Exosomes From Bone Marrow Mesenchymal Stem Cells Alleviate Pulmonary Fibrosis Caused By Senescence Of Alveolar Epithelial Cells Through Endoplasmic Reticulum Stres

Background and objective:Idiopathic pulmonary fibrosis(IPF)is a lethal and fibrotic interstitial lung disease.So far,the pathogenesis of IPF has not been fully elucidated and it lacks effective clinical drugs.Recent studies have demonstrated that alveolar epithelial cell senescence occupies a key position in the progression of pulmonary fibrosis.Endoplasmic reticulum(ER)stress has been shown to accelerate cell senescence in various cell types.However,it is unclear whether ER stress affects alveolar epithelial cell senescence and subsequent pulmonary fibrosis.An in-depth analysis of the pathogenesis of pulmonary fibrosis could help identify new targets for treatment.In recent years,the potential effect of mesenchymal stem cells(MSCs)on treating pulmonary fibrosis has attracted much attention.Exosomes are the main mediators of the therapeutic effects of MSCs.Recent studies have reported that bone marrow mesenchymal stem cells(BMSCs)can significantly relieve pulmonary fibrosis through exosome secretion,but the exact mechanism needs to be further clarified to provide a new theoretical basis for their clinical translation.This study firstly investigated the regulation role of ER stress in alveolar epithelial cell senescence and pulmonary fibrosis to elucidate new pathogenesis mechanisms in pulmonary fibrosis;then further investigated the effects of BMSC-derived exosomes(BMSC-Exos)on ER stress and alveolar epithelial cell senescence to explore their treatment effect and relevant mechanism for pulmonary fibrosis.Method:Part Ⅰ:We firstly validated type 2 alveolar epithelial cell(AEC2)senescence in lung tissue samples from IPF patients and detected the expression levels and distribution sites of senescence-related proteins and ER stress pathway proteins in IPF lung tissues to assess the relationship between AEC2 senescence and ER stress.The ER stress cell model was then constructed using extracted primary mouse AEC2 to detect the expression levels of cell senescence-related proteins and changes of senescence-associated β-galactosidase staining to explore the effects of ER stress on AEC2 senescence.Then,the expression of cellular senescence markers,changes of senescence-associated secretory phenotype(SASP),and its mediated fibroblast activation were examined after pretreatment with 4PBA and down-regulation of C/EBP homologous protein(CHOP)during ER stress,by siRNA transfection of AEC2.Finally,we applied shRNA to knockdown CHOP and investigated the role of ER stress on pulmonary fibrosis and its relationship with AEC2 senescence in the mice models.Part Ⅱ:We firstly cultured and validated human BMSCs,and extracted BMSC-Exos followed by identification.Then,we constructed an ER stress model with successfully validated human AEC2 and examined the expression of ER stress and cell senescencerelated markers to assess the effects of the intervention with BMSCs and BMSC-Exos on ER stress and its resulting AEC2 senescence.We further detected SASP changes and their mediated fibroblast activation.Finally,we investigated the therapeutic effects of BMSCExos on pulmonary fibrosis and AEC2 senescence using the mouse lung fibrosis model.Result:Part Ⅰ:1.Marked AEC2 senescence existed in the lung tissue of IPF patients;2.AEC2 senescence is accompanied by excessive activation of ER stress in lung tissues of IPF patients;3.Both bleomycin and tunicamycin induced mouse AEC2 senescence through activation of ER stress,especially via the combined treatment;4.4-PBA pretreatment or down-regulation of CHOP attenuated mouse AEC2 senescence and SASP secretion after inhibiting ER stress;5.Knockdown of CHOP inhibited ER stress,reduced AEC2 senescence in mice,and ultimately relieved pulmonary fibrosis.Part Ⅱ:1.We successfully isolated,extracted,and identified BMSC-Exos;2.BMSCs attenuated ER stress in human AEC2,dependent on secreting exosomes;3.BMSC-Exos also attenuated human AEC2 senescence after inhibiting ER stress;4.BMSC-Exos attenuated SASP secretion after human AEC2 senescence;5.BMSCs inhibited AEC2 senescence via exosome secretion in mice and ultimately reduced pulmonary fibrosis.Conclusion:1.ER stress accelerates AECs senescence in pulmonary fibrosis,and inhibiting ER stress reduces AECs senescence,thus relieving pulmonary fibrosis.2.BMSC-Exos can exert the therapeutic effect on pulmonary fibrosis by alleviating AECs senescence following inhibiting ER stress.