Involvement Of The P38MAPK Signaling Pathway In The Induction Of Heme Oxygnase-1 Influences Mitochondrial Fusion Protein In Lps-attacked Rat Alveolar Epithelial Type Ⅱ Cells

Sepsis is an organ dysfunction of a crisis that is caused by a reaction of a host to an infection,it affects millions of people around the world every year and the mortality rate is as high as 25%,has become one of the top ten causes of death.Excessive oxidative stress and inflammatory response are considered to be an important cause of multiple organ dysfunction due to sepsis,and the lung is one of the most vulnerable target organs in sepsis.Moreover,in the target cells involved in sepsis lung injury,alveolar epithelial cells are important target effector cells,alveolar neutrophil(PMN),alveolar macrophages and other inflammatory cells adhere to the surface of alveolar epithelial cells and release a large amount of reactive oxygen species(ROS)nd other inflammatory mediators to damage alveolar epithelial cells,so that destroying the intra-pulmonary mechanical barrier,leading to an influx of inflammatory factors,and further aggravate the lung injury.Mitochondria are important organelles in cells,in addition to being able to produce ATP,it also plays an important role in regulation of programmed cell death,immune inflammatory response,calcium signaling and fatty acid oxidation.Meanwhile,mitochondria as a dynamic organelle can change it size,shape,location and function in cells through mitochondrial dynamics.The effect of oxygen free radicals on mitochondria leads to a decrease in mitochondrial fusion and affect mitochondrial function,which in turn impairs the long-term viability of cells.In mammals,proteins that regulate mitochondrial fusion include mitochondrial fusion protein 1(mitofusin 1,Mfn1),mitochondrial fusion protein 2(mitofusin 2,Mfn2),optic atrophy protein 1(OPA1).Among them,Mfn1/2 is a protein located in mitochondrial outer membrane and OPA1 is located in mitochondrial inner membrane.Research has shown that,in cultured cells and mouse tissues,the loss of fusion protein can lead to serious breakage of mitochondrial network,terminated the substance exchange between mitochondria and significantly decreased mitochondrial DNA content.Loss of Mfn1 and Mfn2function resulted in a change in mitochondrial metabolic function,loss of membrane potential and reduced mitochondrial respiratory function.Knockout of OPA1 results in extensive loss of mitochondrial membrane potential and reduction of basal respiratory rate.Heme oxygenase-1(HO-1)is a stress reaction enzyme induced by ROS and cytokines,can catalyze the degradation of heme to carbon monoxide,cholestatin and free iron,and plays an important role in inhibiting and regulating inflammation of tissues and organs,oxidative stress,apoptosis and proliferation.Our previous studies shown that,in the model of LPS attacking rats,up-regulation of HO-1 can promotes mitochondrial fusion,but the mechanism remains unclear.As one of the subfamilies of mitogen-activated protein kinase,p38MAPK is the link of information transduction,can be activated by factors such as extracellular inflammatory stimuli.Activated p38MAPK pathway can stimulate endogenous anti-inflammatory ability of organism,induced HO-1 expression and regulation of cell aging,apoptosis,cellular inflammation and oxidative stress,so that plays an important role in alleviating the inflammatory response of acute lung injury.There is no report on the effect of p38MAPK-mediated HO-1 expression on mitochondrial fusion in alveolar II epithelial cells of rats with LPS infection.Objective To investigate the effect of p38MAPK pathway mediated expression of heme oxygenase 1(HO-1)on mitochondrial fusion in LPS attacked rats alveolar epithelial type II cells.Methods Rat alveolar epithelial type II cells were seeded in 6-well plate with 1x10~6cells/ml density.These cells were randomly divided into 7 groups using a random number table:control group(group C),LPS group(group L),LPS+carbon monoxide-releasing molecules CORM-2 group(group LC),LPS+p38MAPK inhibitor SB203580group(group LS),LPS+DMSO group(group LD),CORM-2 group(group CO),SB203580 group(group S).Group C keeps no special treatment,Group L were stimulated by LPS at 10μg/ml,Group LC were given CORM-2 100μM pretreatment0.5h and then added LPS,Group LS were given SB203580 at 1h before LPS given,Group LD were given 0.1%DMSO at 1h before LPS given,Group CO,group S were added to the culture medium CORM-2 100μM,SB203580 10μM.All cells were incubated for 24h.Cell activity were determined by MTT assay,MDA content and SOD activity in the culture medium were determined by thiobarbituric acid and xanthine oxidase from buttermilk respectively,tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in cell supernatant were determined by ELISA.The expression of p38mitogen activated protein kinase(p38MAPK),phosphorylation of p38mitogen-activated protein kinase(p-p38),hemeoxygenase-1(HO-1),Mitofusin1(Mfn1),Mitofusin2(Mfn2),optical atrophy-1(OPA1)were measured by Western blot.Result Compared with group C,group CO and group S,MDA concentration were increased,SOD activity were reduced,the levels of TNF-αand IL-6 were increased,the activity of cell were decreased,and HO-1,p-p38MAPK protein expression were up-regulated,Mfn1,Mfn2,OPA1 were down-regulated in other groups(P<0.05).Compared with group L,MDA concentration were decreased,SOD activity were increased,the levels of TNF-αand IL-6 were decreased,the activity of cell were increased,and HO-1,Mfn1,Mfn2,OPA1,p-p38MAPK were up-regulated in group LC.MDA concentration were increased,SOD activity were decreased,the levels of TNF-αand IL-6 were increased,the activity of cell were decreased,and HO-1,Mfn1,Mfn2,OPA1,p-p38MAPK were down-regulated and in group LS(P<0.05).Compared with group LC,MDA concentration were increased,SOD activity were decreased,the levels of TNF-αand IL-6 were increased,the activity of cell were decreased,and HO-1,Mfn1,Mfn2,OPA1,p-p38MAPK were down-regulated in group LS.And there was no significant difference in the expression level of total p38protein among the groups(P>0.05).Conclusion Heme oxygenase 1 attentuates the expression of mitochondrial fusion protein in LPS-attacked rat alveolar epithelial type II cells,which may be related to p38MAPK signaling pathway.