Effect Of HDAC Inhibitor LBH589 On Lung Injury And Pulmonary Fibrosis

Background: Lung,as the main gas exchange organ of mammals,are frequently to be infected by multiple pathogens and allergens,vulnerable to injury.The chronic and progressive injury of unknown cause on lung epithelium,followed by the aberrant reparative response leads to the occurrence of idiopathic pulmonary fibrosis(IPF).IPF is a chronic interstitial lung disease characterized by excessive extracellular matrix deposition,inflammatory damage and irreversible remodeling of lung tissue structure.Its pathogenesis involves multifaceted interactions of a variety of cell types,such as abnormally repaired alveolar epithelial cells,activated mesenchymal cells,senescent cells,and activation of macrophage subsets etc.Previous studies have shown that the incidence and prevalence of IPF increases with age(the average age at diagnosis is approximately 66 years),and the median survival after diagnosis is 3-4 years.Although pirfenidone and nintedanib in medical therapy can improve the decline of lung function in most patients,improve the quality of life of patients and delay the progression of the disease,they have many side effects and cannot improve the survival rate of patients.Therefore,the key points in IPF research are in-depth investigation of the pathological mechanism underlying IPF and hunt for novel treatment targets.In recent years,histone deacetylase(HDAC)has been found to be abnormally upregulated in the lung fibroblast lesions of IPF patients.HDAC may affect the biological function of fibroblasts and participate in the pathogenesis of fibrotic diseases by altering the degree of affinity between histones and DNA to change the expression level of downstream genes.According to certain reports,using HDAC inhibitors can reduce the histopathologic changes of pulmonary fibrosis.LBH589,a newly marketed HDAC inhibitor approved by the FDA in 2015 for the treatment of multiple myeloma,exhibits more potent broad-spectrum HDAC inhibitory activity compared to other HDAC inhibitors.LBH589 has been shown to downregulate the expression of antiapoptotic genes and antifibrotic factors in lung fibroblasts.However,these results are based on experiments with fibroblasts only.The regulatory effects of LBH589 on other cells in the pathogenesis of IPF are unclear,and LBH589 has not been shown to alleviate lung injury and fibrosis in vivo.These unanswered scientific questions prompted us to conduct additional in-depth studies.Objective: To investigate the effects of the HDAC inhibitor LBH589 on lung injury and fibrosis at the molecular,cellular and animal level in vivo and in vitro.Methods: In the first part of the in vitro study: At the molecular level,the inhibitory effect of HDAC inhibitor LBH589 on HDAC enzyme activity was detected by fluorescence assay.At the cellular level,the regulatory effects on the following three types of key cells were mainly investigated.(1)Lung fibroblasts:The potential of LBH589 to inhibit fibroblast type cell activation and extracellular matrix accumulation was investigated at the cellular level in lung fibroblast cell line MRC-5 and primary lung fibroblasts by Real-time Quantitative PCR(RT-q PCR),Western Blot and Enzyme linked immunosorbent assay(ELISA),etc.(2)Alveolar epithelial cells: Bleomycin(BLM)was used to induce A549 cell senescence model.The effects of LBH589 on cell senescence phenotype were detected by β-galactosidase staining and RTq PCR.At the same time,alveolar epithelial cells were stimulated with transforming growth factor(TGF-β)to establish an epithelial-mesenchymal transition model,and the effect of LBH589 on the expression of mesenchymal cell markers was detected at the gene transcription level.(3)Macrophage: Lipopolysaccharide(LPS)was used to induce RAW264.7 cells to establish an in vitro inflammatory model,a and the anti-inflammatory efficacy of LBH589 was evaluated at the m RNA level.In the second part of the in vivo study: The absorption,distribution and metabolism of the HDAC inhibitor LBH589 in mice were investigated by pharmacokinetic and tissue distribution experiments.Based on the pharmacokinetic and tissue distribution characteristics of LBH589,the pharmacodynamic evaluation was further performed in vivo.First,the effect of LBH589 on early lung inflammation and injury in IPF was investigated by establishing an acute lung injury model with LPS to simulate the early pathogenesis of IPF.Then,we also used a traditional BLM-induced pulmonary fibrosis mice model to better elucidate the amelioration of lung injury and fibrosis in order to more accurately depict the therapeutic effect of LBH589 in IPF.Results: 1.Molecular data showed that LBH589 exhibited a stronger broad-spectrum HDAC enzyme inhibition activity than the positive control group.2.At the cellular level,LBH589 significantly inhibited the activation of lung fibroblasts,a key effector cell,as well as reduced the accumulation of extracellular matrix.At the same time,LBH589 also acts on alveolar epithelial cells,which are the primary sensors of lung injury.LBH589 regulates the driver of pulmonary fibroblast activation by inhibiting BLM-induced alveolar epithelial cell senescence and TGF-β-stimulated epithelial-mesenchymal transition.In addition,LBH589 exhibited anti-inflammatory activity by downregulating the expression levels of inflammatory factors in immune-related macrophages.3.According to in vivo pharmacokinetic and tissue distribution studies,LBH589 demonstrated high bioavailability and good lung exposure following intraperitoneal injection,4.In the mouse model of LPS-induced acute lung injury,LBH589 significantly and dose-dependently ameliorated LPS-induced alveolar wall thickening,alveolar space enlargement,and severe structural disorder.Meanwhile,it reduced macrophage recruitment to alleviate early lung inflammation and injury in mice.In the classic IPF model induced by BLM,LBH589 treatment also significantly reduced inflammatory cell infiltration and ameliorated lung injury.In addition,it may also prevent the activation of fibroblast and reduce collagen deposition,thereby ameliorating pulmonary fibrosis.Conclusions: Taken together,LBH589 exhibited anti-inflammatory and anti-fibrotic activities in multiple dimensions by targeting fibroblasts,alveolar epithelial cells and macrophages,respectively.Meanwhile,LBH589 was shown to be effective in ameliorating lung injury and fibrosis in IPF in vivo.