Overexpression and pre-treatment of recombinant human Secretory Leukocyte Protease Inhibitor (rhSLPI) reduces an in vitro ischemia/reperfusion injury in rat cardiac myoblast (H9c2) cell

One of the major causes of cardiac cell death during myocardial ischemia is the oversecretion of protease enzymes surrounding the ischemic tissue. Therefore, inhibition of the protease activity could be an alternative strategy for preventing the expansion of the injured area. In the present study, we investigated the effects of Secretory Leukocyte Protease Inhibitor (SLPI), by means of overexpression and treatment of recombinant human SLPI (rhSLPI) in an in vitro model. Rat cardiac myoblast (H9c2) cells overexpressing rhSLPI were generated by gene delivery using pCMV2-SLPI-HA plasmid. The rhSLPI-H9c2 cells, mock transfected cells, and wild-type (WT) control were subjected to simulated ischemia/reperfusion (sI/R). Moreover, the treatment of rhSLPI in H9c2 cells was also performed under sI/R conditions. The results showed that overexpression of rhSLPI in H9c2 cells significantly reduced sI/R-induced cell death and injury, intracellular ROS level, and increased Akt phosphorylation, when compared to WT and mock transfection (p <0.05). Treatment of rhSLPI prior to sI/R reduced cardiac cell death and injury, and intra-cellular ROS level. In addition, 400 ng/ml rhSLPI treatment, prior to sI, significantly inhibited p38 MAPK phosphorylation and rhSLPI at 400–1000 ng/ml could increase Akt phosphorylation

Combination of metformin and p38 MAPK inhibitor, SB203580, reduced myocardial ischemia/reperfusion injury in non‑obese type 2 diabetic Goto‑Kakizaki rats

International audienceDiabetic cardiomyopathy, especially myocardial ischemia reperfusion (I/R) injury, is a major cause of morbidity and mortality in type 2 diabetic patients. The increasing of basal p38 MAP Kinase (p38 MAPK) activation is a major factor that aggravates cardiac death on diabetic cardiomyopathy. In addition, metformin also shows cardio-protective effects on myocardial ischemia/reperfusion injury. In this study, we investigated the effect of the combination between metformin and p38 MAPK inhibitor (SB203580) in diabetic rats subjected to I/R injury. H9c2 cells were induced into a hyperglycemic condition and treated with metformin, SB203580 or the combination of metformin and SB203580. In addition, cells in both the presence and absence of drug treatment were subjected to simulated ischemia/reperfusion injury. Cell viability and cellular reactive oxygen species (ROS) were determined. Moreover, the Goto-Kakizaki (GK) rats were treated with metformin, SB203580, and the combination of metformin and SB203580 for 4 weeks. Diabetic parameters and cardiac functions were assessed. Finally, rat hearts were induced ischemia/reperfusion injury for the purpose of infarct size analysis and determination of signal transduction. A high-glucose condition did not reduce cell viability but significantly increased ROS production and significantly decreased cell viability after induced sI/R. Treatment using drugs was shown to reduce ROS generation and cardiac cell death. The GK rats displayed diabetic phenotype by increasing diabetic parameters and these parameters were significantly decreased when treated with drugs. Treatment with metformin or SB203580 could significantly reduce the infarct size. Interestingly, the combination of metformin and SB203580 could enhance cardio-protective ability. Myocardial I/R injury significantly increased p38 MAPK phosphorylation, Bax/Bcl-2 ratio and caspase-3 level. Treatment with drugs significantly decreased the p38 MAPK phosphorylation, Bax/Bcl-2 ratio, caspase-3 level and increased Akt phosphorylation. In conclusion, using the combination of metformin and SB203580 shows positive cardio-protective effects on diabetic ischemic cardiomyopathy