Protective effect of insulin on lipopolysaccharide-induced impairments of H9c2 cells and corresponding mec-hanism

Abstract

Objective To research the protective effect of insulin(IN)on lipopolysaccharide(LPS)-induced impairments of rat cardiomyocytes H9c2, and the role of uncoupling protein 2(UCP2)in this process. Methods Using randomized controlled grouping, after cultured for 24 h, H9c2 cells were randomly divided into 5 groups as follows: control group, LPS stimulation group (LPS group), LPS+ 70 IU/L IN group(IN 70 IU/L group), LPS+ 350 IU/L IN group(IN 350 IU/L group), and LPS+ 700 IU/L IN group(IN 700 IU/L group). H9c2 cells in IN group were treated with 70 IU/L, 350 IU/L or 700 IU/L IN 15 min before LPS stimulation, and H9c2 cells in control group were treated with an equal volume of saline.After that, cells in group LPS and IN were treated with LPS for 24 h. Lactate dehydrogenase (LDH)in the culture was determined with LDH detecting assay kit.The activity of reactive oxygen species (ROS)and superoxide dismutase (SOD), and content of malonaldehyde (MDA)were determined by colorimetric detection.Cell viability was evaluated by cell count kit-8.The expressions of UCP2 in transcription and translation levels were detected through transcription polymerase chain reaction and Western blot respectively. Results The levels of LDH, MDA, and intracellular ROS in LPS group significantly increased compared with control group[LDH: (829.3±75.3)U/L vs (223.5±23.6)U/L, MDA: (60.90±5.73) nmol/mgprot vs (19.70±1.99)nmol/mgprot, ROS: (410.2±81.6)U/well vs (94.3±18.5)U/well, all P<0.05)], while the cell viability and SOD activity significantly decreased[cell viability: 0.822±0.058 vs 1.012±0.023, SOD: (49.20±5.81)U/mgprot vs (89.80±2.57)U/mgprot, all P<0.05]. And the mRNA and protein expressions of UCP2 in LPS stimulation group were up-regulated(1.867±0.130 vs 1.028±0.097, 0.288±0.018 vs 0.180±0.008, all P<0.05).350 IU/L and 700 IU/L IN intervention significantly decreased the levels of LDH, MDA and intracellular ROS[LDH: (568.2±35.7)U/L, (622.8±27.6)U/L vs (829.3±75.3)U/L, MDA: (29.20±4.20)nmol/mgprot, (42.10±2.32)nmol/mgprot vs (60.90±5.73)nmol/mgprot, ROS: (270.3±46.8)U/well, (301.5±16.9)U/well vs (410.2±81.6)U/well, all P<0.05], increased the cell survival and the levels of SOD activity[cell viability: 0.960±0.029, 0.906±0.039 vs 0.822±0.058, SOD: (75.20±2.21)U/mgprot, (61.20±3.38)U/mgprot vs (49.20±5.81)U/mgprot, all P<0.05]. And IN with 350 IU/L and 700 IU/L increased the mRNA and protein expression of UCP2(3.830±0.265, 2.855±0.215 vs 1.867±0.130, 0.464±0.215, 0.355±0.006 vs 0.288±0.018, all P<0.05). Compared with 70 IU/L and 700 IU/L IN group, 350 IU/L IN group had better results. Conclusions IN attenuates LPS-induced oxidative injury in H9c2 cells, which is probably mediated through up-regulating the expression of UCP2. Key words: Insulin; Lipopolysaccharide; H9c2 cell; UCP2; Oxidative stress