Effects Of HO-1 Mediated By PI3K/Akt Signaling Pathway On The Mitochondrial Fission In Rat Alveolar Type Ⅱ Cells Attacked By LPS

Alveolar epithelial type Ⅱ cell（AECⅡ）belongs to lung secreting epithelial cell that reduces the surface tension of alveoli by producing and releasing dipalmitoyl lecithin.AECⅡ can divide,proliferate and differentiate into type I alveolar epithelial cells.In acute lung injury,AECⅡ restores epithelial integrity through diffusion and migration,which contributes to early damage repairation.Under wound or stress conditions such as infection,macrophage stimulates AECⅡ to produce proinflammatory cytokines such as IL-6,IL-1βand TNF-α^[1],which play an important role in maintaining cell homeostasis.Sepsis-associated ALI has high morbidity and mortality,mainly leads to aggravated oxidative stress and cell damage^[2].A recent report has shown that^[3],mitochondrial dysfunction accelerates the progression of sepsis,and mitochondrial dynamics imbalance,as well as mitochondrial fission plays a key role in mitochondrial dysfunction and cell damage.Previous studies by our group have shown that^[4],heme oxygenase-1/carbon monoxide（HO-1/CO）system can participate in mitochondrial oxidative stress and improve mitochondrial protein expression to reduce mitochondrial fission.The phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt）signaling pathway is an important survival signaling pathway that regulates tissue damage caused by oxidative stress through activating downstream target proteins.In addition,studies have indicated that the PI3K/Akt signaling pathway up-regulates HO-1/CO expression in lipopolysaccharide（LPS）-induced acute lung injury and exerts anti-inflammatory effects^[5].However,it is unclear whether the PI3K/Akt signaling pathway regulates mitochondrial fission in AECⅡ by mediating HO-1 expression.Objective To explore the relationship between HO-1 expression mediated by the PI3K/Akt signaling pathway and mitochondrial protein expression in lipopolysaccharide-induced damage to RLE-6TN cells.Methods In this suject,RLE-6TN cells were subcultured in vitro in a 6-well culture plate at a density of 1×10⁶cells/ml using a random number table method.After24 hours of culture,they were randomly divided into the following 10 groups:Control group（C group）,LPS attack model group（L group）,LPS+exogenous CO release agent CORM-2 group（L+CO group）,LPS+PI3K inhibitor LY294002 group（L+LY group）,LPS+CORM-2+LY294002 group（L+CO+LY group）,LPS+inactive CORM-2（iCORM-2）group（L+iCO group）,LPS+dimethyl sulfoxide（DMSO）group（L+D group）,CORM-2 group（CO group）,LY294002 group（LY group）and CORM-2+LY294002 group（CO+LY group）.Group C was cultured normally;group L was given 10μg/ml LPS to stimulate RLE-6TN cells to prepare endotoxin challenge model;L+CO group was given CORM-2 100μM 1 hour before LPS stimulation;L+LY group was given PI3K inhibitor LY294002 25μM 1 hour before LPS stimulation;L+CO+LY group was given LY294002 25μM and CORM-2 100μM 2 h and 1 h before LPS stimulation;L+iCO group was given iCORM-2 100μM 1 h before LPS stimulation;The L+D group was given 0.1%DMSO 1 h before LPS stimulation;the CO group was given 100μM of CORM-2;the LY group was given25μM of LY294002;the CO+LY group was given 25μM of LY294002 at first,after1 h of incubation,100μM of CORM-2 was given in this group.After pretreated,cells were sequentially incubated for 24 h.Then,cell viability was determined by MTT assay,the concentration of TNF-αand IL-6 in the supernatant of the cells was determined by ELISA,and the expression of phosphorylated Akt（p-Akt）,HO-1,Drp1 and Fis1 were detected in RLE-6TN cells by Western blot.Results Compared with group C,the cell vitality were decreased,IL-6,TNF-αconcentration were increased,p-Akt,HO-1,Drp1 and Fis1 protein expression were up-regulated in group L,group L+CO,group L+LY,group L+CO+LY,group L+iCO,group L+D（P<0.05）.Compared with group L,the cell vitality were increased,IL-6,TNF-αconcentration were decreased,Drp1 and Fis1 protein expression were down-regulated,p-Akt,HO-1 protein expression were up-regulated in group L+CO（P<0.05）;the cell vitality were decreased,IL-6,TNF-αconcentration were increased,Drp1 and Fis1 protein expression were up-regulated,p-Akt,HO-1 protein expression were down-regulated in group L+LY（P<0.05）.There were no significant difference in the parameters mentioned above among group L,group L+iCO and group L+D,and among group C,group CO,group LY and group CO+LY（P>0.05）.Conclusion Increased expression of heme oxygenase-1 inhibits mitochondrial fission in lipopolysaccharide-attacked damage to alveolar epithelial type Ⅱ cells of rats,which mechanism is correlated with the activation of PI3K/Akt signaling pathway.