Research progress on the mechanism of role of podoplanin in sepsis / 中华危重病急救医学

Podoplanin (PDPN) is a small transmembrane mucin-like glycoprotein which is expressed on the surface of lymphatic endothelial cells, glomerular podocytes, type-Ⅰ alveolar epithelial cells and some tumor cells. PDPN plays crucial function in variety of physiological and pathological processes such as embryonic development, immunoreaction, inflammation and cancer. C-type lectin-like receptor 2 (CLEC2) is mainly expressed on the platelet which specific ligand is PDPN. The interaction between PDPN and CLEC2 has received extensive attention. In this review, we summarized recent researches on the role of in sepsis and elaborated the possible mechanisms and some potential therapies for sepsis by targeting PDPN, which may provide theoretical basis for the mechanism and treatment of sepsis.

Extracellular histones are involved in lipopolysaccharide-induced alveolar macrophage injury by activating the TWIK2-NLRP3 pathway / 中华危重病急救医学

Objective:To explore the role and mechanism of extracellular histones involved in lipopolysaccharide (LPS)-induced alveolar macrophage injury.Methods:The mouse alveolar macrophage cell line (MH-S) was cultured in vitro and passaged, and the cells were cultured to 80% of cells for cell proliferation. The cells were stimulated with 1 mg/L LPS for 3 hours and 50 mg/L exogenous histones for 3, 6, 12, and 24 hours, respectively (LPS+histones 3, 6, 12, 24 h groups), and other groups included phosphate buffered saline (PBS) control group (PBS group), LPS alone stimulation group (LPS group), the exogenous histones alone stimulation group (histones group) and heparin pretreatment histones group (heparin+LPS+histones group). The cells in each group were challenged with different reagent, the expression of lactate dehydrogenase (LDH) and inflammatory factors in the supernatant were detected by enzyme linked immunosorbent assay (ELISA), and the change of intracellular K + concentration was detected by FluxOR TMⅡgreen potassium channel. The proteins such as potassium channel protein (TWIK2), inflammasome (NLRP3), and apoptosis associated speck like protein containing a CARD (ASC) were determined by Western Blot. Results:Compared with the PBS group, the levels of LDH and inflammatory factors such as interleukin (IL-1β, IL-18) and tumor necrosis factor-α (TNF-α) were significantly increased after LPS stimulation group. Compared with the LPS group, the levels of LDH and inflammatory factors were significantly increased after the treatment with exogenous histones, and reached a peak after 3 hours of the histones stimulation [LDH (U/L): 123.10±1.83 vs. 85.32±1.66, IL-1β (mg/L): 40.75±2.60 vs. 18.78±1.37, IL-18 (mg/L): 49.94±2.45 vs. 30.19±1.82, TNF-α (mg/L): 36.51±1.56 vs. 20.84±1.61, all P < 0.01]. Western Blot results showed that compared with the LPS group, NLRP3, ASC and TWIK2 protein expression were significantly up-regulated in the LPS+histones group (NLRP3/GAPDH: 0.80±0.02 vs. 0.57±0.02, ASC/GAPDH: 0.57±0.02 vs. 0.38±0.01, TWIK2/GAPDH: 0.65±0.01 vs. 0.41±0.01, all P < 0.01), and the expression of the above proteins were significantly down-regulated after heparin pretreatment (NLRP3/GAPDH: 0.28±0.02 vs. 0.80±0.02, ASC/GAPDH: 0.25±0.02 vs. 0.57±0.02, TWIK2/GAPDH: 0.35±0.01 vs. 0.65±0.01, all P < 0.01), indicating that histones could activate NLRP3 through TWIK2 to participate in inflammatory reaction. In addition, intracellular K + concentration in LPS+histones group decreased significantly compared with the LPS group (fluorescence intensity: 35.48±2.53 vs. 83.92±3.11, P < 0.01). Compared with LPS+histones group, K + concentration increased significantly after pretreatment with heparin (fluorescence intensity: 72.10±1.78 vs. 35.48±2.53, P < 0.01), indicating that extracellular histones could cause K + massive efflux through TWIK2, and thus mediate NLRP3 activation and participate in inflammatory injury of alveolar macrophages. Conclusion:Extracellular histones can cause inflammatory damage in alveolar macrophages, and its mechanism may be related to the activation of NLRP3 by extracellular histones activation of TWIK2 channel to promote K + efflux.

Research progress in lung transplantation / 中华危重病急救医学

Lung transplantation is the only effective treatment for end-stage lung disease. The problems, including acute rejection, infection, primary graft dysfunction (PGD) and ischemia/reperfusion injury after lung transplantation, as well as surgical techniques and anesthesia management, donor, cardiopulmonary bypass or extracorporeal membrane oxygenation (ECMO), and the main pathogenesis of the receptor, that restrict the development of lung transplantation seriously, and affect the prognosis of patients. Through the description of above major bottlenecks related to lung transplantation and the factors that seriously affect the survival of lung transplant patients and the current response measures, new ideas for the clinical treatment of lung transplantation are presented.

Research progress in lung transplantation / 中华危重病急救医学

Lung transplantation is the only effective treatment for end-stage lung disease. The problems, including acute rejection, infection, primary graft dysfunction (PGD) and ischemia/reperfusion injury after lung transplantation, as well as surgical techniques and anesthesia management, donor, cardiopulmonary bypass or extracorporeal membrane oxygenation (ECMO), and the main pathogenesis of the receptor, that restrict the development of lung transplantation seriously, and affect the prognosis of patients. Through the description of above major bottlenecks related to lung transplantation and the factors that seriously affect the survival of lung transplant patients and the current response measures, new ideas for the clinical treatment of lung transplantation are presented.

Extracellular histones aggravate acute respiratory distress syndrome by inducing peripheral blood mononuclear cells pyroptosis / 中华危重病急救医学

Objective@#To explore whether extracellular histones aggravate acute respiratory distress syndrome (ARDS) by inducing peripheral blood mononuclear cell (PBMC) pyroptosis.@*Methods@#Twenty patients with ARDS admitted to Shanghai Pulmonary Hospital, Tongji University School of Medicine from April to September in 2019 were enrolled, and 20 healthy volunteers were enrolled as controls. In vivo experiment: peripheral blood samples of patients with ARDS within 24 hours after diagnosis and healthy volunteers were collected, and the levels of plasma extracellular histone, interleukins (IL-1β and IL-18) and lactic dehydrogenase (LDH) were determined by enzyme-linked immunosorbent assay (ELISA). PBMC were harvested, the expression levels of the pyroptosis associated N terminal-gasdermin-D (GSDMD-N) protein were determined by Western Blot. In vitro experiment: PBMC isolated from healthy volunteers were divided into four groups. Blank control group without any treatment; lipopolysaccharide (LPS) group was treated with 1 mg/L LPS for 4 hours; LPS+histones group was treated with 100 mg/L exogenous histones for 24 hours after LPS treatment; LPS+histone+heparin group was treated with 200 U heparin for 24 hours after LPS and exogenous histones treatment. The GSDMD-N protein expression was determined by Western Blot, and the levels of IL-1β, IL-18 and LDH in cell supernatant were determined by ELISA. Spearman test was used to test the correlation among the parameters.@*Results@#In vivo experiment results: compared with healthy control group, the GSDMD-N protein expression in PBMC of patients with ARDS was significantly increased [GSDMD-N/GAPDH: 0.136 (0.062, 0.246) vs. 0.026 (0.018, 0.036), P < 0.01], as well as the plasma levels of IL-1β, IL-18, LDH and extracellular histones [IL-1β (ng/L): 120.0 (94.2, 213.0) vs. 88.5 (82.3, 105.3), IL-18 (ng/L): 164.5 (70.8, 236.3) vs. 60.5 (52.0, 89.0), LDH (U/L): 30.9 (24.7, 39.5) vs. 19.8 (17.2, 21.5), extracellular histones (mg/L): 73.0 (42.8, 112.9) vs. 12.2 (9.6, 16.9), all P < 0.01], indicating that the PBMC of ARDS patients had significant pyroptosis and release of a large number of inflammatory factors. The oxygenation index (PaO2/FiO2) of ARDS patients was 135.5 (94.5, 196.0) mmHg (1 mmHg = 0.133 kPa). Correlation analysis showed that the expression of GSDMD-N protein in patients with ARDS was negatively correlated with PaO2/FiO2 (r = -0.935, P < 0.01) and positively correlated with IL-1β, IL-18, LDH and extracellular histones (r value was 0.844, 0.843, 0.887, 0.899, respectively, all P < 0.01). In vitro experiment results: compared with blank control group, the expression of GSDMD-N protein in PBMC and the levels of inflammatory mediators in the supernatant of the LPS group were significantly increased [GSDMD-N/GAPDH: 0.035±0.006 vs. 0.028±0.006, IL-1β (ng/L): 39.8±5.5 vs. 22.6±4.7, IL-18 (ng/L): 31.2±4.4 vs. 20.0±2.2, LDH (U/L): 51.2±7.3 vs. 36.6±7.6, all P < 0.05], indicating that LPS stimulation could increase PBMC pyroptosis and the release of inflammatory mediators. Compared with LPS group, the expression of GSDMD-N protein and the levels of inflammatory mediators of the LPS+histones group were further increased [GSDMD-N/GAPDH: 0.114±0.009 vs. 0.035±0.006, IL-1β (ng/L): 119.0±18.7 vs. 39.8±5.5, IL-18 (ng/L): 49.2±8.5 vs. 31.2±4.4, LDH (U/L): 127.8±19.8 vs. 51.2±7.3, all P < 0.01], indicating that the stimulation of LPS on PBMC could be significantly amplified by exogenous histone treatment, GSDMD-N protein expression could be up-regulated and inflammatory factor release could be promoted to further induce PBMC pyroptosis. These adverse effects of exogenous histones on PBMC could be abrogated by heparin, the expression of GSDMD-N protein and the levels of inflammatory mediators were significantly lower than those of LPS+histones group [GSDMD-N/GAPDH: 0.063±0.004 vs. 0.114±0.009, IL-1β (ng/L): 46.8±8.6 vs. 119.0±18.7, IL-18 (ng/L): 33.0±5.1 vs. 49.2±8.5, LDH (U/L): 65.4±11.0 vs. 127.8±19.8, all P < 0.05].@*Conclusion@#Extracellular histones in plasma may aggravate ARDS by mediating PBMC pyroptosis.

Extracellular histones aggravate acute respiratory distress syndrome by inducing peripheral blood mononuclear cells pyroptosis / 中华危重病急救医学

To explore whether extracellular histones aggravate acute respiratory distress syndrome (ARDS) by inducing peripheral blood mononuclear cell (PBMC) pyroptosis. Methods Twenty patients with ARDS admitted to Shanghai Pulmonary Hospital, Tongji University School of Medicine from April to September in 2019 were enrolled, and 20 healthy volunteers were enrolled as controls. In vivo experiment: peripheral blood samples of patients with ARDS within 24 hours after diagnosis and healthy volunteers were collected, and the levels of plasma extracellular histone, interleukins (IL-1β and IL-18) and lactic dehydrogenase (LDH) were determined by enzyme-linked immunosorbent assay (ELISA). PBMC were harvested, the expression levels of the pyroptosis associated N terminal-gasdermin-D (GSDMD-N) protein were determined by Western Blot. In vitro experiment: PBMC isolated from healthy volunteers were divided into four groups. Blank control group without any treatment; lipopolysaccharide (LPS) group was treated with 1 mg/L LPS for 4 hours; LPS+histones group was treated with 100 mg/L exogenous histones for 24 hours after LPS treatment; LPS+histone+heparin group was treated with 200 U heparin for 24 hours after LPS and exogenous histones treatment. The GSDMD-N protein expression was determined by Western Blot, and the levels of IL-1β, IL-18 and LDH in cell supernatant were determined by ELISA. Spearman test was used to test the correlation among the parameters. Results In vivo experiment results: compared with healthy control group, the GSDMD-N protein expression in PBMC of patients with ARDS was significantly increased [GSDMD-N/GAPDH: 0.136 (0.062, 0.246) vs. 0.026 (0.018, 0.036), P < 0.01], as well as the plasma levels of IL-1β, IL-18, LDH and extracellular histones [IL-1β (ng/L): 120.0 (94.2, 213.0) vs. 88.5 (82.3, 105.3), IL-18 (ng/L): 164.5 (70.8, 236.3) vs. 60.5 (52.0, 89.0), LDH (U/L): 30.9 (24.7, 39.5) vs. 19.8 (17.2, 21.5), extracellular histones (mg/L): 73.0 (42.8, 112.9) vs. 12.2 (9.6, 16.9), all P < 0.01], indicating that the PBMC of ARDS patients had significant pyroptosis and release of a large number of inflammatory factors. The oxygenation index (PaO2/FiO2) of ARDS patients was 135.5 (94.5, 196.0) mmHg (1 mmHg = 0.133 kPa). Correlation analysis showed that the expression of GSDMD-N protein in patients with ARDS was negatively correlated with PaO2/FiO2 (r = -0.935, P <0.01) and positively correlated with IL-1β, IL-18, LDH and extracellular histones (r value was 0.844, 0.843, 0.887, 0.899, respectively, all P < 0.01). In vitro experiment results: compared with blank control group, the expression of GSDMD-N protein in PBMC and the levels of inflammatory mediators in the supernatant of the LPS group were significantly increased [GSDMD-N/GAPDH: 0.035±0.006 vs. 0.028±0.006, IL-1β (ng/L): 39.8±5.5 vs. 22.6±4.7, IL-18 (ng/L): 31.2±4.4 vs. 20.0±2.2, LDH (U/L): 51.2±7.3 vs. 36.6±7.6, all P < 0.05], indicating that LPS stimulation could increase PBMC pyroptosis and the release of inflammatory mediators. Compared with LPS group, the expression of GSDMD-N protein and the levels of inflammatory mediators of the LPS+histones group were further increased [GSDMD-N/GAPDH:0.114±0.009 vs. 0.035±0.006, IL-1β (ng/L): 119.0±18.7 vs. 39.8±5.5, IL-18 (ng/L): 49.2±8.5 vs. 31.2±4.4, LDH (U/L): 127.8±19.8 vs. 51.2±7.3, all P < 0.01], indicating that the stimulation of LPS on PBMC could be significantly amplified by exogenous histone treatment, GSDMD-N protein expression could be up-regulated and inflammatory factor release could be promoted to further induce PBMC pyroptosis. These adverse effects of exogenous histones on PBMC could be abrogated by heparin, the expression of GSDMD-N protein and the levels of inflammatory mediators were significantly lower than those of LPS+histones group [GSDMD-N/GAPDH: 0.063±0.004 vs. 0.114±0.009, IL-1β (ng/L): 46.8±8.6 vs. 119.0±18.7, IL-18 (ng/L): 33.0±5.1 vs. 49.2±8.5, LDH (U/L): 65.4±11.0 vs. 127.8±19.8, all P < 0.05]. Conclusion Extracellular histones in plasma may aggravate ARDS by mediating PBMC pyroptosis.

Pivotal role of cell hyperactivation in the pathogenesis of different diseases and its mechanism / 中华危重病急救医学

Inflammasomes are key inflammatory signaling platforms that detect several stimulus derived from microbial substances and sterile environmental insults, eliciting pyroptosis and the release of cytokines. Recent studies have found that inflammasomes could also elicit cell hyperactivation which is defined as living cells that release interleukin-1 (IL-1). Hyperactive cells promote long-term IL-1 release and then activate adaptive immune response, which has a pivotal role in the infection ofSalmonella andStaphylococcus aureus, as well as in non-lethal inflammatory sepsis. So it is of great significance to explore the pathogenesis of cell hyperactivation in several inflammatory diseases. Here, we summarized the possible pathogenesis of cell hyperactivation in different inflammatory diseases, such as infection, sepsis and acute respiratory distress syndrome (ARDS), providing a theoretical basis for clinical treatment of these diseases.