[Mechanism of Extracellular Histone-Induced Endothelial Dysfunction Leading to Sepsis-Induced Acute Respiratory Distress Syndrome]. / å¾ªçŽ¯ç»„è›‹ç™½è¯±å¯¼å† çš®åŠŸèƒ½éšœç¢è‡´è„“æ¯’ç—‡æ€¥æ€§å‘¼å¸çª˜è¿«ç»¼åˆå¾çš„æœºåˆ¶ç ”ç©¶.

Objective: Sepsis-induced acute respiratory distress syndrome (ARDS) is an independent risk factor for mortality in critically ill septic patients. However, effective therapeutic targets are still unavailable due to the lack of understanding of its unclear pathogenesis. With increasing understanding in the roles of circulating histones and endothelial dysfunction in sepsis, we aimed to investigate the mechanism of histone-induced endothelial dysfunction leading to sepsis-induced ARDS and to provide experimental support for histone-targeted treatment of sepsis-induced ARDS. Methods: First of all, in vitro experiments were conducted. Human umbilical vein endothelial cells (HUVEC) were stimulated with gradient concentrations of histones to explore for the optimal stimulation concentration in vitro. Then, HUVEC were exposed to histones at an optimal concentration with or without resatorvid (TAK-242), a selective inhibitor of Toll-like receptor 4 (TLR4), for 24 hours for modeling. The cells were divided into 4 groups: 1) the blank control group, 2) the blank control+TAK-242 intervention group, 3) the histone stimulation group, and 4) the histone+TAK-242 intervention group. HUVEC apoptosis was determined by flow cytometry, VE-Cadherin expression in endothelial cells was determined by Western blot, and the integrity of adhesion connections between endothelial cells was evaluated with confocal fluorescence microscopic images. Male C57BL/6 mice aged 6-8 weeks and weighing 22-25 g were used for the in vivo experiment. Then, the mice were given cecal ligation and puncture (CLP) as well as histone injection at 50 mg/kg via the tail vein for sepsis modeling. The experimental animals were divided into 6 groups: 1) the blank control group, 2) the blank control+TAK-242 intervention group, 3) the CLP model group, 4) the CLP+TAK-242 intervention group, 5) the histone model group, and 6) the histone+TAK-242 intervention group. After 24 h, the concentrations of serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined using ELISA kits. Western blot was performed to determine the expression of vascular endothelial (VE)-cadherin in the lung tissue. Hematoxylin and eosin (HE) staining was performed to observe the pathological changes in the lung tissue of the mice. Evans Blue was injected via the tail vein 30 min before the mice were sacrificed. Lung tissue was collected after the mice were sacrificed. Then, the concentrations of Evans blue dye per unit mass in the lung tissue from mice of different groups were evaluated, the rates of pulmonary endothelial leakage were calculated, and the integrity of the pulmonary endothelial barrier was evaluated. Results: The results of the in vitro experiment showed that, compared with those of the control group, HUVEC apoptosis was significantly increased under histone stimulation (P<0.05), the expression of VE-cadherin was decreased (P<0.05), and the integrity of adherens junctions between endothelial cells was damaged. TAK-242 can significantly inhibit histone-induced HUVEC apoptosis and VE-cadherin expression reduction and maintain the integrity of adherens junctions between endothelial cells. According to the findings from the in vivo experiments, in mice with CLP-induced and histone-induced sepsis, TAK-242 effectively alleviated the increase in serum concentrations of IL-6 and TNF-α, reduced the downregulation of VE-cadherin expression in the lung tissue (P<0.05), decreased endothelial permeability of the lung vessels, and improved pathological injury in the lung tissue. Conclusion: By binding to TLR-4, histone decreases VE-cadherin expression on the surface of vascular endothelial cells, disrupts the integrity of intercellular adherens junctions, and triggers pathological damage to lung tissue. Using TLR-4 inhibitors can prevent sepsis-induced ARDS in histone-induced sepsis.

Magnesium alleviates extracellular histone-induced apoptosis and defective bacterial phagocytosis in macrophages by regulating intracellular calcium signal.

Extracellular histones have been determined as important mediators of sepsis, which induce excessive inflammatory responses in macrophages and impair innate immunity. Magnesium (Mg2+), one of the essential nutrients of the human body, contributes to the proper regulation of immune function. However, no reports indicate whether extracellular histones affect survival and bacterial phagocytosis in macrophages and whether Mg2+ is protective against histone-induced macrophage damage. Our clinical data revealed a negative correlation between circulating histone and monocyte levels in septic patients, and in vitro experiments confirmed that histones induced mitochondria-associated apoptosis and defective bacterial phagocytosis in macrophages. Interestingly, our clinical data also indicated an association between lower serum Mg2+ levels and reduced monocyte levels in septic patients. Moreover, in vitro experiments demonstrated that Mg2+ attenuated histone-induced apoptosis and defective bacterial phagocytosis in macrophages through the PLC/IP3R/STIM-mediated calcium signaling pathway. Importantly, further animal experiments proved that Mg2+ significantly improved survival and attenuated histone-mediated lung injury and macrophage damage in histone-stimulated mice. Additionally, in a cecal ligation and puncture (CLP) + histone-induced injury mouse model, Mg2+ inhibited histone-mediated apoptosis and defective phagocytosis in macrophages and further reduced bacterial load. Overall, these results suggest that Mg2+ supplementation may be a promising treatment for extracellular histone-mediated macrophage damage in sepsis.

Macrophage-1 antigen exacerbates histone-induced acute lung injury and promotes neutrophil extracellular trap formation.

Acute lung injury (ALI), which occurs in association with sepsis, trauma, and coronavirus disease 2019 (COVID-19), is a serious clinical condition with high mortality. Excessive platelet-leukocyte aggregate (PLA) formation promotes neutrophil extracellular trap (NET) release and thrombosis, which are involved in various diseases, including ALI. Macrophage-1 antigen (Mac-1, CD11b/CD18), which is expressed on the surface of leukocytes, is known to promote NET formation. This study aimed to elucidate the role of Mac-1 in extracellular histone-induced ALI. Exogenous histones were administered to Mac-1-deficient mice and wild-type (WT) mice with or without neutrophil or platelet depletion, and several parameters were investigated 1 h after histone injection. Depletion of neutrophils or platelets improved survival time and macroscopic and microscopic properties of lung tissues, and decreased platelet-leukocyte formation and plasma myeloperoxidase levels. These improvements were also observed in Mac-1-/- mice. NET formation in Mac-1-/- bone marrow neutrophils (BMNs) was significantly lower than that in WT BMNs. In conclusion, our findings suggest that Mac-1 is associated with exacerbation of histone-induced ALI and the promotion of NET formation in the presence of activated platelets.

Extracellular histones exacerbate heat stroke-induced liver injury by triggering hepatocyte pyroptosis and liver injury via the TLR9-NLRP3 pathway.

BACKGROUND: Severe heat stroke is often complicated by multiple organ failure, including liver injury. Recent evidence indicates that the underlying mechanism constitutes sterile inflammation triggered by cell damage, in which hepatocyte NOD-like receptor family pyrin domain-containing 3 inflammasome activation and pyroptosis play key roles. As extracellular histones act as damage-associated molecular patterns and mediate tissue toxicity and inflammation, we aimed to investigate whether extracellular histones contribute to inducing hepatocyte pyroptosis following heat stroke, promoting the development of liver inflammation and injury, and elucidate the potential underlying mechanisms. METHODS: Exogenous histones were administered to AML-12 murine hepatocytes or male aged 8-12 week mice following hyperthermic treatment (at 39 °C in a chamber with 60 % relative humidity). Prior to heat exposure, endogenous histones were neutralized using neutralizing antibodies, inflammasomes were inhibited by RNA silencing, and Toll-like receptor 9 was modulated using a pharmacological agonist or antagonist. Inflammasome assembly, caspase-1 activation, histological changes, and liver enzyme levels were measured. Statistical comparison of more than two groups was performed using one-way ANOVA with Tukey's post-hoc testing. The correlations were analyzed using Pearson's correlation test. All experiments were repeated thrice. A p-value < 0.05 was considered significant. RESULTS: Heat stroke induced histone release into the extracellular space at levels correlating with liver injury. Moreover, extracellular histones augmented heat stroke-induced liver injury both in vitro and in vivo in a dose- and time-dependent manner, whereas neutralizing histones conferred protection following heat stroke. Histones mediated NOD-like receptor family pyrin domain-containing 3 inflammasome activation through the Toll-like receptor 9 signaling pathway, which resulted in hepatocyte pyroptosis and liver inflammation. CONCLUSIONS: Our findings show that histones are critical mediators of hepatocyte pyroptosis that aggravate liver injury in a heat stroke setting. Therefore, we suggest extracellular histones as potential therapeutic targets to limit heat stroke-induced cell death and liver injury.

Novel function of transglutaminase 2 in extracellular histone-induced acute lung injury.

Extracellular histones induce endothelial damage, resulting in lung haemorrhage; however, the underlying mechanism remains unclear. Factor XIII, as a Ca2+-dependent cross-linking enzyme in blood, mediates fibrin deposition. As another isozyme, transglutaminase 2 (TG2) has a catalytic activity distributing in most tissues. Herein, we investigated whether TG2 promotes fibrin deposition and mediates the adhesion of platelets to ECs in histone-induced acute lung injury (ALI). We evaluated the lung histology and the adhesion of platelets to endothelial cells (ECs) after injecting histones to wild-type (WT) C57BL/6J and TG2 knockout (TG2-/-) mice, and administered a TG2 inhibitor (NC9) to WT mice. Pulmonary haemorrhage was more severe in TG2-/- mice than that in WT mice. The area of fibrin deposition and the proportion of CD41+CD31+ cells were lower in TG2-/- mice than in WT mice. Pre-treatment of NC9 decreased the area of fibrin deposition and the proportion of CD41+CD31+ cells in WT mice. These results suggest that TG2 prevents from pulmonary haemorrhage in ALI by promoting the adhesion of platelets to ECs and the fibrin deposition.

The Role of Histones and Heparin in Sepsis: A Review.

Septic shock with multiple organ failure is a devastating situation in clinical settings. Through the past decades, much progress has been made in the management of sepsis and its underlying pathogenesis, but a highly effective therapeutic has not been developed. Recently, macromolecules such as histones have been targeted in the treatment of sepsis. Histones primarily function as chromosomal organizers to pack DNA and regulate its transcription through epigenetic mechanisms. However, a growing body of research has shown that histone family members can also exert cellular toxicity once they relocate from the nucleus into the extracellular space. Heparin, a commonly used anti-coagulant, has been shown to possess life-saving capabilities for septic patients, but the potential interplay between heparin and extracellular histones has not been investigated. In this review, we summarize the pathogenic roles of extracellular histones and the therapeutic roles of heparin in the development and management of sepsis and septic shock.

Expression of a Crry/p65 is reduced in acute lung injury induced by extracellular histones.

Acute lung injury (ALI) occurs in patients with severe sepsis and has a mortality rate of 40%-60%. Severe sepsis promotes the release of histones from dying cells, which can induce platelet aggregation, activate coagulation and cause endothelial cell (EC) death. We previously reported that the expression of membrane complement receptor type 1-related gene Y (Crry)/p65, which plays a principal role in defence against abnormal activation of complement in the blood, is reduced in response to peritoneal mesothelial cell injury, and we hence hypothesized that a similar mechanism occurs in pulmonary ECs. In this study, we examined the role of Crry/p65 in histone-mediated ALI using an experimental animal model. In ALI model mice, exposure to extracellular histones induces lung injury and results in a decrease in Crry/p65 expression. The levels of lactic acid dehydrogenase (LDH), a marker of cell damage, were significantly increased in the serum of ALI model compared with vehicle mice. The significant inverse correlation between the expression of Crry/p65 and LDH levels in plasma revealed an association between Crry/p65 expression and cell damage. The levels of complement component 3a (C3a) were also significantly increased in the serum of the ALI model compared with vehicle mice. Notably, a C3a receptor antagonist ameliorated lung injury induced by histones. We hypothesize that extracellular histones induce complement activation via down-regulation of Crry/p65 and that C3a might serve as a therapeutic target for the treatment of ALI.

The relationship of Serum Histone H3.3 and H4 with chronic Hepatitis B.

OBJECTIVE: To determine the role of serum histone H3.3 and H4 in patients with chronic hepatitis B to explore any relationship between the two. METHODS: The prospective controlled clinical pilot study was conducted in the Gastroenterology Clinic of Bezmialem Vakif University, Istanbul, Turkey, from January to October 2017, and comprised biopsy-proven patients with chronic hepatitis B and healthy controls. Demographics, hepatitis B virus deoxyribonucleic acid quantity, hepatitis B e-antigen, aspartate aminotransferase, alanine transaminase, international normalized ratio, total/direct bilirubin, albumin and thrombocyte counts as well as histological activity index and fibrosis scores were noted. Data was analysed using SPSS 22. RESULTS: Of the 140 subjects, 70(50%) each were cases and controls. The overall mean age of the sample was 43.38±15.07 years (range: 18-70 years). There was positive correlation of histone H3.3 with hepatitis B virus deoxyribonucleic acid, aspartate aminotransferase, alanine transaminase and international normalized ratio levels. Histone H4 levels only correlated with hepatitis B virus deoxyribonucleic acid and international normalized ratio. Hepatitis B e-antigen positivity was present in 14(20%) of the cases. CONCLUSIONS: Histone H3.3 levels appeared to be associated with pathophysiological changes in chronic hepatitis B patients, suggesting that future treatments should target H3.3.

Extracellular histones induce calcium signals in the endothelium of resistance-sized mesenteric arteries and cause loss of endothelium-dependent dilation.

Histone proteins are elevated in the circulation after traumatic injury owing to cellular lysis and release from neutrophils. Elevated circulating histones in trauma contribute to coagulopathy and mortality through a mechanism suspected to involve endothelial cell (EC) dysfunction. However, the functional consequences of histone exposure on intact blood vessels are unknown. Here, we sought to understand the effects of clinically relevant concentrations of histones on the endothelium in intact, resistance-sized, mesenteric arteries (MAs). EC Ca2+ was measured with high spatial and temporal resolution in MAs from mice selectively expressing the EC-specific, genetically encoded ratiometric Ca2+ indicator, Cx40-GCaMP-GR, and vessel diameter was measured by edge detection. Application of purified histone protein directly to the endothelium of en face mouse and human MA preparations produced large Ca2+ signals that spread within and between ECs. Surprisingly, luminal application of histones had no effect on the diameter of pressurized arteries. Instead, after prolonged exposure (30 min), it reduced dilations to endothelium-dependent vasodilators and ultimately caused death of ~25% of ECs, as evidenced by markedly elevated cytosolic Ca2+ levels (793 ± 75 nM) and uptake of propidium iodide. Removal of extracellular Ca2+ but not depletion of intracellular Ca2+ stores prevented histone-induced Ca2+ signals. Histone-induced signals were not suppressed by transient receptor potential vanilloid 4 (TRPV4) channel inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels or Toll-like receptor receptors. These data demonstrate that histones are robust activators of noncanonical EC Ca2+ signaling, which cause vascular dysfunction through loss of endothelium-dependent dilation in resistance-sized MAs. NEW & NOTEWORTHY We describe the first use of the endothelial cell (EC)-specific, ratiometric, genetically encoded Ca2+ indicator, Cx40-GCaMP-GR, to study the effect of histone proteins on EC Ca2+ signaling. We found that histones induce an influx of Ca2+ in ECs that does not cause vasodilation but instead causes Ca2+ overload, EC death, and vascular dysfunction in the form of lost endothelium-dependent dilation.

Extracellular Histone Promotes Prostate Cancer Migration and Epithelial-Mesenchymal Transition through NF-κB-Mediated Inflammatory Responses.

INTRODUCTION: This study aims to explore the relationship betweenextracellular histone and prostate cancer and its mechanism. METHODS: Migration of prostate cancer cells was detected by Transwell. Inflammatory factor expression was investigated by ELISA. Epithelial-mesenchymal transition and expression of NF-κB pathway-related proteins were investigated using Western blotting. RESULTS: Under the induction of extracellular histones, the migration rate of prostate cancer cells and the levels of IL-1ß, TNF-α, and IL-6 were notably enhanced. Then, expression of E-cadherin was significantly down-regulated, while levels of N-cadherin, vimentin, ß-catenin, Snail, p-p65 and p-IκBα were significantly up-regulated, which was reversed by PDTC (pyrrolidine dithiocarbamate). CONCLUSION: Extracellular histone significantly promotes the progression of prostate cancer cells via NF-κB pathway-mediated inflammatory responses, which may serve as a novel target for treating prostate cancer.

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.

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.