Lipid Emulsion Inhibits Apoptosis Induced by a Toxic Dose of Verapamil via the Delta-Opioid Receptor in H9c2 Rat Cardiomyoblasts

Abstract

The goals of this study were to investigate the effects of lipid emulsion (LE) on apoptosis induced by a toxic dose of verapamil in H9c2 cells and to elucidate the associated cellular mechanism. The effects of LE alone and combined with an inhibitor on the decreases in cell counts and viability induced by verapamil and diltiazem were examined using the MTT assay. The effects of verapamil alone, combined LE and verapamil treatment, and combined inhibitor, LE and verapamil treatment on cleaved caspase-3, caspase-8 and Bax expression, were examined using Western blotting. The effects of verapamil alone and combined with LE on the number of TUNEL-positive H9c2 cells were also examined. LE attenuated the decreases in cell counts and viability induced by verapamil and diltiazem. However, the magnitude of the LE-mediated attenuation of decreased cell viability was enhanced by verapamil compared with diltiazem treatment. Naloxone, naltrindole hydrochloride, LY294002 and MK-2206 inhibited the LE-mediated attenuation of increased cleaved caspase-3 and caspase-8 expression induced by verapamil. LE attenuated the increase in the number of TUNEL-positive cell induced by verapamil. These results suggest that LE attenuates apoptosis induced by verapamil via activation of the delta-opioid receptor, phosphoinositide 3-kinase and Akt.

Lipid Emulsion Inhibits the Late Apoptosis/Cardiotoxicity Induced by Doxorubicin in Rat Cardiomyoblasts

“…The mechanism associated with the apoptosis observed in doxorubicin-induced cardiotoxicity has been extensively investigated, and multiple pathways are known to be involved in apoptotic cell death [ 16 , 26 ]. Lipid emulsion reversed the cell viability decrease induced by bupivacaine or verapamil in H9c2 cells [ 13 , 14 ]. Similar to these previous reports, the results presented herein demonstrated that lipid emulsion attenuated the decreased cell viability induced by doxorubicin, as determined by the MTT assay ( Figure 1 B) [ 13 , 14 ].…”

“…Lipid emulsion reversed the cell viability decrease induced by bupivacaine or verapamil in H9c2 cells [ 13 , 14 ]. Similar to these previous reports, the results presented herein demonstrated that lipid emulsion attenuated the decreased cell viability induced by doxorubicin, as determined by the MTT assay ( Figure 1 B) [ 13 , 14 ]. α- and γ-linolenic acid, which are long-chain fatty acids, counteract the cardiotoxicity of doxorubicin [ 10 , 27 ].…”

“…H9c2 rat cardiomyoblasts obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, GE Healthcare, Salt Lake City, UT, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin as described previously [ 13 , 14 ]. Cells were cultured in 100-mm dishes at 37 °C in 5% CO 2 and sub-cultured in 1:4 ratios upon reaching 90% confluence.…”

“…Cell viability was estimated colorimetrically using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as previously reported [ 13 , 14 ]. Cells were seeded on a 24-well plate at a density of 10 5 cells/well and were treated with doxorubicin (10 −6 , 3 × 10 −6 and 10 −5 M) alone for 24 h, treated with lipid emulsion (0.125, 0.25, 0.75 and 2%) alone for 25 h, or pretreated with lipid emulsion (0.125, 0.25, 0.75 and 2%) for 1 h followed by doxorubicin (10 −5 M) for 24 h. After incubation, the cells were washed twice with phosphate buffered saline (PBS, pH 7.4), treated with MTT in PBS (0.5 mg/well), and then incubated for 4 h at 37 °C in the dark.…”

“…Following treatment, proteins were extracted, and the total protein concentrations were determined using the Bradford method as previously reported [ 13 ]. The sample protein from the cell lysate was mixed with 5× sodium dodecyl sulfate sample buffer (0.1 M Tris-HCl, 20% glycerol, 4% sodium dodecyl sulfate and 0.01% bromophenol blue).…”