Role Of PI3K/Akt Signaling Pathway In CO Up-regulation Of Mitochondrial Fusion Proteins In Rats Alveolar Epithelial Cells Attacked By LPS

In the early stages of sepsis,pulmonary microvascular endothelial cells and alveolar epithelial cells are mainly involved.Direct damage to the external factors and excessive inflammation can destroy alveolar epithelial cells.Damage to the alveolar epithelium leads to decreased barrier function,increased alveolar exudate,and decreased pulmonary surfactant.In contrast,the inhibition of alveolar epithelial cell apoptosis during ARDS can significantly alleviate the progression of the disease course,which indicates that lung epithelial cells may play an important role in the course of ARDS.ARDS is characterized by pulmonary oxidative stress and increased apoptosis,and mitochondria are considered to be the main targets of oxidative damage.In the state of oxidative stress in sepsis,mitochondrial fusion and fission play a key role in maintaining functional mitochondria.In mammals,mitochondrial fusion is regulated by Mfn1/2 located in the outer membrane of mitochondria and OPA1 located in the inner membrane of mitochondria.Studies have shown that endogenous or low concentrations of exogenous CO can produce lung protection by promoting mitochondrial fusion.The PI3K/Akt signaling pathway is an important signaling pathway in the body and is involved in the regulation of cell functions such as cell proliferation,differentiation,apoptosis,and glucose transport.Studies have shown that activating the PI3K/Akt signaling pathway can produce lung protective effects,but whether its specific mechanism is related to mitochondrial fusion is unclear.Objective To evaluate the role of PI3K/Akt signaling pathway in CO-induced up-regulation of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells of rats.Methods The rat alveolar epithelial cells were cultured in a 5%CO₂ cell culture incubator at 37℃with F12K complete medium containing 10%fetal bovine serum and 1%green chain double antibody,and divided into 10 groups（n=5）according to the random number table method:C group,L group,L+CO group,L+LY group,L+CO+LY group,L+i CO group,L+D group,CO group,LY group and CO+LY group.LPS 10μg/ml was added to the culture medium in group L.CORM-2 100μM was added to the culture medium and 1 h later 10μg/ml LPS was added in L+CO group.LY294002 25μM was added to the medium,and 1 h later LPS 10μg/ml was added in L+LY group.In L+CO+LY group,25μM LY294002 was added to the culture medium,CORM-2 100μM was added 1 h later,and then LPS 10μg/ml was added 1 h later.i CORM 100μM was added to the culture medium and 1 h later LPS 10μg/ml was added in L+i CO group.In L+D group,the equal concentration of DMSO was added to the culture medium and 1 h later LPS 10μg/ml was added.CORM-2 100μM was added to the culture medium in CO group.LY294002 25μM was added to culture medium in LY group.LY294002 25μM was added to the culture medium,and 1 h later CORM-2 100μM was added in CO+LY group.Cells were harvested after 24 h of incubation for measurement of the MDA content,SOD activity and expression of HO-1,p-Akt,Mfn1,Mfn2 and OPA1 by Western blot.Results Compared with group C,the MDA content was significantly increased and SOD activity was decreased in L,L+CO,L+LY,L+CO+LY,L+i CO,L+D groups,and the expression of HO-1,Mfn1,Mfn2,OPA1 protein and p-Akt was significantly up-regulated in group L（P<0.05）.Compared with group L,MDA content was significantly decreased and SOD activity was increased in group L+CO,MDA content was significantly increased and SOD activity was decreased in group L+LY,the expression of HO-1,Mfn1,Mfn2,OPA1 protein and p-Akt was significantly up-regulated in group L+CO,and the expression of HO-1,Mfn1,Mfn2,OPA1protein and p-Akt was significantly down-regulated in group L+LY（P<0.05）.Compared with group L+CO,the MDA content was significantly increased,SOD activity was decreased,and the expression of HO-1,Mfn1,Mfn2,OPA1 and p-Akt was down-regulated in group L+CO+LY（P<0.05）.Conclusion CO can up-regulate the expression of mitochondrial fusion protein when endotoxin attacks rat alveolar epithelial cells.The mechanism is related to the activation of the PI3K/Akt signaling pathway.