| The skin flap is formed by the skin with blood supply and its attached subcutaneous tissue.Flap transplantation is a common method for repairing tissue defect and organ reconstruction.In the clinical application,whether the random pattern flap or axial pattern flap is undergoing the stage of ischemia and re-establishment of blood circulation during the transplantation,the transplantation of free flap is more so.During the skin repair operation,the flap tissue ischemia reperfusion process cannot be avoided.Flap ischemia reperfusion injury is a complex pathophysiological process.Many experimental studies have shown that the lesion of vascular endothelial injury,inflammatory response and other lesions caused by perfusion of oxygen free radical damage and calcium overload play an important role in the process of ischemia reperfusion injury.Vascular endothelial cells play an important role in various physiological activities,such as synthesizing and secreting a variety of biological active substances,keeping contraction state of blood vessels,regulating blood flow,activition of blood coagulation,white blood cells,regulating effect on platelet aggregation.In skin flap of ischemia reperfusion,endothelial cell morphology and functional abnormalities,main expression in endothelial cell metabolic disorders,cell apoptosis,the synthesis of various vascular active substances and cytokine imbalance,can cause local vascular resistance increases,capillary contraction congestion,caused "no reflow phenomenon,no reflow phenomenon and increase of vascular endothelial cell injury and so on.IL-1 is the central mediator of immune and inflammatory responses in vivo,and is one of the most powerful inflammatory factors.IL-1 is mainly activated by activating the pattern recognition receptor interleukin-1 receptors in the body.Interleukin-1 receptor(IL-1R)mediated nuclear factor-?B(NF-?B)signaling pathway is an important component of immune and inflammatory systems in the body.IL-lr mediated NF-the B signaling pathway is divided into the myeloid differentiation primary-response protein 88(MyD88)dependent signaling pathway and MyD88 non-dependent signaling pathway according to the intracellular signal transduction.TIR/BB ring analogue AS-1 is similar to the structure domain of MyD88 TIR/BB,and studies show that AS-1 can inhibit the downstream signal transduction caused by i1-1.And a preliminary study has found small molecule compounds TIR/BB ring quasi AS-1 can be mediated by inhibition of IL-1 beta MyD88 signaling pathway to improve myocardial ischemia-reperfusion injury,although the pathological and physiological processes of myocardial ischemia-reperfusion injury skin flap graft ischemia-reperfusion injury has certain similarities,but different diseases,different stages,different organizations,the same material intervention is unlikely to cause the same outcome and ending,and participate in the regulation of biological effect mechanism may also vary.In particular,whether AS-1 can improve vascular endothelial cell injury in skin flap ischemia and reperfusion injury needs further research and confirmation.[Aimed]1.To observe the protective effect of as-1 on ischemia reperfusion injury of endothelial cells;2.To investigate the protective mechanism of as-1 for ischemia reperfusion injury in endothelial cells.[Methods]1.Grouping of experiments:Cell experiment:human umbilical vein endothelial cells were divided into four groups:control group(Con);Experimental group(I/R);Drug treatment group(I/R+as-1);Solvent control group(I/R+DMSO).2.Experimental model:Cell experiment:after pretreatment with as-1 or solvent,human umbilical vein endothelial cells received anoxia for 4 hours,and the complex oxygen 1h was stimulated.The extraction protein is carried out in the next step.3.Detection indicatorsThe repair ability of vascular endothelial cells was detected by cell scratch test.CCK8 test showed cell activity.Cell apoptosis was detected by cell loss.Western-blot detection of related protein expression;Real-time PCR detection of related gene expression.[Results]In order to achieve the experimental purpose,this study is divided into three parts:Part ?:the protective effect of as-1 on ischemia reperfusion injury of endothelial cells.1.AS-1 did not affect vascular endothelial cell activity.The vascular endothelial cells gave different concentrations of AS-1 stimulation 72h.Cell activity was detected by CCK8 kit.It was found that different concentrations of as-1 had no effect on vascular endothelial cell activity.2.AS-1 can improve vascular endothelial cell injury induced by ischemia reperfusion.Anoxia can significantly inhibit the activity of vascular endothelial cells compared with the control group.However,AS-1 could significantly improve the cellular activity induced by hypoxic oxygenation,while the solvent control group had no similar effect.3.AS-1 can protect the apoptosis of vascular endothelial cells caused by H/R.H/R stimulation can significantly promote apoptosis of vascular endothelial cells compared with control group.However,AS-1 could significantly improve the apoptosis induced by hypoxia complex oxygen,while the solvent control group had no similar effect.4.H/R treatment can inhibit the repair of vascular endothelial cells,which can be alleviated by AS-1.It was found that H/R stimulation can significantly inhibit the repair of endothelial cells,and the repair time of hypoxic-oxygenated cells was significantly reduced.The repair function of vascular endothelial cells was significantly enhanced by the as-1 pretreatment,while the solvent control group did not see this effect.Part ii:effects of TIR/BB ring analogue AS-1 on the expression of inflammatory factors in vascular endothelial cells induced by H/R.1.AS-1 can inhibit the expression of inflammatory cytokines il-1 in H/R treatment.H/R treatment can significantly promote the synthesis of inflammatory cytokines il-1 in vascular endothelial cells compared with the control group.The pretreatment of as-1 before hypoxia can significantly inhibit the expression of inflammatory cytokines il-1,while the solvent control group did not see this effect.2.AS-1 can inhibit the expression of inflammatory cytokines TNF-alpha under H/R treatment.H/R treatment can significantly promote the synthesis of inflammatory cytokines TNF-alpha in vascular endothelial cells compared with the control group.However,the pretreatment of as-1 before hypoxia can significantly inhibit the expression of inflammatory cytokines TNF-alpha,while the solvent control group did not see this effect.3.AS-1 can inhibit the expression of chemokine MCP-1 induced by H/R treatment.H/R treatment can significantly promote the synthesis of inflammatory cytokines in vascular endothelial cells,MCP-1,compared with the control group.The pretreatment of as-1 before hypoxia can significantly inhibit the expression of inflammatory cytokines MCP-1,while the solvent control group did not see this effect.Part iii:the mechanism of protecting effect of AS-1 involved in vascular endothelial cells injury caused by H/R treatment.1.AS-1 can inhibit the phosphorylation of P38 protein in vascular endothelial cells induced by H/R treatmentThe phosphorylation of P3 8 protein was obviously induced by hypoxia complex oxygen injury,compared with the control group.However,after the pretreatment of vascular endothelial cells AS-1,the expression of p-p38 protein was significantly inhibited by hypoxic reoxygenation,while the solvent control group did not.2.AS-1 can inhibit the phosphorylation of the I?B protein in vascular endothelial cells induced by H/R treatment.The H/R treatment can obviously induce the phosphorylation of I?B,compared with the control group.However,after pretreatment of vascular endothelial cells with AS-1,the expression of p-I?B induced by H/R treatment was significantly inhibited,while the solvent control group did not.[Conclusion]TIR/BB ring analogue AS-1 can protect the injury of vascular endothelial cells induced by H/R treatment,and inhibit the expression of inflammatory factors caused by H/R.The mechanism was related to the inhibition of IL-1R on the activation of downstream signal MAPK and NF-?B. |
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