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. nontreatment, menadione, and menadione-melatonin treatment, 4.0 × 10(5) each), while in vivo study used 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/6/8 hr after reperfusion), IR-Mito (mitochondria 15,000 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 found to be lower, whereas mitochondrial cytochrome c were found to be 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 and 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-hr 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), and 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 an additional benefit of alleviating hepatic IR injury.

Melatonin biosynthesis in plants: multiple pathways catalyze tryptophan to melatonin in the cytoplasm or chloroplasts

“…A recent report regarding the possible involvement of 2‐hydroxymelatonin in the defense response against combined cold and drought stress suggested that melatonin metabolites play pivotal roles in plant growth and development as functional substances . Melatonin synthesis and catabolism are closely coupled to regulation of the levels of melatonin and its metabolites, all of which act as biostimulators or signaling molecules for a series of biological functions in plants, as well as acting as a health‐promoting molecule in humans . Further in‐depth studies on melatonin synthesis and regulation in plants will provide effective metabolic engineering strategies to generate melatonin‐rich transgenic crops and microbes, by either overexpressing the key melatonin biosynthetic genes or suppressing the negative regulatory genes for melatonin synthesis …”

Intravenous administration of xenogenic adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes markedly reduced brain infarct volume and preserved neurological function in rat after acute ischemic stroke

“…The procedure and protocol of IF and IHC staining were based on our previous reports [36–38]. In detail, frozen sections (4 μm thick) were obtained from the brain infarct zone (BIZ) of each animal, permeated with 0.5% Triton X-100, and incubated with antibodies against glial fibrillary acid protein (GFAP) (1:500, DAKO), aquaporin4 (AQP4) (1:200, Abcam), vascular endothelial growth factor (VEGF) (1:400, Abcam), CD31 (1:100, Abcam), von Willebrand factor (vWF) (1:200, Abcam), CXCR4 (1:100, Abcam), stromal cell-derived factor (SDF)-1α (1:100, Santa Cruz), CD11 (1:200, Abcam), CD68 (1:100, Abcam), and γ-H2AX (1:5000, Abcam), p53BP1/CD90 (1:200, Novus Biologicals/1:100, Abcam), and XRCC1/CD90 (1:200, Abcam/1:100, Abcam) at 4 ° C overnight.…”

“…The procedure and protocol for identifying small vessels in the BIZ were based on our previous reports [32, 36–38]. In detail, IHC staining of small blood vessels was performed with α-SMA (1:400) as primary antibody at room temperature for 1 hour, followed by washing with PBS thrice.…”

Reduced delivery of epididymal adipocyte‐derived exosomal resistin is essential for melatonin ameliorating hepatic steatosis in mice

“…Previous studies demonstrate that the overload of energy substrate‐mediated MAMs reorganization results in mitochondrial Ca 2+ dysbolism and oxidative stress in hepatocytes, indicating that ER‐mitochondrial coupling is a significant regulatory pathway during the development of obesity. Although, in this investigation, we do not deeply study the role of mitochondria in this regulatory circuit, previous reports indicate that melatonin can alleviate diabetes‐induced hepatic mitochondrial dysfunction, suggesting that the reversion of overload of energy substrates‐induced mitochondrial imbalance may be another research interest.…”