Costunolide Ameliorates Lipoteichoic Acid (LTA)-induced Acute Lung Injury And Molecular Mechanisms

Acute lung injury(ALI)severely threatens human health and leads patients to death.It is common that Gram positive bacterial infection induces acute lung injury.The compounds from pathogen not only directly induce cell damage,but also induce severe inflammation response that would lead to multiple organ damage and finally trigger multiple organ failure.Therefore,inhibition of these pathogen's compounds is alternative approach to reducing pulmonary inflammation and alleviating acute lung injury.Lipoteichoic acid(LTA)is a pathogenic composition and belongs to Gram positive bacteria.Treatment with LTA can induce mouse acute lung injury which including induction of cytokine storm,lung injury,lung edema and pulmonary neutrophil infiltration.Tissue resident macrophage is one of innate immune cells,which is first line immune cell to defense against bacterial invasion.Upon stimulation with LTA,pulmonary macrophages produce amount of cytokines and chemokines that directly induce epithelial cell and endothelial cell damage as well as drive neutrophils intro lung tissue.Pulmonary macrophages also generate iNOS and produce NO,which increases pulmonary permeability and induces lung injury.Toll-like receptor 2(TLR2)is expressed on macrophages and belongs to the family of pathogen recognition receptors(PRRs).LTA is TLR2 ligand and binding between LTA and TLR2 can trigger activation of myeloid differentiation primary-response protein 88(MyD88)and in turn recruit tumor necrosis factor receptor associated factor 6(TRAF6)and protein kinase transforming growth factor beta-activated kinase 1(TAK1).Phosphorylation of TAK1 will activate MAPK signaling including p38 MAPK,ERK and JNK as well as NF-k B signaling,which finally actives transcriptional factors cAMP response element binding protein(CREB)and activator protein 1(AP1)that initiating the expression of cytokines and chemokines.Challenge with LTA induces cytokine storm and enhances pro-inflammatory cytokine expression including tumor necrosis factor-alpha(TNF-?),interleukin-6(IL-6)and keratinocyte chemoattractant(KC),which leads to sepsis-induced acute lung injury.Therefore,blockage of these signaling molecules can specifically inhibit inflammation and treat acute lung injury.This study focuses on the protection role of costunolide in protection of LTA-induced acute lung injury by determining animal model,macrophage function and molecule mechanisms.Costunolide is a sesquiterpene lactone and extracted from the Compositae and the Magnoliaceae,which has multiple pharmacological activities,including inhibition of tumor cell proliferation,protection from viral and fungal infections,as well as reduction of inflammatory response.However,it is still unknown whether costunolide has effect on protecting against gram positive bacteria-induced inflammatory response and whether costunolide inhibits LTA-TLR2-mediated cell signaling.Our research was carried out in following three steps.Part ?: Costunolide protects against LTA-induced acute lung injury.The first part aims to determine whether costunolide protects against LTA-induced acute lung injury.By using LTA-induced mouse acute lung injury model,histology technologies,flow cytometry assay and biochemistry measurement,wefound that costunolide attenuated LTA-induced pulmonary pathologic damage,reduced lung edema and pulmonary neutrophil infiltration.The LTA-induced expression of TNF-?,IL-6 and KC were dramatically decreased.The in vivo data indicated that costunolide alleviates LTA-induced acute lung injury.Part ?: Costunolide inhibits LTA-induced macrophage activation.The second part aims to determine whether costunolide decreases LTA-induced classic macrophage activation.By using ELISA and immunoblot approaches,in response to LTA stimulation,we found that costunolide reduced LTA-induced production of TNF-?,IL-6 and KC in bone marrow derived macrophages(BMDMs).LTA-induced expression of iNOS was also attenuated upon treatment with costunolide.These results suggested that costunolide can target macrophage and inhibit macrophage activation.Part ?: Costunolide attenuates LTA-induced MAPK signal pathway.The third part aims to determine whether costunolide has effect on MAPK and NF-k B signaling pathways.By using immunoblot and immunoprecipitation technologies,we found that costunolide inhibited LTA-induced p38 MAPK and ERK phosphorylation but didn't affect JNK phosphorylation and NF-kB signaling pathway.In addition,in response to LTA treatment,costunolide attenuated TAK1 phosphorylation and blocked the interaction between TAK1 and Tab1 which was required for MAPK activation,cytokine production and macrophage activation.Therefore,our data suggested that costunolide is a potential agent for treating acute lung injury.