Melatonin pretreatment enhances the therapeutic effects of exogenous mitochondria against hepatic ischemia–reperfusion injury in rats through suppression of mitochondrial permeability transition

Abstract

We tested the hypothesis that melatonin (Mel) enhances exogenous mitochondria (Mito) treatment against rodent hepatic ischemia-reperfusion (IR) injury. In vitro study utilized three groups of hepatocytes (i.e., non-treatment, menadione, and menadione-melatonin treatment, 4.0 x 10(5) each), while in vivo study involved adult male Sprague-Dawley rats (n=40) equally divided into sham-control (SC), IR (60-min left lobe ischemia + 72-hr reperfusion), IR-Mel (melatonin at 30 min/6h/8h after reperfusion), IR-Mito (mitochondria 15000 μg/rat 30 min after reperfusion), and IR-Mel-Mito. Following menadione treatment in vitro, oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (cleaved caspase-3/PARP), DNA-damage (γ-H2AX/CD90/XRCC1), mitochondria-damage (cytosolic cytochrome-C) biomarkers and mitochondrial permeability transition were lower, whereas mitochondrial cytochrome-C were higher in hepatocytes with melatonin treatment compared to those without (all p<0.001). In vivo study demonstrated highest liver injury score and serum AST in IR group but lowest in SC group, higher in IR-Mito group than that in groups IR-Mel and IR-Mel-Mito, and higher in IR-Mel group than that in IR-Mel-Mito group after 72-hour reperfusion (all p<0.003). Protein expressions of inflammatory (TNF-α/NF-κB/IL-1β/MMP-9), oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (caspase-3/PARP/Bax), mitochondria-damage (cytosolic cytochrome-C) biomarkers displayed an identical pattern, whereas mitochondria integrity marker (mitochondrial cytochrome-C) showed an opposite pattern compared to that of liver injury score (all p<0.001) among five groups. Microscopically, expressions of apoptotic nuclei, inflammatory (MPO(+) /CD68(+) /CD14(+) cells) and DNA-damage (γ-H2AX(+) cells) biomarkers exhibited an identical pattern compared to that of liver injury score (all p<0.001) among five groups. Melatonin-supported mitochondria treatment offered additional benefit of alleviating hepatic IR injury. This article is protected by copyright. All rights reserved.