Study On Monocyte Function In Acute Respiratory Distress Syndrome Induced By Sepsis

Objective:Acute respiratory distress syndrome（ARDS）is a clinical syndrome caused by pulmonary and/or extrapulmonary causes.As an acute respiratory disease,the clinical presentation is bilateral chest X-ray opacity with severe hypoxemia due to non-cardiogenic pulmonary edema.Sepsis refers to systemic inflammatory syndrome caused by infection,which represents a response pattern of the immune system to injury.The pathogenic factors of ARDS are varied,including direct injury factors and indirect influence factors.Common direct injuries include pneumonia,inhalation of toxic substances,near drowning or lung contusion;And indirect mechanisms such as sepsis,burns,shock,drug overdose pancreatitis,gynecological injury（placental abruption,amniotic embolism,eclampsia）,multiple fractures,severe extra-thoracic trauma,or massive blood transfusion.Sepsis is one of the most common causes of ARDS.ARDS induced by sepsis has the characteristics of faster onset and higher mortality.However,at present,there is no clear conclusion on its pathogenesis and more effective diagnosis and treatment measures are lacking.Monocytes are a kind of important innate immune cells.As the precursor cells of macrophages,they are generally considered to recruit to the lungs in large numbers during the occurrence of lung inflammation and undergo differentiation process,and play a role in lung inflammation as macrophages.However,its further role in the occurrence and development of sepsis-induced ARDS remains to be clarified.This study aims to explore more roles and mechanisms of monocytes in the occurrence and development of ARDS,in order to provide new ideas for the diagnosis and treatment of sepsis induced ARDS.Methods:1)Destiny of monocyte/macrophage cells in acute and chronic lung inflammations:a)The ARDS model of mice was established by cecal ligation and puncture（CLP）method,and the changes of monocyte/macrophage in the lungs of ARDS mice were observed by single-cell transcriptome sequencing;b)Tracheal instillation of silicon dioxide（Si O₂）7-day silicosis model was established in mice,and single-cell transcriptome sequencing was used to observe the changes of monocyte/macrophage cells in the lungs of mice with silicosis;c)Bioinformatics analysis of single-cell transcriptome sequencing results,collecting conditioned medium of macrophages stimulated by Si O₂,and sending them to the mice.LPS and conditioned medium were added to THP-1,respectively,and the expression changes of macrophage marker protein F4/80 were detected by WB.2)The function of monocytes in ARDS:a)Bioinformatics analysis of single-cell transcriptome sequencing and spatial transcriptome sequencing results,real-time quantitative PCR（q RT-PCR）detection of inflammation-related genes in THP-1 under LPS stimulation The expression changes of Il1b,Tnf and Tgfb1;b)The effect of conditioned medium after LPS-stimulated THP-1was collected on HUVEC of human umbilical vein endothelial cells,and the change of HUVEC adhesion ability was determined by adhesion experiment.Endothelial cadherin CDH5 level,trans-endothelial electrical resistance（TEER）assay and dextran assay were used to detect the permeability of HUVEC cells,and Matrigel gel-forming tube assay was used to detect the tube-forming ability of HUVEC cells.3)The mechanism of monocyte involvement in ARDS:a)Bioinformatics analysis of monocyte subpopulation in single cell transcriptome sequencing.Cell count was used to measure changes in THP-1 cell number under LPS stimulation,and CCK8 was used to measure changes in cell viability;b)Bioinformatics analysis of TOP30 differentially expressed genes was performed to determine the target protein CCNB2.QRT-PCR was used to detect ccnb2 expression in THP-1 under LPS stimulation at different time points,and WB was used to detect CCNB2 protein expression in THP-1;c)After fluorescence detection of LV3 lentivirus infected THP-1,the knockdown efficiency of Ccnb2 in THP-1 was detected by q RT-PCR and WB;d)The morphological changes of THP-1 after LPS-stimulated Ccnb2 knockdown by Lentivirus were observed by fluorescence.WB detected the expression changes of M1-type macrophage marker protein NOS2,M2a type macrophage marker protein ARG-1 and M2c type macrophage marker protein SOCS3 in THP-1 after LPS stimulation of LV3 lentivirus knockdown Ccnb2.The expression of inflammatory genes Il4,Il10,Il1b,Tnf,Tgfb1 in THP-1 after LPS-stimulated Ccnb2 knockdown by LV3 lentivirus was determined by q RT-PCR;e)LV3 lentivirus knockdown Ccnb2 was collected and HUVEC was treated with LPS-stimulated THP-1 conditioned medium.The protein expression changes of TJP1 and CDH5 were detected by WB,the permeability of HUVEC cells was detected by TEER and glucan methods,and the tube-forming ability of HUVEC cells was detected by Matrigel gel tube assay.Results:1)The sequencing results of the pulmonary single-cell transcription group sequencing of the ARDS model of the CLP method showed that the number of monocytes increased,the number of macrophages decreased;while the tracheal drop Si O₂ was established to establish a pulmonary single-cell transcription group sequencing result of the silicone model of mice.It shows that the number of monocytes is not changed,the number of macrophages is increased;the time-sequential analysis obtains little correlation between monocyte cells and macrophages in ARDS,and the analysis results between monocytes and macrophages in the silicon lung model are transformed,and no differentiation was found in THP-1 stimulated by LPS in vitro.2)GO enrichment biological analysis showed that monocytes in ARDS were involved in inflammatory response,secreting inflammatory factors IL-1β,TNF-α,and transforming growth factor TGF-β1.After LPS stimulation,the expression of the above genes Il1b,Tnf,Tgfb1 increased in THP-1.After HUVEC was treated with LPS-stimulated THP-1 conditioned medium,the adhesion ability of HUVEC cells was decreased,the levels of tight junction protein TJP1 and vascular endothelial cadherin CDH5 protein were decreased,the permeability of HUVEC cells was enhanced,and the tubulogenesis ability was weakened.3)The monocytes in ARDS were subdivided into two subgroups by bioinformatics analysis.After GO biological process analysis,it was found that monocyte group MONocyte-2 had proliferation characteristics.After LPS stimulation in vitro,the number of THP-1 cells was increased and cell viability was significantly increased.After that,the expression of CCNB2,a cell cycle regulating protein,was specifically increased in the monocyte subgroup of Monocyte-2 by GO string analysis.After LPS stimulation in vitro,the m RNA expression and protein level of CCNB2 in THP-1 were increased.Subsequently,Ccnb2 in THP-1 was silenced successfully by LV3 lentivirus,and th P-1 of Ccnb2 was silenced by LPS stimulation.The phenotype of macrophage was found in THP-1 morphology,and the expression of M2c macrophage marker protein SOCS3 was increased by WB.Qrt-pcr showed that the expression level of IL-10,a landmark anti-inflammatory factor of M2c macrophages,was increased.Endothelial HUVEC cells were treated with l PS-stimulated th P-1 with low CCNB2 expression on conditioned medium,and the levels of tight junction protein TJP1 and vascular endothelial cadherin CDH5 were found to be increased,the permeability of endothelial HUVEC cells was reduced,and the tubulogenesis ability was significantly enhanced.Conclusion:This study showed that CCNB2 in monocytes can inhibit inflammatory response by regulating the differentiation of monocytes into M2c macrophages in sepsis induced ARDS,thus alleviating the storm of lung inflammation,providing a new idea for the clinical treatment of sepsis induced ARDS.