Chemoattractant-receptor-On-TH2-Cells(CRTH2) Antagonist,CT-133,Effectively Alleviates Lipopolysaccharide-and Cigarette Smoke-induced Acute Lung Injury In Mouse

Acute lung injury(ALI)and acute respiratory distress syndrome(ARDS),characterized by overwhelming lung inflammation,are life-threatening conditions,and no single effective pharmacological therapy is currently available to treat ALI/ARDS.Accumulated evidence has revealed that prostaglandin D2(PGD2)exhibits a key role in orchestrating lung inflammation,and high expression of PGD2 and CRTH2 has been observed in ALI.However,the protective effects of CRTH2 antagonists against ALI have not been explored yet.Hence,this study aimed to explore the protective effect of CT-133,a newly discovered selective and potent CRTH2 antagonist,against ALI at both in vivo and in vitro levels.Initially,two types of ALI murine models including lipopolysaccharide(LPS)-induced ALI models and cigarette smoke(CS)-exposed ALI models were prepared.For LPS-induced ALI models preparation,CT-133(10 and 30 mg/kg)and Dex(1 mg/kg)were intragastrically administrated one hr prior to intratracheal LPS challenge and twelve hr after LPS challenge.Similarly,for the preparation of CS-induced ALI models,CT-133(10 and 30 mg/kg)and Dex(1 mg/kg)were intragastrically administrated one hr prior to whole-body CS-exposure for seven consecutive days.Twenty-four hr after LPS challenge and last CS-exposure,P02 of all mice was measured using the moor VMS-OXYTM monitor and then mice were killed to collect the broncho-alveolar lavage fluid(BALF).Collected BALF was used to measure the inflammatory cells,neutrophils and macrophages count via Wright-Giemsa staining,albumin contents using albumin determination kit,and cytokines(TNF-?,IL-1?,IL-6,KC,and IL-10)level through respective enzyme-linked immunosorbent assay(ELISA)kits.Isolated lungs were harvested for the determination of lung weight coefficient myeloperoxidase(MPO)activity via MPO kit,and histological examination by hematoxylin-eosin(H&E)staining.Pulmonary vascular permeability was assessed by measuring the extravasations of Evans blue dye(EBD)into the lungs.Next,peritoneal neutrophils were isolated from euthanized mice four hr after intraperitoneal injection of 1.5%glycogen and the effect of CT-133 on PGD2-induced neutrophils migration was assessed by using the Boyden chamber assay kit.Meanwhile,thioglycollate(4%)was intraperitoneally injected to mice for three consecutive days;peritoneal macrophages were isolated forty-eight hr after the last injection and then treated with different concentration of LPS and cigarette smoke extract(CSE)to measure the PGD2 secretion via PGD2 ELISA kit.Afterward,cytotoxic effect of CT-133 alone and in a combination of LPS,CSE,and PGD2 on RAW 264.7 macrophages and isolated peritoneal macrophages were observed using MTT assay.Further,ELISA and RT-PCR were performed to investigate whether CT-133 has impact on the secretion of TNF-?,IL-1?,IL-6,KC and IL-10 from LPS-,CSE-and PGD2-stimulated RAW 264.7 macrophages and isolated peritoneal macrophages.Finally,WB was performed to investigate the possible underlying protective mechanism of CT-133 at both in vivo and in vitro levels.Here,we found that LPS-challenge and CS-exposure strikingly provoked the key characteristics of ALI;such as decreased PO2 and increased inflammatory cells,macrophages and neutrophils count,lung weight coefficient,BALF albumin contents and proinflammatory cytokines level,lung MPO activity,EBD exudation into lungs,and lung histopathological changes.Moreover,protein and mRNA levels of TNF-?,IL-1?,IL-6,and KC were remarkably increased while IL-10 was strikingly decreased from LPS-,CSE-and PGD2-stimulated RAW 264.7 macrophage and isolated peritoneal macrophages.Importantly,PGD2 expression was significantly increased from LPS-and CSE-stimulated isolated peritoneal macrophages;supporting the participation of PGD2 in ALI.Meanwhile,CRTH2 antagonism with CT-133 significantly attenuated LPS-and CS-induced ALI through suppressing infiltration of inflammatory cells,particularly macrophages and neutrophils,in the BALF,reducing pulmonary vascular permeability and edema,attenuating TNF-?,IL-1?,IL-6,and KC and augmenting IL-10 production in BALF,ameliorating lung MPO activity and improving PO2 and lung histopathological changes.Additionally,CT-133 pretreatment not only reversed the uncontrolled secretion of TNF-?,IL-1?,IL-6 and KC from LPS-,CSE-and PGD2-stimulated macrophages but also augmented IL-10 production.Furthermore,CT-133 alleviated in vitro neutrophil migration chemoattracted by PGD2.Moreover,CT-133 treatment significantly blocked LPS-and CS-induced nuclear factor-kappa B(NF-?B p65)activation at both in vivo and in vitro levels.In conclusion,we reported for the first time that protective effect of CT-133 is most probably related to the reduction of alveolar macrophages and neutrophils infiltration,pulmonary vascular permeability,pro-inflammatory cytokines production and augmentation of IL-10 secretion through inhibition of NF-?B p65 activation,and CRTH2 could be a new potential therapeutic option to treat ALI.