Effect And Its Potential Mechanism Of Alpha-linolenic Acid In Mice With Lipopolysaccharide-induced Acute Lung Injury

ObjectiveAcute lung injury(ALI)and its severe form,acute respiratory distress syndrome(ARDS),are the leading causes of mortality in critically ill patients.The occurrence of ALI can be triggered by numerous factors,such as shock,sepsis,trauma,burns,severe pneumonia,and acute pancreatitis.Alpha-linolenic acid(ALA),an important component of polyunsaturated fatty acids(PUFAs),possesses potent anti-inflammatory properties.To date,the effects of ALA on acute lung injury(ALI)remains unknown.This study was designed to assess the potential protective effects of ALA against LPS-induced ALI and the underpinning mechanisms were further explored.Materials and Methods1.An animal model of ALI was established via i.t.(intratracheally)injection of lipopolysaccharide(LPS,1mg/kg).Wide-type male C57BL/6 mice were randomized divided into four groups.Sham group,in which mice were intratracheally administrated with normal saline 50μl.Lipopolysaccharide(LPS)group,in which mice were intratracheally administrated with LPS(1mg/kg,50μl).Low-dose of ALA group,in which mice were intraperitoneally injected with alpha-linolenic acid(360mg/kg)and 1h later were intratracheally administrated with LPS(1mg/kg,50μl).High-dose of ALA group,in which mice were intraperitoneally injected with alpha-linolenic acid(1800mg/kg)and 1h later were intratracheally administrated with LPS(1mg/kg,50μl).2.After the mice(n=24)in four groups were injected with LPS for 6h,bronchoalveolar lavage fluid(BALF)was collected to measure the protein concentrations,the levels of TNF-α,IL-6,IL-1β,IL-10,and the inflammatory cell counts,including the total cells,neutrophils and macrophages.Meanwhile,the lungs were also harvested to detect the apoptosis via TUNEL staining.3.After the mice(n=24)in four groups were injected with LPS for 6h,the lungs were harvested to detect the lung dry/wet ratio.4.After the mice(n=24)in four groups were injected with LPS for 6h,the upper right lobe of lung tissues were captured and stained with the hematoxylin and eosin(HE)reagent according to standard protocols.The rest of lungs were used to detect the levels of myeloperoxidase(MPO),malondialdehyde(MDA),superoxide dismutase(SOD)andglutathione(GSH).5.After the mice(n=24)in four groups were injected with LPS for 6h,the lungs were also harvested to detect the expression of caspase-3,IκBα,and NF-κB(p65).6.RAW264.7 was treated with different concentrations of ALA for 1h prior to the LPS(1μg/ml)stimulation.Then the supernatants were collected,and levels of TNF-α,IL-6,IL-1β and IL-10 were determined by ELISA.Total proteins were collected to determine the expression of caspase-3 and the phosphorylation of IκBα and NF-κB(p65).Results1.ALA significantly inhibited LPS-induced TNF-α,IL-1β and IL-6 secretion in BALF(P<0.05),and increased the production of IL-10(P<0.01).The protein concentration and inflammatory cells infiltration in ALI were substantially alleviated by ALA pretreatment(P<0.01).Additionally,TUNEL positive cells in lung tissues were markedly reduced by ALA pretreatment(P<0.01).2.Lung dry/wet ratio in ALI were substantially alleviated by ALA pretreatment.3.The histopathological changes induced by LPS were also mitigated by ALA pretreatment(P<0.05).The levels of MPO and MDA were highly increased while the activities of SOD and GSH were decreased in lung with the injection of LPS,which was reversed by ALA pretreatment(P<0.05).4.ALA markedly suppressed the expression of caspase-3 and the phosphorylation of IκBα and NF-κB(p65)activated in ALI.5.In RAW264.7 culture condition,ALA pretreatment significantly inhibited LPS-induced the secretion of TNF-α,IL-1β and IL-6(P<0.05),but the increased IL-10 production was not observed in vitro(P>0.05).Besides,ALA pretreatment markedly suppressed the expression of caspase-3 and the phosphorylation of IκBα and NF-κB(p65).ConclusionsALA effectively protected mice against LPS-induced ALI.NF-κB pathway was likely to be involved in ALA mediated protective effects.