Obeticholic Acid Alleviate Lipopolysaccharide-induced Acute Lung Injury Via Its Anti-inflammatory Effects In Mice

BackgroundAcute lung injury(ALI)is a common disease leading to acute respiratory failure and death.In the past decades,the morbidity and mortality of ALI have remained high,and there is no effective therapeutic method and drugs.FXR,a ligand-dependent transcription factor,plays an important role in regulating bile acid metabolism.Obeticholic acid(OCA)is a synthetic FXR agonist which has been shown that it also has anti-inflammatory effects.LPS-induced ALI models is a widely used animal model of acute lung injury.This experiment investigated whether OCA activating-FXR can reduce LPS-induced acute lung injury in mice,and whether it can repress acute lung injury by alleviating several inflammatory signaling pathways.Objective(1)Whether OCA alleviates LPS-induced acute lung injury in mice;(2)Whether OCA can repress LPS-induced acute lung injury through inhibiting several inflammatory signals in mice.MethodsSixty male mice were randomly divided into six group with each 10 mice,Control group,OCA pretreatment group,LPS-2h group,the LPS-6h group,LPS+OCA-2h group and LPS+OCA-6h group.Mice in the OCA group,LPS+OCA-2h group and LPS+OCA-6h group were supplementation with OCA(OCA,5mg/kg,dissolved in corn oil)by gavage at 72 h,48h and 24 h before LPS injection.Mice in the LPS group were intraperitoneally injected with LPS(1.0mg/kg,dissolved in normal saline),while mice in the Control group and OCA group were injected with equal volume normal saline.Mice were sacrificed respectively at 0h,2h and 6h after LPS injection,and lung tissues were collected.The lungs coefficient and pathological changes were observed.Alveolar lavage fluid was prepared.The inflammatory cytokines in serum and bronchoalveolar lavage fluid were detected by ELISA.The levels of Tnf-α,Il-1β,Tgf-β,Kc,Il-10 and Mcp-1 mRNA in lung tissues were measured by RT-PCR.The protein expression of NF-κB(Nuclear factor-kappa B,NF-κB)signaling,MAPKs signaling and PI3K/Akt signaling pathways were evaluated using Western-Blotting.The positive nuclear localization of FXR,NF-κB p65 and NF-κB p50 subunits were detected through immunohistochemistry.Results(1)OCA treatment aleviated LPS-induced acute lung injury in mice.The model of acute lung injury in mice was successfully established by intraperitoneal LPS injection,and oral OCA supplementation could significantlly decrease LPS-induced acute lung injury in mice.The absolute lung weight and relative lung coefficient in mice were markedly higher than Control group at 6 h after LPS exposure.Pathology showed that oral OCA supplementation could effectively inhibit LPS-induced pulmonary edema and inflammatory cell infiltration,and oral OCA supplementation prominently decreased LPS-induced up-regulation of the pathological score of acute lung injury in mice.These results showed that OCA treatment could appreciably attenuate LPS-induced acute lung injury in mice.(2)OCA treatment reduced LPS-induced up-regulation of pulmonary pro-inflammatory cytokines and chemokines expression in the lungsThe levels of Tnf-α,Il-1β,Tgf-β mRNA in the lungs of mice were remarkably increased at 2 h and 6 h after LPS injection.Oral OCA supplementation notably repressed LPS-induced up-regulation of pro-inflammatory cytokines in the lungs.Pulmonary chemokines,Kc and Mcp-1 mRNA,were significantlly increased after LPS exposure,while oral OCA supplementation could antagonize LPS-induced of up-regulation pulmonary chemokines.The previous study also found that the anti-inflammatory factors,IL-10 mRNA in the lung,was significantlly increased at 2 h and 6 h after LPS exposure,and the level of IL-10 mRNA was further increased at 6 h after OCA pretreatment.These results showed that OCA not only inhibited LPS-induced up-regulation pulmonary pro-inflammatory cytokines and chemokines in mice,but also promoted the release of anti-inflammatory cytokine.(3)OCA treatment decreased LPS-induced the release of pro-inflammatory cytokine and chemokines in serumThe level of pro-inflammatory cytokine TNF-α was significantlly increased after LPS exposure in serum.Oral OCA supplementation could effectively block LPS-induced up-regulation of pro-inflammatory cytokine in serum.Serum chemokine KC was significantlly increased after LPS exposure,and OCA pretreatment could suppress LPS-induced up-regulation of chemokine in serum.The results showed that oral OCA supplementation could inhibit LPS-induced the releases of pro-inflammatory cytokine and chemokines in serum.(4)OCA treatment attenuated LPS-induced pro-inflammatory cytokines and chemokines in broncho alveolar lavage fluidThe expression of pro-inflammatory cytokine TNF-α in BALF was significantlly increased after LPS exposure.Oral OCA supplementation could discard LPS-induced up-regulation of pro-inflammatory cytokine in BALF.Meanwhile,oral OCA supplementation also could block LPS-induced release of chemokine KC in BALF.Simultaneously,the number of cells in BALF was counted.These results showed that OCA could repress LPS-induced increase of inflammatory cells in BALF.The levels of neutrophils and macrophages in BALF were elevated after LPS exposure.LPS-induced increase of inflammatory cells was repressed when0 mice were pretreated with OCA before LPS injection.These results suggest that oral OCA supplementation could reduce LPS-induced release of pro-inflammatory cytokines and chemokines in BALF.(5)OCA pretreatment alleviated LPS-activated pulmonary NF-κB signaling pathway in mice.LPS exposure activated NF-κB signaling pathway in the lungs.The levels of NF-κB p65 and NF-κB p50 nuclear translocation were significantlly enhanced in the LPS group.Oral OCA supplementation effectively inhibited LPS-induced nuclear translocation of NF-κB p65 and NF-κB p50 in the lungs.The number of NF-κB p65 and NF-κB p50 positive nucleus were analyzed by immunohistochemistry.OCA reduced LPS-induced up-regulation of the number of NF-κB p65 and p50 positive nucleus in the lungs.These results indicated that supplementation with OCA relieved LPS-activated pulmonary NF-κB signaling pathway in mice.(6)OCA pretreatment attenuated LPS-activated pulmonary MAPKs signaling pathway in mice.LPS exposure could activate pulmonary MAPKs signaling pathway in mice.The phosphorylation levels of ERK1/2,JNK and p38 were significantlly increased after LPS exposure.Oral OCA supplementation significantlly attenuated the phosphorylation levels of ERK1/2,JNK and p38.These results revealed OCA pretreatment obviously attenuated LPS-activated pulmonary MAPKs signaling pathway in mice.(7)OCA treatment inhibited LPS-activated pulmonary PI3K/Akt signaling pathway in miceLPS exposure could activate pulmonary PI3K/Akt signaling pathway in mice.The level of pulmonary p-Akt in mice was significantlly upregulated after LPS exposure,and OCA pretreatment evidently alleviated LPS-induced pulmonary Akt phosphorylation in mice.These results demonstrated that supplementation with OCA inhibited LPS-activated pulmonary PI3K/Akt signaling pathway in mice.(8)OCA treatment activated pulmonary FXR signaling in miceOCA had no effect on pulmonary Fxr mRNA in mice.FXR positive nucleus was then analyzed.Oral OCA supplementation promoted pulmonary FXR nuclear translocation,LPS exposure inhibited OCA-induced pulmonary FXR nuclear translocation in mice.These results suggested that OCA pretreatment activated pulmonary FXR signaling in mice.ConclusionThe previous research analyzed the antagonistic effect of the activation of pulmonary FXR on LPS-induced acute lung injury in mice.The following conclusions were drawn based on the above results:(1)Activation of pulmonary FXR obviously alleviated LPS-induced acute lung injury in mice.(2)Activation of pulmonary FXR significantlly reduced LPS-induced the release of pro-inflammatory cytokines and chemokines in mice.(3)Activation of pulmonary FXR clearly attenuated LPS-activated pulmonary NF-κB,MAPKs and PI3K/Akt signaling pathways in mice.These conclusions suggest that FXR agonists can be used as potential therapeutic drugs for acute lung injury caused by inflammation in clinical practice,which provides certain theoretical basis for the application of drugs in the treatment and prevention of acute lung injury.