Hyperbaric oxygen-assisted melatonin therapy protects the heart from acute ischemia-reperfusion injury

In this study we have examined whether hyperbaric oxygen (HBO)-assisted melatonin (Mel) therapy effectively preserves the heart function against ischemia (40-min)-reperfusion (IR) injury. First, the in vitro study has been performed by use of cell culture. H9C2 cells were treated as groups: A (H9C2) (without any treatment), B (H9C2+IR), C (H9C2+IR+HBO), D [H9C2+IR+Melatonin (50 µM)] and E (H9C2+IR+Melatonin+HBO). The result showed that the protein expressions of oxidative-stress (NOX-1/NOX-2)/inflammatory (TNF-α/NF-κB)/apoptotic (mitochondrial-Bax/cleaved-caspase-3/cleaved-PARP) and cellular levels of DNA/mitochondrial-damaged (γ-H2AX/XRCC1-CD90+/cytosolic-cytochrome-C) biomarker were significantly increased in group B compared to control group A. These biomarkers were significantly reduced in group C, D and E compared to groups B with the significantly higher reduction in group E than that in groups C and D (p<0.001). In the in vivo study, adult-male SD rats (n=40) were equally divided into group 1 (sham-operated-control), group 2 (IR), group 3 (IR+HBO therapy at 1.5/24/48h after IR procedure), group 4 [IR+Mel (50mg/kg at 1.5h, followed by 20mg/kg and at days 1/2/3 after IR)] and group 5 (IR+HBO-Mel) and the heart was harvested at 72h after IR. The result showed that at 72h, the circulating levels of endothelial-progenitor-cells (c-kit-CD31+/CD31-sca-1+/KDR-CD34+/VE-Cadherin-CD34+) were lowest in group 2, highest in group 5 and followed by groups 3 > 4 >1. The significant differences were present between each two matched groups (p<0.0001). The protein expressions of angiogenic factors (SDF-1α/CXCR4/VEGF/HIF-α) were progressively increased from groups 1 to 5 with significant differences. The protein expressions of apoptosis (mitochondrial-Bax/cleaved-caspase-3/cleaved-PARP)/fibrosis (TGF-ß/Smad3)/oxidative-stress (NOX-1/NOX-2/oxidized protein)/inflammation (TNF-α/IL-1ß/MMP-9) and infarct/fibrotic areas were significantly increased in group 2 compared to the control group 1. All these parameters were significantly reduced in groups 3-5 compared to group 2 with significantly lowest level in group 5 among groups 3-5 (p < 0.01), whereas the left-ventricular-ejection-fraction exhibited an opposite pattern compared to the inflammatory factors. In conclusion, HBO-Mel therapy offered a synergic benefit for protecting the heart from IR injury.