Role Of PI3K/Akt Pathway In The Effect Of CO On The Expression Of MFN1 And FIS1 In LPS-induced Rats Alveolar Macrophages

Acute respiratory distress syndrome(ARDS)is a critical illness syndrome,which has a high mortality.The mechanisms of ARDS is complex,involved in inflammation,oxidative stress,the formation of water channel protein,blood coagulation/fibrinolysis system imbalance,apoptosis,in which oxidative stress and inflammation are the vital role in the development and outcome of the ARDS.widely studied.Mitochondria are the main place of oxygen free radicals production,which is closely associated with inflammation.Mitochondria fusion and fission is one of the regulator of maintainning mitochondrial function.ARDS can lead to mitochondria fusion fewer and fission more [1].Carbon Monoxide,the product of heme catalyzed by HO-1,have anti-inflammatory anti oxidative stress effect in ARDS[2-3].Meanwhile,Our previous studies have shown that CO could relieve the injury of the alveolar macrophages induced by endotoxin damage,as well as promoted mitochondria fusion protein 1(MFN1)expression and inhibited the expression of fission protein FIS1[4-5].1-phosphatide acyl inositol-3-kinase serine/threonine kinase protein kinase(PI3K/Akt)signal pathway is widely exists in cells,involved in the growth and survival,proliferation and apoptosis,carbohydrate metabolism,gene transcription,cell migration and cell cycle regulation [6-7],which is also one of the mechanisms of ARDS caused by sepsis [8-10].This study intends to explore the effect of PI3K/Akt signaling pathway in CO on MFN1 and FIS1 expression in LPS-induced alveolar macrophage injury.Objective To evaluate the the role of PI3K/Akt pathway in the effect of CO on MFN1 and FIS1 in LPS-induced rats alveolar macrophages.Methods Primarily cultured rats alveolar macrophages NR8383,aged 12-20 weeks,were seeded into the laminin pre-treated 96-well plates at a density of 4×104/ml and cultured for 24 h.And then we use a random number table to distribute this cells into 10 groups(n=10 each)using: control group(group C),LPS group(group L),LPS+CORM-2 group(group L+O),LPS+CORM-2+LY294002 group(group L+O+Y),LPS+DMSO group(L+D group),LPS+iCORM-2 group(L+iC group),LPS+LY294002 group(L+Y group),CORM-2 group(O group),LY294002 group(Y group),CORM-2 +LY294002 group(O+Y group).Cells keeps no special treatment in group C.Cells were treatmented by LPS at 10 μg/ml in group L,group L+O and group L+O+LY.Group L+O were conducted with CORM-2 100 μmol 1 h before LPS given.Group L+O+Y were given LY294002 20 μm and CORM-2 100 μmol at 1.5 h,1 h before LPS treatment.All of the indicators were determined at 24 h after treatment.TNF-α and IL-10 in the supernatant were determined by ELISA.The level ROS,MDA,SOD,ATP were detected using relevant kit.The expression of MFN1,FIS1,HO-1,Akt,p-Akt were determined by Real-Time-PCR and Western blot.Results LPS could upregulate the expression of FIS1,but downregulate the expression of MFN1.CO could further upregulate MFN1 expression but downregulate FIS1,meanwile,the level of inflammation and oxidative stress were limited.When PI3 K /Akt were blocked,the expression of MFN1 were downregulated,FIS 1 expression were upregulated.Conclusion CO up-regulated the expression of MFN1 and downregulated FIS1 in LPS-induced rats alveolar macrophages,and PI3K/Akt pathway activation is involved in it.