Exploring The Pathological Mechanisms And Potential Therapeutic Targets Of Silicosis Based On Multiple Omics

Objective:Silicosis is related to long-term inhalation of silica particles in occupational activities.The particles accumulate in the lungs,causing alveolar epithelial cell injury,inflammatory cell infiltration and inflammatory factor secretion,and then stimulating fibroblast proliferation and collagen secretion,leading to chronic lung inflammation,diffuse pulmonary fibrosis and lung insufficiency.Silicosis currently remains a serious public health problem worldwide.Previous studies showed that blocking the development of chronic inflammation to pulmonary fibrosis is crucial to retard the progress of silicosis,but there are limited drugs to attenuate this process.Therefore,further study of the important pathways and gene targets affecting progression of the disease is of profound significance for the treatment of silicosis.Materials and Methods:Lung tissue samples from silicosis patients and healthy donors were collected for transcriptomics analysis.Mouse models of silicosis were prepared by a single intratracheal instillation of silica suspension using C57BL/6J mice,and control mice were instillation with equal amount of phosphate-buffered saline(PBS).After 3,6 and 9 weeks,mouse lung function and right heart function were examined and the inflammation and fibrosis status were assessed.Mouse lung tissues of silicosis at different stages were then taken for transcriptomics analysis,combining with transcriptomics analyses in patients to explore of key targets or pathway in silicosis progression.Untargeted metabolomics was used to define the metabolic pathways that changed in silicosis,and the key metabolites were further quantified using targeted metabolomics method.The omics results were verified by quantitative real-time PCR(qPCR),protein immunoblotting(Western blotting),and immunohistochemistry(IHC).Selecting clinical drugs to key targets,giving therapeutic drug in the mouse model of silicosis after 3 weeks of modeling,after 3 weeks of treatment,mouse lung function and right heart function were detected,and the inflammation and fibrosis status of mice lungs were evaluated.Results:Transcriptomic results showed significant changes in transcript levels in silicosis patients compared with normal healthy donors,with differential genes significantly enriched in "immunity","metabolism","signaling transduction" and"protein digestion and absorption" pathways.Mice exposed to silica showed significant pulmonary dysfunction,pulmonary inflammation and fibrosis as compared to normal mice.The inflammation score of lung tissues increased significantly after 3 weeks of modulation,rising to the highest at 6 weeks and remaining abnormal to 9 weeks.Lung fibrosis occurred after 3 weeks of exposure,and fibrosis score rising to its peak at 6 weeks and maintained to 9 weeks.Transcriptomics analysis of lung tissues from mice at different time points divided differential genes into five different expression patterns based on their trends at different time points,among which genes that continuously changed throughout the disease were mainly enriched in metabolism-related pathways.Untargeted metabolomics analysis of normal and silicosis mice suggested significant activation of the arachidonic acid(AA)metabolic pathway in silicosis mice.Quantification of metabolites by targeted metabolomics showed that AA pathway metabolites,prostanoid D2(PGD2)and Thromboxane A2(TXA2)were significantly upregulated in silicosis mouse lung tissues.The experimental validation results confirmed that the mRNA and protein levels of PGD2 and TXA2 synthetase were significantly up-regulated in the lung tissues of silicosis patients and mice.Further therapeutic experiments were performed in a mouse model of silicosis by using Ramatroban,which is a clinical antagonist of PGD2 and TXA2 receptors.The results showed that Ramatroban significantly reduced silica-induced cardiopulmonary dysfunction,pulmonary inflammation and fibrosis compared with the control group.Ramatroban significantly inhibited silica-induced activation of NOD-like receptor protein 3(NLRP3)inflammasomes in silicosis mice.Conclusions:Our results reveal the importance of the metabolic reprogramming,especially PGD2 and TXA2 in silicosis progression.By blocking the receptors of both prostaglandins,Ramatroban could be a novel potential therapeutic agent to inhibit silicosis progression.