Regulating Mechanism Of Endoplasmic Reticulum Stress On Lung Mesenchymal Stem Cell Differentiation And Epithelium-mesenchyme Crosstalk In Pulmonary Fibrosis

Background and objectivesPulmonary fibrosis is a group of fibroproliferative diseases within the lung mesenchyme caused by multiple etiologies,but the pathogenesis is currently poorly understood.Despite the intricacy of the mechanisms involved,the current agreement regards the development of pulmonary fibrosis as a process of excessive healing in response to lung epithelial damage and regeneration failure.This excessive healing involves two processes.Firstly,myofibroblasts responsible for collagen synthesis accumulates in the mesenchyme,but the origin of myofibroblasts in pulmonary fibrosis is still controversial.Recent evidence has shown that lung resident mesenchymal stem cells(LR-MSC)can transform into myofibroblasts;Secondly,multiple pro-fibrotic factors secreted from alveolar epithelial cells after injury disrupt the epithelium-mesenchyme crosstalk,leading to activation and proliferation of fibroblasts.The SHH/Hedgehog signaling pathway plays a critical role in maintaining epithelium-mesenchyme homeostasis.In addition,studies have confirmed that endoplasmic reticulum(ER)stress is significant in pulmonary fibrosis and can promote the development of pulmonary fibrosis.In this study,we will investigate the role of ER stress in these two processes to discover new mechanisms of ER stress involvement in pulmonary fibrosis and identify potential therapeutic targets to intervene in the development of pulmonary fibrosis.MethodsPart 1:The levels of ER stress and myofibroblast differentiation in LR-MSC of patients with idiopathic pulmonary fibrosis(IPF)were measured separately using immunofluorescence.Bleomycin-induced mouse model of pulmonary fibrosis was established,to analyze the level of ER stress and differentiation in LR-MSC during pulmonary fibrosis.Isolated primary mouse LR-MSC was cultured,then stimulated with tunicamycin to evaluate the impacts of ER stress on epithelium-reparative function;LRMSC was induced to myofibroblast differentiation in vitro using TGFβ1,and in this setting,investigating the impacts of the intervention of the ER stress-downstream transcript factor C/EBP homologous protein(CHOP)on the behavior or fate of LR-MSC;In vivo,intratracheally adoptive transplantation of LR-MSC was conducted to BLM mice and observe the behavioral changes of LR-MSC during pulmonary fibrosis and alteration of severity of pulmonary fibrosis,as well as the effect of CHOP on these processes.Part Ⅱ:Levels of ER stress and SHH expression in AEC2 of IPF patients were analyzed.Tunicamycin induces ER stress of human type 2 alveolar epithelial cells(AEC2),then observes the SHH secretion of AEC2 and its effect on fibroblast;ER stress-strengthened mouse pulmonary fibrosis model induced by tunicamycin combined with bleomycin was established,then investigates the effect of the intervention on the level of CHOP on the severity of pulmonary fibrosis in vivo.ResultsPart Ⅰ:1.LR-MSC from IPF patients undergo endoplasmic reticulum stress and differentiate into myofibroblasts;2.The sorting strategy of LR-MSC has been optimized,and Scal+CD45-CD31-EpCamcan be used to sort LR-MSCs more accurately;3.LR-MSC undergo ER stress and differentiate into myofibroblasts during pulmonary fibrosis;4.The epithelial reparative function of LR-MSC can be destroyed by tunicamycininduced ER stress;5.Overexpression of CHOP,a downstream factor of ER stress,promotes TGFβ1-induced myofibroblast differentiation via TGFβ/SMAD signaling pathway;6.Down-regulation of CHOP of LR-MSC inhibits myofibroblast differentiation during pulmonary fibrosis and alleviates pulmonary fibrosis.Part Ⅱ:1.AEC2 in IPF patients undergoes endoplasmic reticulum stress with disrupted epithelial-mesenchymal dialogue caused by activation of the Hh signaling pathway;2.Tunicamycin induces SHH secretion from AEC2,which is regulated by CHOP;3.The conditional medium of AEC2 stimulated with tunicamycin can activate the Hh signaling pathway of fibroblasts and promote the proliferation and collagen synthesis of fibroblasts;4.Knockdown of CHOP can alleviate the pulmonary fibrosis induced by tunicamycin combined with bleomycin.Conclusion1.LR-MSCs undergo ER stress during pulmonary fibrosis.In this process,ER stress induces the overexpression of CHOP,which promotes its transfer to myofibroblasts by sensitizing the TGFβ/SMAD signaling pathway involved in the progression of pulmonary fibrosis;2.AEC2 undergoes ER stress during pulmonary fibrosis and induces SHH secretion.The secreted SHH activates the Hh signaling pathway of interstitial fibroblasts,leading to the activation of fibroblasts,which are involved in pulmonary fibrosis.