| Pulmonary fibrosis(PF)is a kind of severe chronic respiratory disease caused by repeated alveolar and parenchymal lung damage caused by multiple reasons.Persistent inflammation leads to repeated injury-repair processes in lung tissue,eventually replacing normal lung tissue with scarring formed by abnormal repair,resulting in the progressive decline of lung function,and even respiratory failure.Among them,idiopathic pulmonary fibrosis(IPF)is the most representative type of fibrosis.Due to the lack of effective therapeutic targets,the two existing marketed drugs,pirfenidone and nintedanib,could only partially alleviate the symptoms of mild and moderate patients.Therefore,it is of great significance for the prevention and treatment of pulmonary fibrosis to search for new drug targets and new drugs targeting the pathogenesis of pulmonary fibrosis.Sphingosine-1-phospho-1 receptor(S1PR1),a G-protein-coupled receptor encoded by endothelial differentiation gene 1(EDG1),plays an important role in regulating lymphocyte transport,regulating cytoskeletal rearrangement,and improving vascular endothelial cell barrier function.Compared with other cell types,S1PR1 was highly expressed in immune cells and vascular endothelial cells.Studies have shown that the integrity and function of the alveolar barrier play a role in the occurrence and development of fibrosis.Vascular endothelial cells and alveolar epithelial cells are important components of the alveolar barrier.Based on this,we conducted relevant research on the regulation mechanism of S1PR1 on alveolar barrier and the influence of fibrosis.IMMH002,also known as Ethoximod,is a highly selective S1PR1 agonist independently developed by the Institute of Materia Medica,Chinese Academy of Medical Sciences.IMMH002 can treat a variety of autoimmune diseases by inducing lymphocyte homing,and it was approved by the CFDA for the treatment of psoriasis in 2016.Previous studies have shown that IMMH002 had a good therapeutic effect on pulmonary fibrosis in mice.Based on this,we conducted a study on the mechanism of S1PR1 regulating alveolar barrier homeostasis to improve pulmonary fibrosis.This research is mainly divided into the following three parts:(1)Regulation mechanism of vascular endothelial S1PR1 on pulmonary fibrosis.The single cell sequencing and transcriptome sequencing databases reported in the literature were analyzed.Our study found that in single-cell sequencing and transcriptome sequencing databases,the total lung and alveolar vascular endothelial S1pr1 in IPF patients were significantly reduced compared to healthy people.We also built the vascular endothelium-specific S1pr1 knockout transgenic animals {S1pr1+/-Tek-CreERT2,S1pr1+/-),these animals presented obvious endothelial barrier damage,a large number of inflammatory cells in filtration and obvious collagen deposition and bleomycin stimulation could exacerbate this phenomenon.Transcriptome analysis has also demonstrated that the genes related to collagen deposition,such as Col1a1,Lum,and Has were significantly upregulated.It suggested that vascular endothelial barrier could damage promote fibrosis.In addition,specific knockout of S1pr1 could reduce the expression of lung tight junction Tjp1、Cldn3、Cldn18、Ocln and enhance fibrosis-related protein Fibronectin.In vitro studies have demonstrated that the active form of IMMH002-P,a selective agonist of S1PR1,can significantly increase the transendothelial resistance of EOMA cells and reduce thrombine-induced FITC-BSA barrier leakage.Transcriptome studies of vascular endothelial cells(EA.hy 926 cells)have also shown that IMMH002-P can significantly reduce inflammatory response and enhance cell connection-related pathways.(2)Mechanism of S1PR1 on alveolar epithelial mesenchymal transformation.We demonstrated that the highly selective S1PR1 agonist IMMH002-P inhibited TGF-βinduced epithelial mesenchymal transition(EMT)by morphological observation,assessment of migratory capacity by scratch assay,and determination of mesenchymalassociated marker-changes at mRNA and protein levels.High concentration of IMMH002P(1 nM)inhibited the fibroblastic transformation of epithelial cells and also showed great inhibition of TGF-β-induced cell migration.Immunoblotting experiments also demonstrated that IMMH002-P had a strong inhibitory effect on mesenchymal markers like N-Cadherin,α-SMA,and Fibronectin,providing an experimental basis for further research on the mechanism of treating lung fibrosis by improving the EMT process in IMMH002.(3)In vivo pharmacodynamic study of IMMH002 on animal model of pulmonary fibrosis.The therapeutic effect of S1PR1 agonist IMMH002 on pulmonary fibrosis was evaluated by mouse bleomycin pulmonary fibrosis model and silica induced pulmonary fibrosis model.In the study of silica-induced fibrosis model,the results showed that:1)IMMH002 can improve the lung function of mice with fibrosis in a dose-dependent manner;2)IMMH002 could significantly reduce peripheral blood inflammatory cells in model mice;3)IMMH002 could significantly improve lung collagen deposition and lung tissue structure of model mice.In the study of bleomycin-challenged lung fibrosis model,the results showed that:1)IMMH002 could improve the survival rate and lung function of mice with fibrosis;2)IMMH002 reverses the bleomycin-induced increase in lung index and reduces the number of inflammatory cells;3)IMMH002 had an effect on the improvement of lung tissue structure,inflammatory infiltration and fiber deposition.In conclusion,bioinformatics,transcriptomics,and molecular pharmacology were used in this study to demonstrate that activation of S1PR1 could effectively protect the integrity of the alveolar barrier and improve pulmonary fibrosis by increasing endothelial cell tight junctions and reversing EMT.S1PR1 is expected to be a potential drug target for PF and IMMH002 is expected to be a potential new drug for PF. |
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