Regulation Of ENaC-mediated Alveolar Fluid Clearance By Insulin Via PI3K/Akt Pathway In Acute Lung Injury

Objective To observe the effect of insulin on the regulation of alveolarfluid clearance (AFC) and epithelial sodium channel (ENaC) expression，and to explore the role of insulin in acute lung injury (ALI) through theestablishment of animal model of ALI with non-hyperglycemia;to furtherclarify the possible signaling pathway and mechanism involved thataffected the expression of ENaC by insulin through culture of alveolarepithelial cell line A549,whcih may provide a theoretical evidence and newidea for its future clinical application.Methods (1) Adult healthy SD rats were anesthetized by intraperitonealadministration of sodium pentobarbital (50mg/kg) and were performed byinsertion of an internal jugular vein catheter.①Insulin Group(LPS+Insulin)： Lipopolysaccharide(LPS)(5mg/kg)was given via jugular veincatheter.Insulin(0.1U/kg/h)was administered via micro-osmotic pumps16hours before LPS exposure.②Wortmannin (PI3K inhibitor) Group(LPS+Insulin+Wortmannin): LPS(5mg/kg)was given via jugular veincatheter.Insulin(0.1U/kg/h)was administered via micro-osmotic pumps16 hours before LPS exposure.Wortmannin(0.06mg/kg) were injectedintravenously three times at-90,+90, and+360minutes relative to the LPSinjection.③LPS Group:LPS(5mg/kg)was given via jugular veincatheter.An equivalent volume of saline was administered viamicro-osmotic pumps16hours before LPS exposure.④Control Group:Anequivalent volume of saline was given via jugular vein catheter.Anequivalent volume of saline was administered via micro-osmotic pumps16hours before jugular vein injection.Glucose levels and insulin levels in eachgroup were determined by Glucometer and enzyme-linked immunosorbentassay (ELISA) at0,1hours,4hours,8hours after jugular vein injectionrespectively.Blood samples were obtained and lung tissues were collectedafter SD rats in each group were sacrificed.The detection of AFC andbronchoalveolar lavage fluid (BALF) were done in the right lung,whiletotal lung water content (TLW) was analyzed in the left lung. Pathologicalchanges of lung tissue were observed by HE staining and were comparedby lung injury score.The changes of ENaC expression in lung tissue weredetermined by immunocytochemistry.ENaC mRNA and protein expressionin lung tissue were determined by reverse transcriptase polymerase chainreaction (RT-PCR) and western blot respectively.The levels of Aktphosphorylation and the protein expressions of Nedd4-2in lung tissue wereanalyzed by western blot.(2) A549cells were divided into①ControlGroup: the cells were incubated in serum-free medium for2hours;② Insulin Group: cells were incubated in medium containing insulin (200mU/L) for2hours;③LY294002(PI3K inhibitor) Group: the cells werepre-incubated with LY294002(10μM) for30minutes before insulin(200mU/L) treatment for2hours;④Akt inhibitor Group: the cells werepre-incubated with Akt inhibitor(100nM) for30minutes before insulin(200mU/L) treatment for2hours. ENaC mRNA and protein expression in A549cells were determined by RT-PCR and western blot.The levels of Aktphosphorylation and the protein expressions of Nedd4-2in A549cells wereanalyzed by Western blot.The relationship between Nedd4-2and eachsubunit of ENaC were analyzed by co-immunoprecipitation.Results (1) Glucose levels and insulin levels in each group showed nosignificant differences at0,1h,4h,8h respectively.Interleukin-6(IL-6),tumor necrosis factor-α (TNF-α), myeloperoxidase (MPO) activity andprotein content in BALF were significantly increased in LPS Groupcompared with that in Control Group.IL-6, TNF-α, MPO activity andprotein content in BALF were significantly decreased by insulin,the effectsof which were blocked by wortmannin.Pathological changes ofLPS-induced lung injury were significantly improved by insulintreatment,of which lung injury score was significantly lower than that inLPS Group,which was blocked by wortmannin. AFC was significantlydecreased and TLW was significantly increased in LPS Group comparedwith that in Control Group.AFC was significantly increased and TLW was significantly decreased by insulin treatment,which was blocked bywortmannin.α-ENaC, β-ENaC and γ-of ENaC mRNA and proteinexpression and the levels of Akt phosphorylation were up-regulated withdown-regulation of Nedd4-2protein expression by insulin compared withthat in LPS Group.α-ENaC, β-ENaC and γ-of ENaC mRNA and proteinexpression and the levels of Akt phosphorylation were decreased withup-regulation of Nedd4-2protein expression by wortmannin intervention.(2)α-ENaC, β-ENaC and γ-of ENaC mRNA and protein expression and thelevels of Akt phosphorylation were significantly up-regulated withdecrease of Nedd4-2protein expression in A549cells by insulin.α-ENaC,β-ENaC and γ-of ENaC mRNA and protein expression and the levels ofAkt phosphorylation were decreased with up-regulation of Nedd4-2proteinexpression by LY294002and Akt inhibitor intervention respectively.Theexpressions of Nedd4-2binding to α-ENaC, β-ENaC and γ-ENaC weredecreased by insulin through co-immunoprecipitation.The expressions ofNedd4-2binding to α-ENaC, β-ENaC and γ-ENaC were increased byLY294002and Akt inhibitor intervention respectively.Conclusions (1)Insulin can promote alveolar edema fluid clearance, soas to reducing the accumlation of pulmonary edema fluid and lung watercontent in ALI.(2)Insulin regulated the ENaC expression via PI3K/Aktsignaling pathway,which had an effect on alveolar edema fluid clearance.(3)Insulin inhibited Nedd4-2binding to the ENaC subunits, which activated ENaC and increased the expressions of each ENaC subunit via PI3K/Aktsignaling pathway.