Analysis Of Related Mechanisms Of Post-conditioning With Inhaled High Concentration Hydrogen Attenuates Skin Ischemia/reperfusion Injury Through The Necrotic Pathway

Objectives:To analyze the protective effect of high concentration hydrogen(HCH)inhalation on the flap of rat flap ischemia/reperfusion(I/R)injury model and the potential mechanism of inhibiting necrotic pathway.Methods:The experimental animals were divided into three groups:sham operation group(SH),ischemia/reperfusion injury control group(IR),and ischemia-reperfusion injury group after high concentration hydrogen treatment(HCH),15 SD rats in each group,8 weeks,weighing 280-320 g.After 3 hours of ischemia-reperfusion treatment,the SH group and the IR group were placed in normal atmospheric environment,and the HCH group was placed in high-concentration hydrogen for 1 hour.On the third postoperative day,the blood perfusion of different groups of rat flaps was measured by laser Doppler flowmeter and laser speckle contrast analysis.The expressions of necroptosis-related molecules RIP1,RIP3,MLKL,PGAM5 and Drpl in different groups of rat flaps were determined by HE staining,immunohistochemistry staining,Western Blot and RT-qPCR.Results:In this experiment,we found that high concentration hydrogen post-conditioning can significantly improve the survival rate of ischemia-reperfusion injury flaps.Compared with the IR group(22.74±5.41%;29.35±7.15 PU),the HCH group(42.81 ±7.08%,p<0.001;54.15±7.98 PU,p<0.001)significantly increased the flap survival ratio and blood perfusion.After HE staining,it was found that the inflammatory cells mainly accumulated in the dermis layer after ischemia-reperfusion injury,and the inflammatory infiltration of the flap tissue decreased after high-concentration hydrogen treatment.The results of immunohistochemistry showed that the expression of RIP-1,RIP-3,MLKL,PGAM5 and Drpl in the IR group was strongly positive,while the HCH group was weakly positive.Western Blot and RT-qPCR analysis showed that the HCH group was significantly lower than the IR group in terms of RIP-1,RIP-3,MLKL,PGAM5,Drp1 protein and RNA expression after HCH treatment.Conclusions:HCH can significantly improve the survival area and blood flow of the ischemic reperfusion injury flap;after HCH treatment,the inflammatory infiltration of the flap tissue decreases,and the expression levels of necrotic related factors,RIP-1,RIP-3,MLKL,PGAM5 and Drpl,were all significantly decreased.