| Research BackgroundAcute lung injury(ALI)/acute respiratory distress syndrome(ARDS)is caused by severe infection,trauma and other internal and external factors,which is a common clinical severe disease characterized by refractory hypoxemia.It is now widely believed that the essence of ALI / ARDS is inflammation and the incontrollable inflammation is closely related to the disease.In recent years,with the deepened research of ARDS,the diagnosis and treatment technology has improved significantly.However,the mortality rate remains at a high level[1-2].Therefore,reserching more in-depthly of its pathogenesis,seeking new effective therapeutic targets and developing new drugs and treatment to extenuate excessive inflammation to reduce morbidity and mortality is particularly important.Death-related protein kinase 1(DAPK1)is a calmodulin(CaM)-modified serine/ threonine protein kinase that contains multiple functional regions,which is widely involved in multiple pathways-induced apoptosis as one of the positive regulators of apoptosis[3-4].It also participates in the regulation of inflammation and autophagy[5-6].Still other studies have found that DAPK1 participates in the kidney and brain ischemic acute injury disease process[7-8].However,whether DAPK1 is also involved in the pathogenesis of acute lung injury,or is associated with the out of control of inflammation has not been reported.Therefore,this study aims to construct acute lung injury model and macrophage inflammation model by using LPS to observe the expression change of DAPK1.DAPK1 expression was down-regulated to detect cell autophagy level,proinflammatory gene expression and inflammatory factor expression level thus investigating the role and mechanism of DAPK1 in the pathogenesis and regulation of inflammation in acute lung injury.Reserch Object:1.To elucidate the expression of DAPK1 and its role of uncontrolle inflammation in acute lung injury.2.To investigate the potential mechanism of DAPK1 in the control of inflammatory response.Methods:1.Expression of DAPK1 in lung tissue of mice with acute lung injury.The acute injury model was established by intraperitoneal injection of LPS(10mg/kg).The lung injury was observed by HE staining,and the expression and distribution of DAPK1 in lung tissue of acute lung injury was confirmed by immunohistochemistry,Western blot and Real-time PCR.The level of plasma proinflammatory cytokines was measured by ELISA.2.Effect and mechanism of DAPK1 on LPS-induced inflammatory response in mouse macrophages.Western blot and Real-time PCR were used to observe the expression of DAPK1 with the change of time and concentration in RAW264.7 macrophages after LPS stimulation.DAPK1 shRNA adeno-associated virus were contributed;After macrophages were infected,the effects on NF-?B promoter activity,downstream p38-mTOR signaling pathway and autophagy-associated protein LC3 and p62 were observed by Western blot,the expression changes of IL-6,TNF-a and IL-1? was detected by Real-time PCR.3.Effects of down-regulation of DAPK1 expression on inflammatory reaction in acute lung injury.The acute injury model was established by intraperitoneal injection of LPS.The expression of DAPK1 was selectively inhibited by pretreatment of DAPK1-specific inhibitor TC-DAPK6.Western blot was used to detect the inhibitory effect of TC-DAPK6 on DAPK1.HE staining was used to observe the pathological changes of LPS induced lung injury after DAPK1 inhibition.Western blot and Real-time PCR were used to observe the level of NF-?B P65 and proinflammatory cytokines after DAPK1 inhibition.Kaplan-Meier survival analysis was used to study the survival time of mice in different treatment groups.Results:1.The model of acute lung injury was successfully constructed.DAPK1 expression was low in lung tissues of normal mice.The expression of DAPK1 was higher than the control group at 3h after acute lung injury(P <0.05),and then reached the highest level at 24h(P <0.05).The expression of TNF-? and IL-6 in acute lung injury were significantly higher than that in control group(P <0.05),and maintained at a high level at 12-24h(P <0.05).With the prolongation of LPS treatment time,the wet to dry weight ratio(W/D)of lungs increased gradually(P <0.05).2.With the increasing of LPS concentration to stimulate RAW264.7 macrophages,the expression of DAPK1 gradually increased(P<0.05).The expression of DAPK1 began to rise at 6h after LPS stimulating(P<0.05),and reached the highest level at 24 h and then decreased at 48h(P<0.05).In addition,the expression of NF-?B promoter and inflammatory factors IL-6,TNF-?and IL-1? were obviously increased after treatment with LPS on RAW264.7 cells.After down-regulation of DAPK1 expression in macrophages by AAV-DAPK1 shRNA transfection,NF-?B promoter activity declined significantly(P <0.05),while P38-mTOR expression was inhibited,LC3 II/I up-regulated and p62 expression decreased(P <0.05),autophagy is activated.Moreover,the expression of proinflammatory cytokines IL-1?,IL-6 and TNF-? were also decreased(P <0.05).3.After the specifically inhibition of DAPK1 expression by TC-DAPK6,the pathological damage of lung tissue in mice were significantly attenuated than that in LPS alone group(P <0.05),and the expression of NF-?B P65 was obviously decreased after DAPK1 inhibition(P <0.05).Plasma proinflammatory(TNF-??IL-6)level was significantly lower than that in LPS group(p <0.05).The lung tissue W/D is decreased obviously.KaplanMeier survival analysis showed that the survival time of DAPK1-inhibited mice was clearly longer than that of LPS group(p <0.01).Conclusions:1.The expression of DAPK1 in LPS-induced acute lung injury was significantly increased,may be involved in the pathogenesis of acute lung injury.2.DAPK1 participates in the process of acute lung injury by regulating P38-mTOR signaling pathway to inhibit macrophage autophagy and activating NF-?B inflammatory signaling pathway.3.In vivo studies have further confirmed that DAPK1 can promote the development of ARDS by activating NF-?B inflammatory signaling pathway. |
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