Effect Of Excessive Endoplasmic Reticulum Stress In Alveolar Epithelial Cells And Release Of Exosomal Tenascin-C On Acute Lung Injury Induced By Sepsis

[Background]:Sepsis is a systemic inflammatory response syndrome caused by the invasion of pathogenic microorganisms,which can lead to septic shock and multiple organ dysfunction syndrome in severe cases.The lung is the most vulnerable target organ to excessive inflammatory response in sepsis.About 25%-50%of patients with sepsis will develop acute lung injury(ALI)or even Acute Respiratory Distress Syndrome(ARDS).However,the pathogenesis of acute lung injury in sepsis remains unclear.The transmission of inflammatory information between lung structural cells and immune cells is an important link in the process of acute lung injury in sepsis.Exosomes play an important role in inflammatory injury as a medium of intercellular information transmission.The donor cells load certain active substances,such as proteins,into exosomes,which then transport these active substances to the recipient cells,thereby changing the phenotype of the recipient cells.However,previous studies have not fully elucidated the specific molecular mechanism of exosome signal transduction in acute lung injury caused by sepsis and its potential clinical application value.TNC(Tenascin-C)is an extracellular matrix protein,which is a glycoprotein encoded by the human TNC gene.Studies have shown that TNC gene mediates a variety of immune inflammation involved in tissue damage,repair and cancer.In acute lung injury models,TNC is significantly overexpressed at the early stage of inflammation,mainly in the alveolar interwall of the damaged tissue,and TNC is essential for the chemotaxis of immune cells.However,the role of TNC in the pathophysiology of acute lung injury in sepsis remains unclear.Endoplasmic reticulum stress(ER stress)is a self-protection mechanism of cells.Moderate ER stress can restore endoplasmic reticulum homeostasis and maintain cell survival.Excessive and persistent ER stress remove damaged cells by activating the endoplasmic reticulum apoptotic pathway.Endoplasmic reticulum stress mediated apoptosis is an important pathogenesis of acute lung injury in sepsis.[Objective]:This project is an in-depth study of the pathogenesis and development of acute lung injury in sepsis.In this study,the proteomic method was used to screen and discover the significantly different protein TNC expressed in plasma exosomes of sepsis patients,and to further explore the molecular mechanism of how endoplasmic reticulum stress upregulates TNC expression,which is helpful to clarify the role and mechanism of endoplasmic reticulum stress,TNC and inflammatory reaction in the occurrence and development of sepsis ALI,explore its feasibility as a therapeutic target of sepsis acute lung injury,and explore its potential clinical application value.[Method]:1.The High throughput proteomic method was used to screen out the significantly differentially expressed protein TNC in plasma exosomes of patients with sepsis,and bioinformatics analysis such as GO function and KEGG signaling pathway was performed to clarify the biological functions of differentially expressed proteins.2.Mass spectrometry results were tested by targeted proteomics(PRM)was used to confirm the differential protein TNC which was significantly highly expressed in plasma exosomes of patients with acute lung injury caused by sepsis.The ROC curve showed a clear correlation between TNC and sepsis ALI.3.LPS stimulates alveolar epithelial cells to establish sepsis acute lung injury model and verify that activation of endoplasmic reticulum stress signaling pathway PERK-eIF2α-CHOP could up-regulate TNC expression.4.siRNA technique was used to reveal the transcriptional regulation relationship between CHOP and TNC in sepsis ALI.(Result]:1.Screening of exosomal TNC significantly associated with sepsis induced ALI.2.The expression level of TNC was significantly increased in plasma exosomes of patients with acute lung injury caused by sepsis.3.LPS induced the expression of TNC in alveolar epithelial cells and inhibited cell proliferation and induced apoptosis in a time and dose-dependent manner,in vitro.4.The expression of TNC in exosomes was high in the supernatant of alveolar epithelial cells after LPS stimulation.5.LPS induces ER stress in vitro.Inhibition of ER stress protects the viability and apoptosis of LPS-induced alveolar epithelial cells,alleviates oxidative stress,reduces the production of inflammatory cytokines and nuclear translocation of NF-κB p65.6.Endoplasmic reticulum overstress pro-apoptotic factor CHOP transcriptionally regulates the expression of TNC.[Conclusion]:In sepsis,the endoplasmic reticulum stress signaling pathway PERK-eIF2α-ATF4-CHOP is activated,and pro-apoptotic factor CHOP is activated to regulate the expression of transcriptional TNC and promote its secretion into the circulating immune microenvironment by means of exosomes.This project proposes a new mechanism for the regulation of endoplasmic reticulum stress pathway and TNC expression.TNC plays a key role in mediating immune response and organ damage as an inflammatory chemokine,which is expected to provide a new target and a new strategy for the diagnosis and treatment of acute lung injury in sepsis.