The Protective Mechanism Of Insulin In H9c2 Against High Glucose Induced Injury

OBJECTIVE:To establish a model of H9c2 injury induced by high glucose; To observe the influence of insulin on cell apoptosis and the expression of endoplasmic reticulum stress marker in cultured myocardial cells exposed to high glucose; To investigated the role of PI3K/Akt signal pathway on high glucose induced damage in H9c2 cells.METHODS:1. Myocardial cell culture and the model of high glucose induced injuryEmbryonic rat heart-derived H9C2 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 5.5 mmol/L glucose, 10% FBS (fetal bovine serum). To establish a model of H9c2 injury induced by high glucose, H9c2 cells were cultured in 35mmol/L high glucose for different times(6h,12h,24h,48h,72h).2. Measurement of cell viability and apoptosisH9c2 cells were divided into 4 group randomly:control group(5.5 mmol/L low glucose), HG group(35 mmol/L high glucose), INS group(100nmol/L insulin), HG+INS group(35 mmol/L high glucose and 100nmol/L insulin). MTT assay was employed to assess the viability of the H9c2 cardiac cells at different times(0,24h,48h,72h and 96h). According to the data of cell viability, the apoptotic cell observed by Hoechst staining at 48 hour, and cell grouping was the same as above.3. Determination of the expression of endoplasmic reticulum stress marker by RT-PCRH9c2 cells were treated with high glucose for different times, and total cellular RNA was extracted from cells for detection the time effect of GRP78mRNA expression. According to the data of time effect, H9c2 cells were divided into 4 group randomly:control group(5.5mmol/L low glucose), HG group(35 mmol/L high glucose), HG+INS group(35 mmol/L high glucose and 100nmol/L insulin), LY294002+INS+HG group(treatment of the cells with LY294002 for 30min prior to exposure to HG and INS). The expression of GRP78mRNAwere detected at 48 hour point.4. Western blot analysis.H9c2 cells were divided into 6 group randomly:control group(5.5 mmol/L low glucose), HG group(35 mmol/L high glucose), INS group (100nmol/L insulin), LY294002 group(LY294002 50μmol/L), HG+INS group (35 mmol/L high glucose and 100nmol/L insulin), LY294002+INS+HG group (treatment of the cells with LY294002 for 30min prior to exposure to HG and INS). We detected the t-Akt and p-Akt protein expression level using Western blotting.RESULTS:After the cells were exposure to HG for 48h, there were an increase in the number of apoptotic cells and a reduction of cell viability (P<0.5), compared to the control group. Notably, treatment of the H9c2 cells with insulin significantly increased the cell viability, as well as a decrease in the number of apoptotic cells, compared to the HG group. Hochest staining showed that in high glucose cell nucleus appeared karyopycnosis, chromatin gathered to peripheral, or nucleus splintered into apoptosis body. The expression of GRP78 mRNA was induced by HG in time dependent manner. Compared to the HG group, the p-AKT expression was elevated significantly in INS+HG group (P<0.01), and the effect was attenuated by LY294002 (P<0.01).CONCLUTION:The protective effect of insulin against high glucose induced H9C2 injury may involve PI3K/AKT signaling pathway activation.