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

Abstract

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.

Endothelial-Cell-Derived Human Secretory Leukocyte Protease Inhibitor (SLPI) Protects Cardiomyocytes against Ischemia/Reperfusion Injury

“…Previous studies reported that SLPI acts as an ROS scavenger by reducing intracellular ROS production in many cells during sI/R [11,12,13,14]. To prove that SLPI secreted from EA-SLPI cells reduced intracellular ROS production, in vitro intracellular ROS production was determined under simulated ischemia (sI), hypoxia, and H 2 O 2 .…”

“…The cytoprotective effect of secretory leukocyte protease inhibitor (SLPI) has been reported in different cardiac resident cells, including cardiomyocytes, cardiac fibroblasts, and vascular endothelial cells [11,13,14]. Moreover, SLPI has also been shown to provide cardioprotection against I/R injury [10,12].…”

“…Simulated ischemia (sI) was performed following the method mentioned in previous studies [11,12]. Wild-type or SLPI-overexpressing EA.hy926 cells were seeded into a 24-well tissue culture plate at a density of 1.5 × 10 4 cells/well and incubated with simulated ischemic basic buffer (137 mM NaCl, 3.8 mM KCl, 0.49 mM MgCl 2 , 0.9 mM CaCl 2 , 4.0 mM HEPES) containing 20 mM 2-deoxyglucose, 20 mM sodium lactate, and 1 mM sodium dithionite at pH 6.5.…”

“…After H/R, H9c2 cells cultured in wild-type or SLPI-overexpressing EA.hy926 media were collected for protein concentration by adding 2× SDS sample buffer containing β-mercaptoethanol as previously described [11]. Cells were scraped and transferred to a new pre-cooled microcentrifuge tube.…”

“…Our previous reports also demonstrated the cardioprotective effect of SLPI. Overexpression of human SLPI in rat cardiac myoblasts (H9c2) or treatment with rhSLPI in both H9c2 and adult rat ventricular myocytes (ARVMs) reduced cell death and injury from an in vitro I/R injury [11,12], as well as decreasing the infarct size [12]. Furthermore, SLPI also protected non-cardiomyocytes such as adult rat cardiac fibroblast cells (ARCFs) and human umbilical vein endothelial cells (HUVECs) from an in vitro simulated I/R (sI/R) injury [13,14].…”