关键词: 糖尿病心肌病, 肌细胞, 心脏, 细胞凋亡, 氧化性应激, 原癌基因蛋白质c-akt, 棕榈酸, 吡格列酮

Abstract: Objective To investigate the effect and underlying mechanisms of pioglitazone (PGZ) on apoptosis of rat H9C2 cardiomyocytes induced by high glucose (HG) /palmitic acid (PA). Methods H9C2 cells were cultured and stimulated with different concentrations of PA and HG for 12, 24 and 48 h to determine the best concentration by CCK-8 assays. The cell model of hyperglycemia and hyperlipid injury was established by co-cultivation of 0.1 mmol/L PA and 50 mmol/L HG. The injury cells stimulated with different concentrations of PGZ for 12, 24 and 48 h were determined the best intervention time and concentrations. H9C2 cells were divided into the five groups: the control group (25 mmol/L glucose), the solvent group (PA solvent+DMSO), the hyperglycemia and hyperlipid group (HGPA), the H-PGZ group (10 μmol/L PGZ+HGPA) and the L-PGZ group (5 μmol/L PGZ+HGPA). The apoptosis rate was detected by fluorescent probe of AnnexinV-FITC/PI. The level of reactive oxidative species (ROS) was detected by DCFH-DA with flow cytometry. The MDA content and SOD level were assayed by specific kits. The protein expression levels of AKT, P-AKT, Caspase3, C-Caspase3, BCL-2 and BAX were detected by Western blot assay. After 24 h of NAC (1 mmol/L N-acetylcysteine) +HGPA and HGPA treatment, the above AKT pathway and apoptosis-related protein expression levels were detected again. Results CCK-8 showed that H9C2 cell viability decreased successively in the PA group after treatment with 0, 0.05, 0.1, 0.2 and 0.4 mmol/L PA for 48 h（P＜0.05). In 12 h, compared with the 25 mmol/L glucose group, the cell proliferation increased in the 50, 75 and 100 mmol/L HG groups, and the cell proliferation increased successively in 24 h and 48 h in the 25, 35, 50, 75 and 100 mmol/L HG groups. Compared with the control group, the cell viability decreased within 48 hours in the HGPA group (P＜0.05). Compared with the HGPA group, the cell viability decreased at 12 h in the 80 μmol/L PGZ treatment group (P＜0.05). The cell viability increased at 24 h in the 10 μmol/L PGZ group, while cell viability decreased in the 40 and 80 μmol/L groups (P＜0.05). At 48 h, the cell viability increased in the 5, 10 and 20 μmol/L groups, while the cell viability decreased in the 40 and 80 μmol/L groups (P＜0.05). Compared with the control group, the apoptosis rate, ROS level, MDA content, C-Caspase3 expression and BAX expression increased in the HGPA group, while SOD level, P-AKT expression and BCL-2 expression decreased (P＜0.05). The apoptosis rate, ROS, MDA, C-caspase3 and Bax decreased in turn in the HGPA group, the L-PGZ group and the H-PGZ group, while the protein expression levels of SOD, P-AKT, Caspase3 and BAX increased in turn (P＜0.05). Compared with the HGPA group, C-Caspase3 expression decreased in the NAC+HGPA group, while P-AKT increased (P＜0.05). Conclusion PGZ has an inhibitory effect on ROS, which can promote activation of the AKT pathway, and reduce the apoptosis of H9C2 cardiomyocytes induced by HG/PA.

Key words: diabetic cardiomyopathies, myocytes, cardiac, apoptosis, oxidative stress, proto-oncogene proteins c-akt, palmitic acid, pioglitazone