| Objective:1.To study whether there is necroptosis in skin flaps after ischemia/reperfusion injury.2.To investigate the influence of Nec-1 in skin flaps after ischemia/reperfusion injury.3.To explore whether the protect effect of hydrogen molecule in skin flaps after ischemia/reperfusion injury is related to its inhibition of necroptosis.4.By comparing the effects of molecular hydrogen and Nec-1 on the necroptosis in skin flaps after ischemia/reperfusion injury,also the effects of molecular hydrogen and Nec-1 on the condition of survival rate and blood perfusion in skin flaps after ischemia/reperfusion injury are researched,we hope to put feasible suggestion forward the research direction of the prevention and treatment of skin flap ischemia/reperfusion injury.Methods:1.The experiment was designed with 2 different groups,CTL group and Nec-1 group.By using the laser Doppler flowmeter,we detected the survival rate and blood perfusion condition at different positions of skin flaps after ischemia/reperfusion injury.Then H&E staining,TUNEL staining and electron microscope observation were performed to observe the morphology of skin flap tissues.IHC staining and WB were used to detect the expression level of RIP1,a key factor of necroptosis.2.This part of our research includes 3 different groups,SH group(sham group),Nec-1 group and CTL group according to different treatment methods.24h after the ischemia/reperfusion of skin flaps,survival rate and blood perfusion were detected.Then the morphology of tissues and cells were observed by staining the flap tissue by using different methods.The expression level of RIP1 was detected to determine whether Nec-1 can protect the flap tissue and reduce necrosis by inhibiting necroptosis in the ischemia-reperfusion flap tissue.3.45 male SD rats were randomly divided into 3 groups,HCH group(molecular hydrogen group),CTL group and SH group.The survival rate and blood perfusion of skin flaps were detected by laser Doppler blood flowmeter.Morphological characteristics of the flap tissue were observed by H&E staining.RIP1 expression level was detected by immunohistochemical staining and Western Blot technology.The results were obtained to prove that the protective effect of molecular hydrogen on the ischemia/reperfusion injury flap is achieved by inhibiting necroptosis.4.According to different treatment,randomly divide 60 male SD rats into HCH group,Nec-1 group,CTL group and SH group.24h after the ischemia/reperfusion of skin flaps,survival rate and blood perfusion were detected.Then the morphology of tissues and cells were observed by staining the flap tissue by using different methods.The expression level of RIP1 in flaps were detected by immunohistochemical staining.Finally,compare the data between the groups to determine whether molecular hydrogen and Nec-1 have any difference in flap survival rate,blood perfusion and necroptosis level.Results:1.By watching the morphological characteristics,skin flap tissue in Nec-1 group has less inflammatory cell infiltration than CTL group.Flap survival rate and blood perfusion was higher in Nec-1 group.In terms of the level of necroptosis,as the distance between flap tissue and vascular pedicle increased,the necroptosis level gradually raised,differences were detected of the data of RIP 1 expression level between two groups in the distal flap region.2.The morphological observation results showed that inflammatory cell infiltration condition were rarely seen in SH group and Nec-1 group,which is significantly different in CTL group.On the condition of flap survival rate and blood perfusion,Nec-1 group was worse than SH group,but it was significantly better than CTL group.RIP1 expression level in CTL group was the highest compared to SH group,which is the lowest group,Nec-1 group was between those two.3.After the application of molecular hydrogen treatment,the level of necroptosis in skin flaps of HCH group was significantly lower than that in the CTL group but still higher than SH group.The skin flap survival rate and blood perfusion condition of HCH group were better than CTL group but worse than SH group.4.Compared to Nec-1 group,the survival rate of flap and blood perfusion of HCH group keeps a similar level.However,in terms of RIP 1 expression level,it shows a significant lower level in HCH group.Conclusions:1.In the ischemia/reperfusion injury flap,necroptosis can play an important role in the process of its ischemia/reperfusion injury.2.Nec-1 can reduce skin flap ischemia/reperfusion injury by inhibiting necroptosis via RIP1 access.3.Molecular hydrogen can inhibit the effect of RIP1 to reduce the necroptosis level in ischemia/reperfusion skin flaps.It can also protect skin flap to increase the skin flap survival rate and blood perfusion.4.Molecular hydrogen and Nec-1 have similar protective effects on skin flaps after ischemia-reperfusion injury.However,molecular hydrogen has a stronger inhibitory effect on necroptosis. |
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