Study On The Mechanism And Inhibitory Effects Of Hydrogen Molecule On Skin Flap Cell Apoptosis Induced By Ischemia/Reperfusion

Ischemia/reperfusion injury (IRI) often occurs in free or axial flap transplantationduring clinical surgery, such as plastic surgery, orthopedics and craniofacial surgery.After years of research, survival and the success rate of skin flap transplantation hasimproved, but there are still cases of partial or all flap necrosis. Multiple treatmentsaimed at preventing flap necrosis have been investigated, of which hyperbaric oxygenpreconditioning has been widely used. But we still need to explore a more secure andconvenient treatment. Hydrogen molecule has been found to selectively reducingcytotoxic oxygen radicals, and the anti-inflammation of hydrogen molecule has beendetected in skin flap ischemia/reperfusion (I/R) model. Many studies have shown thathydrogen molecule can regulate apoptotic signals, so the anti-apoptosis effect ofhydrogenmolecule on skin IRI is worth studying,This project studies the anti-apoptosis effect of hydrogen molecule in ratsabdominal skin island flap I/R model. Thirty-six male Sprague-Dawley rats wererandomly divided into three groups:(1) SH: sham surgery group,(2) I/R-P:6-hischemia group treated with physiological saline (PS),(3)I/R-H:6-h ischemia grouptreated with hydrogen-rich saline (HRS). We mainly achieved the following results:1. We successfully builded rat abdominal skin flap ischemia/reperfusion (I/R)model with ligating the left superficial epigastric artery and clamping the rightsuperficial epigastric artery for6h. Rats in ischemia/reperfusion (I/R) groups wereintraperitoneally injected with physiological saline or hydrogen-rich saline (HRS),and the flap tissues were taken from the proximal along the vascular axis of the flap.2.72h after skin flap ischemia, Laser doppler flowmeters was used to measurethe blood perfusion and the survival rate of skin flap, TUNEL staining was used toobserve early apoptosis in skin flap. The survival rate and average blood perfusion oftotal skin flap in I/R-H group was higher than I/R-P group (p<0.05), on the contrary,the rate of cell apoptosis was weaker than I/R-P group(p<0.05).3.24h after skin flap ischemia, the level of pASK-1, pJNK, Bcl-2and Bax andwere examined by immunohistochemistry and immunoblotting, in addition Caspase-3activity were measured by fluorometric assay, and the expression of Bcl-2, Bax and Caspase-3mRNA were detected by RT-qPCR. I/R-H group has a lower protein levelof pASK-1and pJNK (p<0.05, p<0.05, respectively), a lower Caspase-3activity(p<0.01), a higher expression of Bcl-2protein (p<0.05), and a decreased Bax/Bcl-2radio in protein and mRNA (p<0.001, p<0.01respectively) than those of I/R-P group.Bcl-2, Bax and Caspase-3mRNA expression in I/R group was higher than those in SHgroup. Bcl-2mRNA expression in I/R-H group was upregulated much more than thatin I/R-P group, but Bax and Caspase-3mRNA expression was on the contrary.These results show that hydrogen molecule can significantly relieve skin flap IRI,improve flap survival rate, and inhibit cell apoptosis through preventing the activationof ASK-1/JNK pathway, downregulating Bax/Bcl-2radio, and upregulating activationlevel of Caspase-3.