Effect Of Circulating Exosomes On Endoplasmic Reticulum Stress In Lung Tissue Of Lps-induced ARDS Mice

BackgroundAcute respiratory distress syndrome?ARDS?is a critical clinical syndrome with high mortality rate,and few effective therapies have been found in the past 50 years,indicating that the pathogenesis of ARDS remains unclear.Exosomes,a novel cross-communication mechanism,are involved in critical diseases.However,the role of circulating exosomes in the development of ARDS remains poorly understood.ObjectiveTo observe the serum GRP78 levels in patients with acute respiratory distress syndrome?ARDS?,and its role in the development of ARDS.To investigate the effect of circulating exosomes on Endoplasmic Reticulum Stress in Lung Tissue of LPS-induced ARDSMethods?1?A retrospective investigation was conducted.52 patients with ARDS were selected as the study subjects and divided into the non-survivor group and the survivor group according to the outcomes of the patients at 28 days.Collecting their basic clinical information,body mass index?BMI?,acute physiology and chronic health evaluation system??APACHE II?score,oxygenation Index?FiO₂/PO₂?,hospitalization length,invasive mechanical ventilation length,and blood examination results such as white blood cell counts?WBC?,procalcitonin?PCT?and c-reactive protein?CRP?,etc.At the same time,20 pneumonia patients and20 healthy volunteers were involved as the control group,and 2ml of venous blood sample from each subject was collected for further study.Serum GRP78 levels were detected and compared among those groups.?2?Mice were randomly divided into control group,LPS group,EXO group and SER group using random number table.The circulating exosomes were collected from LPS-induced ARDS mice at Day 2.The exosomes and exosome-depleted serum from ARDS mice were injected into naive mice via the tail vein as the EXO group and SER group,respectively.The mice were sacrificed 48 h after intratracheal or tail vein injection.The pathological changes in lung tissue were evaluated by HE staining;The concentrations of total protein in bronchoalveolar lavage fluid?BALF?were assessed by BCA?bicinchoninic acid?.In BALF,the levels of IL-6 and TNF-?were determined by ELISA and myeloperoxidase?MPO?activities were measured by MPO assay kit.The total cell counts and polymorphonuclear neutrophil?PMN?counts of BALF were detected by Giemsa staining.The protein levels of GRP78,CHOP,?-Catenin and VE-cadherin were measured by Western blot.?3?Mice were randomly divided into control group,LPS group and CEXO group using random number table.The circulating exosomes were collected from naive ARDS mice and the exosomes were injected into ARDS mice via the tail vein 24h after LPS intratracheal injection as the CEXO group.The mice were sacrificed 24 h after tail vein injection.The pathological changes in lung tissue were evaluated by HE staining;The concentrations of total protein in BALF were assessed by BCA.The serum levels of IL-6 and TNF-?were determined by ELISA.The total cell counts and PMN counts of BALF were detected by Giemsa staining.The protein levels of GRP78 were measured by Immunohistochemistry and Western bloting.Results?1?The serum GRP78 level of ARDS patients was significantly higher than that of pneumonia patients and healthy subjects?P<0.05?.Compared with the survivor group,the invasive mechanical ventilation lengths and serum GRP78 levels of patients in non-survivor group were significantly increased?P<0.05?,while BMI and FiO₂/PO₂ were significantly decreased?P<0.05?.Moreover,the serum GRP78 levels of ARDS patients were negatively correlated with the FiO2/PO2?r=-0.55,P<0.01?,and positively correlated with APACHEII scores?r=0.39,P<0.01?.?2?Compared with mice in control group,classic ARDS pathological changes were observed in mice of LPS group,manifesting by severe pathological lung injury?P<0.05?,increased in W/D weight ratio,total protein levels,cell counts,MPO activities,IL-6 and TNF-?levels in BALF?P<0.05?,accompanied by up-regulated levels of GRP78 and CHOP,down-regulated levels of?-Catenin and VE-cadherin in lung tissues?P<0.05?.Compared with mice in control group,injured pathological changes were observed in mice of EXO group,manifesting by pathological lung injury?P<0.05?,increased in W/D weight ratio,total protein levels,cell counts,MPO activities,IL-6 and TNF-?levels in BALF?P<0.05?,accompanied by up-regulated levels of GRP78 and CHOP,down-regulated levels of?-Catenin and VE-cadherin in lung tissues?P<0.05??3?Compared with mice in control group,classic ARDS pathological changes were observed in mice of LPS group,manifesting by severe pathological lung injury?P<0.05?,increased in W/D weight ratio,total protein levels and cell counts in BALF,higher IL-6 and TNF-?levels in serum?P<0.05?,accompanied by up-regulated levels of GRP78 in lung tissues?P<0.05?.However,compared with mice in LPS group,alleviatived pathological changes were observed in mice of CEXO group,manifesting by alleviatived pathological lung injury?P<0.05?,decreased in W/D weight ratio,total protein levels and cell counts in BALF,lower IL-6 and TNF-?levels in serum?P<0.05?,accompanied by down-regulated levels of GRP78in lung tissues?P<0.05?.Conclusion?1?Endoplasmic reticulum stress is involved in the development of ARDS,and the increased serum GRP78 indicated the poor prognosis for patients.?2?These data demonstrate that circulating exosomes from LPS-induced ARDS mice trigger ER stress in lung tissue,facilitating the development of ARDS,at least partly by promoting endothelial dysfunction.?3?circulating exosomes from na?ve mice alleviated ER stress in lung tissue,protecting against the development of ARDS.