability and apoptosis of H9c2 cardiac cell:Cell viability was assessed by methyl thiazolyl tetrazolium (MTT) assay. Cell apoptosis was detected by flow cytometry. The second part of the experiment was to explore the potential mechanisms:Reactive oxygen species (ROS) was assessed by flow cytometry. The protein expressions of cleaved-cysteinyl aspartate specific protease-3 (c-caspase-3), phosphor (p)-JNK, p-p38 MAPK, glucose regulated protein 78 (GRP78) and C/EBP homologus protein (CHOP) were determined by Western blotting.ResultsPart 11. Cell viabilityThe H9c2 cardiac cells in control group were considered 100% viability. Viability of cells treated with HG decreased to 62.06%±3.58%(P<0.01). When the cells were pretreated with 1 ng/mL IL-33 (P<0.05) or 5 ng/mL IL-33 (P<0.01) prior to exposure to HG, the cell viability increased significantly compared to HG treated group. Pre-treatment of cells with NAC, SP600125 or SB203580 before exposure to HG also prevented HG-induced decreases in cell viability (P<0.01)2. Quantitation of apoptosisCompared with the control group (1.21%±0.48%,), HG treated group had a significantly higher cell apoptotic ratio (22.01%±3.09%, P<0.01). However, the increased percentage of the apoptotic cells was reduced by the pre-treatment with IL-33, SP600125, SB203580 or NAC (P<0.01)Part 21. Measurement of intracellular ROS accumulation There was a marked increase in production of ROS in the HG group compared to the control group (P<0.01). However, the increased amount of ROS induced by HG was reduced by IL-33. NAC exerted a similar ROS-decreasing effect (P<0.01).2. Expression of c-caspase-3 Exposure of H9c2 cardiac cells to HG resulted in an increase in c-caspase-3 expression (P<0.01), while the expression of c-caspase-3 was decreased in the IL-33, SP600125, SB203580 or NAC pre-treatment group compared with that in the HG group (P<0.05).3. Expression of p-JNK and p-p38 MAPKIn the HG group, expression levels of p-JNK and p-p38 MAPK were significantly increased compared to those in the control group. However, pre-treatment with IL-33 or NAC decreased the phosphorylation of JNK and p38 MAPK (P<0.05).4. Expression of GRP78 and CHOPIn the HG group, expression levels of GRP78 and CHOP were significantly increased compared to those in the control group (P<0.01). However, pre-treatment with IL-33 or NAC decreased the expression levels of GRP78 and CHOP (P<0.05).Conclusion1. HG can induce a decreased viability and a increased apoptosis in H9c2 cardiac cells. IL-33 can inhibit HG-induced cardiomyocyte injury.2. IL-33 protects H9c2 cardiac cell from HG-induced injury by inhibiting oxidative stress, endoplasmic reticulum stress and phosphorylation of JNK and p38 MAPK.