Protective Effects And Mechanisms Of Bufexamac On Lipopolysaccharide-induced Acute Lung Injury In Mice

Acute lung injury(ALI)is caused by all kinds of non-cardiac severe internal and external pulmonary factors leading to pulmonary capillary diffuse injury,increased permeability to pulmonary edema,transparent membrane formation and atelectasis as the main pathological characters.The main clinical symptoms of ALI are progressive dyspnea and refractory hypoxemia,and further development can evolve into acute respiratory distress syndrome(ARDS).The essence of the ALI pathogenesis is the failure of the inflammatory response,leading to the diffused lung injury and respiratory dysfunction.At present,the clinical treatment of ALI/ARDS is through protective mechanical ventilation,and no drug approved by FDA to treat ALI/ARDS is available.Therefore,the development of new drugs or new targets has becoming a very important issue.Bufexamac is a non-steroidal anti-inflammatory drug that has been in market for many years.It is used to treat rheumatoid arthritis,eczema and other inflammation dermatoses,but its anti-inflammatory mechanism is still unclear.Neutrophil chemotaxis is the main cause of inflammation out of control in ALI.Production of leukotriene B4(LTB4)from leukotriene A4(LTA4)catalyzed by leukotriene A4 hydrolase(LTA4H)is one of the important chemokines that mediates neutrophil chemotaxis,therefore,LTA4H inhibitors can be used as a candidate drug to cure ALI.In the present study,the listed market drug library was screened through leukotriene A4 hydrolase(LTA4H)inhibitor high throughput screening platform.It was found that the Bufexamac functioned as a LTA4H inhibitor.Bufexamac effectively inhibits LTA4 hydrolysis to LTB4,which affects the migration of neutrophils and thus protects against acute lung injury.Using structural biological technique and ALI mice model platform,we studied the inhibition specificity and inhibitory sites of Bufexamac on LTA4H,and its protective effect on LPS-induced acute lung injury in mice.Furthermore,we investigated the correlation between the protective effect of Bufexamac on ALI and the activations of key proteins in nuclear factor κB(NF-κB)and mitogen activated protein kinase(MAPK)signaling pathways.The main contents and research results of this thesis are as follows:(1)We screened the listed market drug library through the LTA4H inhibitor high-throughput screening platform and found that the Bufexamac could act as a LTA4H inhibitor.We used X-ray diffraction method to analyze the crystal structure of the LTA4H-Bufexamac complex.We found that Bufexamac could bind to the L stenotic active pouch of LTA4H.The carbonyl and hydroxy groups of Bufexamac specifically bind with three key amino acid residues(Glu296,Glu318 and Tyr383)in the active sites of LTA4H and Zn2+.This result provided molecular basis for the specific inhibitory effect of Bufexamac on LTA4H.(2)We used ELISA and cell migration assay to investigate the effect of Bufexamac on LTB4 production and neutrophil migration.We found that Bufexamac could effectively decrease LTB4 production by inhibiting LTA4H activity,thereby inhibiting the neutrophil chemotaxis.(3)We established a mice model of acute lung injury induced by LPS to study the protective effect of Bufexamac on ALI mice.We found that Bufexamac could inhibit over-activation of NF-κB in NF-κB signaling pathway and was involved in blocking the three key protein kinases(p38MAPK,ERK,JNK)in MAPK signaling pathway in LPS-induced ALI mice model.(4)We used computer aided drug design(CADD)to optimize the chemical structure of Bufexamac to improve the inhibitory activity to LTA4H,which laid the foundation for further study.In conclusions,we confirmed that the protective effect of Bufexamac on the acute lung injury in mice was proved through inhibiting LTA4H activity.NF-κB and MAPK signaling pathways was involved in the protective mechanism of Bufexamac on acute lung injury in mice.Our results indicated that Bufexamac could be developed as a candidate drug to cure the acute lung injury.