Protective Effects And Mechanisms Of S-(+)-ketamine On Sepsis-induced Lung Injury

Part Ⅰ Effects of S-（+） ketamine on lung injury and local immune microenvironment in sepsisObjective: To explore the mechanisms of over-activation of pulmonary immune response in the early stage of sepsis and to evaluate the role of S-（+）ketamine（S-KT）in septic lung injury.Methods: Sepsis mouse model was induced by cecal ligation and puncture（CLP）.Adult male C57BL/6J mice were randomly divided into 5 groups:（1）Sham operation + saline group（Sham+saline）: Normal saline（10 ml/kg）was injected intraperitoneally 24 hours before and immediately after sham operation;（2）CLP+saline group: 0.9% Saline（10 ml/kg）was injected intraperitoneally 24 hours before CLP,and 0.9% saline（10 ml/kg）was injected intraperitoneally immediately after CLP;（3）CLP+S-KT combined administration group: Intraperitoneal injection of S-KT（15mg/kg）24 hours before CLP,and intraperitoneal injection of S-KT（15mg/kg）immediately after CLP;（4）CLP+S-KT prophylaxis group: Intraperitoneal injection of S-KT（15 mg/kg）24 hours before CLP,and intraperitoneal injection of normal saline（10 ml/kg）immediately after CLP;（5）CLP+S-KT alone treatment group: Normal saline（10 ml/kg）was injected intraperitoneally 24 hours before sham operation,and S-KT（15 mg/kg）was injected intraperitoneally immediately after CLP.24 hours after sham operation or CLP,the survival rate,body weight,anal temperature and murine sepsis score were observed to evaluate the prognosis of sepsis,hematoxylin-eosin（HE）staining and corresponding pathological injury score were used to evaluate the pathological injury of lung.The degree of lung injury was evaluated by lung wet-dry weight ratio,lung bactierial load,detection of tight junction protein by Western blotting and pulmonary vascular permeability by Evans Blue extravasation.Differential signaling pathways were screened by proteomic sequencing;inflammatory factors in lung tissue were detected by Western blotting and Cytometric Bead Array（CBA）;the levels of pulmonary neutrophils and extracellular traps,alveolar macrophages and pulmonary interstitial macrophages and polarization were detected by flow cytometry and immunofluorescence staining;IL-17 A overexpression adenovirus intratracheal injection and flow cytometry were used to detect the effect of IL-17 A on pulmonary immune cells.Results: S-KT combined-administration significantly increased the 21-day survival rate and improved the prognosis of septic mice;S-KT reduced the destruction of lung structure,inflammatory cell infiltration and pulmonary barrier damage induced by sepsis;the results of protein sequencing and Western blotting showed that IL-17 signaling pathway was significantly active after sepsis.S-KT significantly suppressed the elevation of IL-17 A and other inflammatory factors in the lungs in the early stage of sepsis.In the early stage of sepsis,the levels of neutrophils and extracellular traps in the lungs increased significantly,the proportion of alveolar macrophages and interstitial macrophages increased,and the M1 polarization of the above macrophages increased significantly,while S-KT could significantly inhibit the increase of neutrophils and extracellular traps and the proportion of alveolar macrophages and M1 polarization after sepsis.The overexpression of IL-17 A promoted the expansion of pulmonary neutrophils and the M1 polarization of alveolar macrophages.Conclusion: S-KT combined-administration can inhibit the overactivation of pulmonary immune response by reducing the level of IL-17 in the lung in the early stage of sepsis,thus attenuating the lung injury induced by CLP.Part Ⅱ Migration of small intestinal γδ T cells into lung mediates pulmonary immune overactivation after sepsisObjective: To explore the cell source of lung IL-17 A in the early stage of sepsis and the role of "gut-lung" axis in septic lung injury.Methods:（1）The source of IL-17A-producing cells in lung tissue was detected by flow cytometry;（2）The change of intestinal barrier permeability was evaluated by FITC-dextran permeation test,and the levels of tight junction proteins MUC-2,Occludin and ZO-1 were detected by Western Blotting to explore the reasons for the increase of intestinal permeability after sepsis;（3）The changes of intestinal immune cells in 24 hours after sepsis were observed by intestinal single-cell RNA sequencing,the proportion of intestinal γδ T cells was detected by flow cytometry;（4）The migration of small intestinal γδ T cells to the lung was verified by using Kaede-transgenic mice.Kaede-transgenic mice were randomly divided into the following three groups:（1）Sham+saline group;（2）CLP+saline group;（3）CLP+S-KT combined-administration group.The fluorescence conversion of Kaede transgenic mice after UV irradiation was verified by immunofluorescence staining,and the proportion of Kaede red+ CD45+ IL-17A+ CD3+ γδ TCR+ cells in lung tissue was detected by flow cytometry to analyze the proportion of intestinal IL-17A-producing γδ T cells in the lung.（5）Small intestinal single-cell RNA sequencing and flow cytometry were used to explore the possible γδ T cell subsets of small intestinal that traffick to the lungs.Results:（1）γδ T cells increased significantly in the lung at 24 hours after sepsis,and became the main cells secreting IL-17 A in lung tissue;（2）The decrease of intestinal tight junction protein MUC-2 led to a significant increase in intestinal permeability at 24 hours after sepsis,while S-KT combined-administration significantly attenuated the destruction of intestinal barrier caused by sepsis;（3）Single-cell RNA sequencing of immune cells in the small intestine of septic mice showed that 24 hours after sepsis,neutrophils and macrophages increased significantly,while natural killer cells and dendritic cells decreased significantly,and γδ T cells increased significantly;（4）The results of small intestinal lymphocyte migration in Kaede transgenic mice showed that γδ T cells migrated to the lungs and became the main source of IL-17 A cells in the lungs.S-KT significantly inhibited the migration of small intestinal γδ T cells to the lungs;（5）The significant increase of γδ T cells in the lung in the early stage of sepsis may be mainly attributable to IL-7R^highCD8^low γδ T cell subsets in the small intestine.Conclusion: 24 hours after sepsis,intestinal tight junction protein MUC-2 decreased significantly,resulting in severe damage to the intestinal barrier.Small intestinal γδ T cells significantly expanded and migrated to the lung,becoming the main source of IL-17 Aproducing cells in the lungs.Part Ⅲ The role of pulmonary activated Wnt signaling pathway in the migration of small intestinal γδ T cells into the lungObjective: To explore the mechanisms of the migration of γδ T cells from small intestine to the lung in the early stage of sepsis.Methods:（1）Detecting the changes of various chemokines in the lung 24 hours after sepsis by Luminex multifactor detection technique.Verifing the level of CCL1 in the lung of septic mice by Western blotting;（2）The in vitro model of acute lung injury induced by lipopolysaccharide（LPS）stimulated-A549 pulmonary epithelial cell line,RAW264.7 macrophage line and MH-S alveolar macrophage line was used to explore the cellular source of CCL1 in lung tissue in the early stage of sepsis by Western blotting;（3）γδ T cells were isolated by magnetic beads sorting,and the chemotactic effect of CCL1 on γδ T cells was verified by transwell migration assay in vitro;（4）On the basis of proteomes,differential proteins were further analyzed,and the levels of key proteins in Wnt signaling pathway were further detected by Western blotting;（5）The transcription factor Lef1 of MH-S Wnt signaling pathway was tested by chromatin co-immunoprecipitation high throughput sequencing（ChIP-seq）to verify the specific binding between Lef1 and CCL1;（6）Through the in vivo experiment of intraperitoneal injection of iCRT14,an inhibitor of Wnt signal pathway in septic mice,and the in vitro experiment of iCRT14 pre-stimulation of MH-S cell line before LPS stimulation,exploring the regulatory relationship between Wnt signaling pathway and CCL1 in the lung of septic mice.Results:（1）The expression of CCL1 in pulmonary parenchyma was significantly upregulated at 24 hours after sepsis,and S-KT significantly decreased the expression of CCL1 in the lungs of septic mice;（2）LPS stimulation could not induce significant elevation of CCL1 in A549 pulmonary epithelial cell line and RAW264.7 macrophage line,while the protein level of CCL1 in MH-S mouse alveolar macrophage line increased significantly after LPS stimulation;（3）The results of transwell migration assay showed that CCL1 had chemotactic effect on γδ T cells in spleen single cell suspension and γδ T cells sorted by magnetic beads;（4）Proteomic sequencing of lung tissue after sepsis showed that the protein level of FZD5 in the lung of the CLP model group was significantly higher than that of the sham operation group（ratio = 5.928）.The results of Western blotting showed that the upstream ligand protein Wnt5 a,downstream protein β-catenin and transcription factor Lef1 of Wnt signaling pathway increased significantly at 24 hours after sepsis.Administration of S-KT significantly inhibited the activation of Wnt signaling after sepsis;（5）ChIP-seq results showed that Lef1 specifically binds to the distal region of CCL1 gene,indicating that Lef1 may have a transcriptional regulatory effect on CCL1;（6）After administration of iCRT14 to inhibit Wnt signaling pathway,the level of CCL1 in the lung of septic mice decreased significantly at 24 hours after operation.The inhibition of Wnt signal pathway by iCRT14 in MH-S cell line could significantly reduce the increase of CCL1 in MH-S cell line after LPS stimulation;（7）iCRT14 significantly inhibited the migration of γδ T from the small intestine to the lungs,decreased the level of inflammatory factors in the lungs of septic mice,and reduced the destruction of pulmonary structure.Conclusion: The Wnt signaling pathway is significantly active in the lung at 24 hours after sepsis and upregulates the expression level of CCL1 in lung tissues through the transcriptional regulation by the transcription factor Lef1.CCL1 in post-septic lung tissue was chemotactic for small intestinal γδ T.In vivo administration of S-KT decreased the level of CCL1 in the lung through downregulation of the Wnt/β-catenin signaling pathway in septic mice,which in turn inhibited the migration of small intestinal γδ T cells into the lung after sepsis.Inhibition of the Wnt/β-catenin signaling pathway in the lung of septic mice attenuated sepsis-induced lung injury by reducing lung immune hyperactivation in the early stages of sepsis and thereby improving the local immune microenvironment.