OBJECTIVE: To investigate the distribution characteristics of polymorphonuclear neutrophil (PMN) in the lungs during the early stage of severe burns and the mechanism of neutrophil elastase (NE) promoting lung injury. METHODS: 6-8-week-old male C57BL/6J mice were selected for the experiments. A 30% total body surface area (TBSA) III degree burn mouse model was established (severe burn group); the Sham-injury group was treated with 37 centigrade water. In the sodium sivelestat intervention group (SV intervention group), NE competitive inhibitor, sivelestat, 100 mg/kg, was injected via tail vein immediately after injury, while other groups received an equal volume of saline. Ten mice were harvested from each group to observe survival for 72 hours. Respiratory function tests were tested at 0 (immediate), 3, 6, 12, and 24 hours after molding. hematoxylin-eosin (HE) and immunohistochemical staining were used to observe lung tissue structure, inflammatory changes and PMN infiltration. The PMN absolute count in mice lung tissue was detected buy flow cytometry. At 6, 12, and 24 hours after molding, PMN counts and the concentration of NE [enzyme linked immunosorbent assay (ELISA)] in peripheral blood plasma, lung tissue, and bronchoalveolar lavage fluid (BALF) were detected. RESULTS: (1) HE staining results showed that compared with the Sham-injury group, the lungs of mice in the severe burn group showed inflammatory changes and PMN infiltration, with more significant changes at 6 hours. Immunohistochemistry results also confirmed that the expression of NE protein released from PMN significantly increased after 6 hours of severe burn injury [(3.79±0.62)% vs. (0.18±0.05)%, t = 11.56, P < 0.01]. (2) Compared with the Sham-injury group, the number of PMN and the concentration of NE in the peripheral blood and lung tissues in the severe burn group were significantly increased (F values were 13.709, 55.350 and 29.890, 13.286, respectively, all P < 0.01), peaking at 6 hours [plasma PMN count (×10⁹/L): 2.92±1.01 vs. 0.92±0.29, lung tissue PMN absolute count (cells): 48 788.03±11 833.91 vs. 1 516.72±415.35, plasma NE (ng/L): 24 522.71±3 842.92 vs. 7 009.34±4 067.86, lung tissue NE (ng/L): 262 189.04±9 695.13 vs. 65 026.03± 16 016.31, all P < 0.01]. The number of PMN in the lung of severely burned mice was highly correlated with NE concentration (r = 0.892, P < 0.001). There was no significantly difference in the PMN absolute count in the BALF of mice between the Sham-injury group and severe burn group (F = 1.403, P > 0.05). The Sham-injury group and severe burn group contained a small amount of NE in the BALF, and the concentration of NE in the BALF of the severely burned 6 hours and 12 hours groups were significantly higher than those of the Sham-injury group (ng/L: 328.58±158.10, 415.30±240.89 vs. 61.95±15.80, both P < 0.05). (3) Kaplan-Meier survival curve showed that the 72-hour survival rate of mice in the SV intervention group was significantly higher than that in the severe burn group (100% vs. 10%, Log-Rank test: χ² = 19.12, P < 0.001). (4) Compared with the Sham-injury group, all lung function indices of the severe burn group decreased significantly. All lung function indices of SV intervention group improved gradually over time, which were significantly better than those of the severe burn group. (5) Compared with the Sham-injury group, the PMN absolute count in lung tissue and the concentration of NE in plasma and lung tissue were significantly higher in the SV intervention group (F values were 46.709, 3.535, 32.701, respectively, all P < 0.05), with a peak at 6 hours. Compared with the severe burn group, the SV intervention group had a higher PMN absolute count in lung tissue (cells: 8 870.80±7 013.89 vs. 25 974.92±22 240.8, P < 0.05), and higher plasma and lung tissue NE concentrations (ng/L: 14 955.94±3 944.41 vs. 21 972.75±4 573.05, 81 956.87±38 658.35 vs. 168 182.30±83 513.91, both P < 0.01) were significantly decreased. CONCLUSIONS In the early stage of severe burns, there is a significant infiltration of PMN into the lungs. The NE promotes lung injury in the early stage of severe burn, and improve lung injury by inhibiting the action of NE.
Animals ; Burns/metabolism* ; Leukocyte Elastase/metabolism* ; Male ; Mice, Inbred C57BL ; Mice ; Neutrophils/metabolism* ; Lung/metabolism* ; Disease Models, Animal ; Neutrophil Infiltration ; Lung Injury/metabolism* ; Glycine/analogs & derivatives* ; Sulfonamides

Sepsis is the most common complication of severe burns and a primary risk factor for endangering the lives of critically severe burn patients. The mortality rate of burn sepsis patients was up to 75%. Neutrophils are one of the innate immune cells and are the earliest and most recruited immune cells at the site of infection, which play a key role in the removal of local infection and the repair of damaged tissue. Many studies have deeply revealed the pathogenesis and progression mechanism of burn sepsis, in which the role of neutrophils has gradually become clear. This paper elaborated on the key mechanisms of neutrophils in the occurrence and evolution of burn sepsis, explored the value of neutrophils in the early warning and prognosis judgement, and the potential therapeutic methods of burn sepsis based on the unique biological behavior of neutrophils in burn sepsis.

Objective:To investigate the regulatory effects and mechanism of Nocardia rubra cell wall skeleton (Nr-CWS) on the biological function of human neutrophils. Methods:The experimental research method was used. Fifteen healthy adult volunteers (7 males and 8 females, aged 24 to 45 years) were recruited from Suzhou Physical Examination Center for physical examination from May to October 2022, the peripheral venous blood was collected, and neutrophils were extracted by immunomagnetic bead sorting. The cells were divided into normal control group without any treatment, Nr-CWS alone group treated with Nr-CWS of final mass concentration 60 ng/mL alone, endotoxin/lipopolysaccharide (LPS) alone group stimulated with LPS of final mass concentration 1 μg/mL alone, and LPS+Nr-CWS group stimulated with LPS first and then treated with Nr-CWS as before. After 1 h of culture, the chemotaxis distance, chemotactic cell percentage, chemotactic index, maximum chemotactic speed, and chemotactic function score of neutrophils were detected using the modified agarose chemotactic model; the proportion and fluorescence intensity of phagocytosis cells, the level of reactive oxygen species (ROS), the protein expression levels of granular protein CD35, CD66b, and CD63, and the concentrations of inflammatory cytokines of interleukin 2 (IL-2), IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor alpha (TNF-α), and interferon-γ in cell culture supernatant were detected by flow cytometry. The number of samples in each group in the above experiments was 15. Data were statistically analyzed with analysis of variance for factorial design and independent sample t test. Results:After 1 h of culture, the chemotactic function score of cells in normal control group, Nr-CWS alone group, LPS alone group, and LPS+Nr-CWS group were 15.0, 14.5±0.5, 1.5±0.5, 12.0±1.5, respectively. Compared with those in normal control group, the chemotaxis distance, chemotactic cell percentage, chemotactic index, maximum chemotactic speed, and chemotactic function score of cells were significantly decreased in LPS alone group and LPS+Nr-CWS group (with t values of 18.36, 18.88, 54.28, 18.36, 46.77, 10.58, 14.74, 6.84, 10.58, and 4.24, respectively, P<0.05); compared with those in LPS alone group, the five chemotactic function indexes as above in LPS+Nr-CWS group were significantly increased (with t values of 11.47, 14.65, 11.62, 11.47, and 13.75, respectively, P<0.05). After 1 h of culture, compared with those in normal control group, the proportion and fluorescence intensity of phagocytosis cells were significantly increased in Nr-CWS alone group (with t values of 6.86 and 6.73, respectively, P<0.05), and the above two indexes were significantly decreased in LPS alone group (with t values of 7.35 and 22.72, respectively, P<0.05) and LPS+Nr-CWS group (with t values of 21.37 and 13.10, respectively, P<0.05). After 1 h of culture, compared with that in normal control group, the level of ROS of cells in LPS alone group was significantly increased ( t=6.64, P<0.05); compared with that in LPS alone group, the level of ROS of cells in LPS+Nr-CWS group was significantly decreased ( t=5.46, P<0.05). After 1 h of culture, compared with those in normal control group, the protein expressions of CD35, CD66b, and CD63 of cells were significantly increased in LPS alone group and LPS+Nr-CWS group (with t values of 16.75, 17.45, 10.82, 5.70, 19.35, and 15.37, respectively, P<0.05); compared with those in LPS alone group, the protein expressions of CD35, CD66b, and CD63 of cells were significantly decreased in LPS+Nr-CWS group (with t values of 4.92, 5.72, and 3.18, respectively, P<0.05). After 1 h of culture, compared with those in normal control group, the concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly increased in LPS alone group (with t values of 22.10, 9.50, 7.21, 10.22, 24.88, 8.43, and 47.48, respectively, P<0.05), and the concentrations of IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly increased in LPS+Nr-CWS group (with t values of 4.68, 5.12, 8.02, 5.58, and 7.13, respectively, P<0.05); compared with those in LPS alone group, the concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly decreased in LPS+Nr-CWS group (with t values of 5.39, 2.83, 5.79, 2.90, 5.87, 4.88, and 39.64, respectively, P<0.05). Conclusions:Nr-CWS can enhance the phagocytosis ability of neutrophils in normal condition and improve the chemotactic function, ROS level, degranulation protein level, and inflammatory factor level of human neutrophils in infectious condition. Nr-CWS can enhance the anti-infection ability of human neutrophils by regulating its biological behavior in innate immunity.

Objective:To investigate the changes and clinical significance of multiple cytokine levels in exhaled breath condensate (EBC) in patients undergoing tracheotomy with severe inhalation injury.Methods:A prospective study was conducted. A total of 32 patients with severe burn combined with severe inhalation injury admitted to the department of burns and plastic surgery of Affiliated Suzhou Hospital of Nanjing Medical University from May 2021 to August 2022 were enrolled. Twenty healthy volunteers from the same period were served as controls. EBC of patients at 12 hours after burn and the samples of healthy controls were collected. The levels of 27 cytokines in EBC, including tumor necrosis factor-α (TNF-α) and interleukins (IL-1β, IL-6, IL-8, IL-10, and IL-17), were determined by liquid phase chip technology. Meanwhile, plasma of patients at 12 hours after burn and the plasma of volunteers were collected, and the levels of inflammatory cytokines were detected by liquid chip technology, and the differences between the levels in plasma and those in EBC were analyzed. Plasma and EBC of patients with aspiration injury were collected at 12 hours and 3, 7, 14 and 21 days after burn, and TNF-α levels were determined by enzyme-linked immunosorbent assay (ELISA).Results:Finally, 32 patients were enrolled, and the total burned area was (40±16)% of total body surface area (TBSA). The time of admission was (4.2±2.3) hours after injury. ① Twenty-seven cytokines in EBC: 18 kinds of cytokines including macrophage inflammatory protein-1β (MIP-1β), IL-6, IL-5, IL-2, IL-1β, IL-8, IL-10, IL-15, IL-9, interferon-γ (IFN-γ), IL-1 receptor antagonist (IL-1ra), TNF-α, chemotactic factor for eosinophil (Eotaxin), basic fibroblast growth factor (bFGF), platelet derived growth factor-BB (PDGF-BB), interferon-inducible protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), granulocyte colony-stimulating factor (G-CSF) were significantly increased in patients with severe aspiration injury compared with health controls. Eotaxin was not detected in EBC of healthy controls. Five cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine ligand 5 (CCL5/RANTES), IL-13, IL-4 and MIP-1α, were not detected in EBC of severe inhalation injury patients and healthy controls. Vascular endothelial growth factor (VEGF) and IL-12 p70 in EBC of severe aspiration injury patients were slightly decreased as compared with healthy controls, while IL-7 and IL-17 were slightly increased, but the differences were not statistically significant. ② Six inflammatory cytokines in plasma: the levels of IL-6 and IL-8 in the severe aspiration injury group were significantly increased as compared with healthy controls [IL-6 (ng/L): 18.51 (10.87, 26.21) vs. 0.22 (0.10, 0.36), IL-8 (ng/L): 10.75 (8.58, 18.79) vs. 1.06 (0.81, 2.14), both P < 0.01]. The plasma levels of TNF-α, IL-1β and IL-10 were slightly increased in patients with severe aspiration injury as compared with healthy controls, and IL-17 was slightly decreased, but the difference was not statistically significant. In the EBC collected during the same period, five inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-8 and IL-10, in patients with severe inhalation injury were significantly increased as compared with healthy controls [TNF-α (ng/L): 16.42 (12.57, 19.21) vs. 7.34 (6.11, 8.69), IL-1β (ng/L): 15.57 (10.53, 20.25) vs. 0.99 (0.67, 1.41), IL-6 (ng/L): 13.36 (9.76, 16.54) vs. 0.70 (0.42, 0.85), IL-8 (ng/L): 1 059.29 (906.91, 1 462.37) vs. 10.36 (8.40, 12.37), IL-10 (ng/L): 2.69 (1.54, 3.33) vs. 1.54 (1.18, 2.06), all P < 0.05]. ③ Dynamic changes of TNF-α in plasma and EBC: the level of TNF-α in EBC of patients with severe aspiration injury was lower than that in plasma. Plasma TNF-α level was increased gradually with the extension of time after injury, and was significantly higher than that of healthy controls on day 3 [ng/L: 30.38 (24.32, 39.19) vs. 22.94 (17.15, 30.74), P < 0.05], and reached the peak on day 14, then fell back. The level of TNF-α in EBC at 12 hours after injury was significantly higher than that in healthy controls [ng/L: 15.34 (11.75, 18.14) vs. 6.99 (6.53, 7.84), P < 0.01], and reached the peak on 3 days after injury, and then gradually decreased. Conclusion:There are changes in the expression of multiple cytokines in EBC of patients with severe inhalation injury, and the changes of many inflammatory cytokines including TNF-α are more sensitive than those in plasma, which can be used to monitor and evaluate the condition of patients with inhalation injury.

Objective:To analyze the changes of intestinal microflora and to predict the metabolic function of intestinal microflora in severe burn patients at early stage by 16S ribosomal RNA ( rRNA) high-throughput sequencing. Methods:In this prospective observational study, 48 patients with severe burns who met the inclusion criteria were admitted to Department of Burns and Plastic Surgery of Affiliated Hospital of Jiangsu University from January 2018 to December 2019 were included in burn group, and 40 healthy volunteers who met the inclusion criteria and underwent physical examination at the Physical Examination Center of Affiliated Hospital of Jiangsu University in the same period were included in healthy group. Fecal samples were collected from patients in burn group in about 1 week after admission and from volunteers in healthy group on the day of physical examination. The 16S rRNA V4 gene sequencing was performed in the feces of patients in burn group and volunteers in healthy group to analyze the relative abundance of various bacteria. The operational classification unit (OTU) was divided by Mothur software to analyze the dominant bacteria. The OTU number, Chao1 index, Ace index, and Shannon index of fecal microflora were analyzed by QIIME1.9.0 software. The principal component analysis for relative abundance of fecal microflora was performed by Canoco Software 5.0. The metabolic function of fecal microflora was predicted by Kyoto Encyclopedia of Genes and Genomes. Data were statistically analyzed with independent sample t test, and Mann-Whitney U test, and Bonferroni correction. Results:The relative abundance of Bacteroides, Enterococcus, Acinetobacter, Macrococcus, and Staphylococcus in feces of patients in burn group was significantly higher than that of volunteers in healthy group ( Z=-5.20, -2.37, -5.17, -4.41, -6.03, P<0.05 or P<0.01), and the relative abundance of unclassified-Helicobacillae, Prevotella, Cecobacteria, unclassified-Rumencocci, Pseudobutyrivibrio, Brautia, and unclassified-Digiestive Streptococcaceae ( Z=-8.03, -3.21, -7.63, -5.88, -8.05, -8.05, -6.77, P<0.01) and other 12 species of bacteria in the feces of volunteers in healthy group was significantly higher than that of patients in burn group. The diversity of fecal microflora of volunteers in healthy group was better than that of patients in burn group, the main dominant microflora of volunteers in healthy group were Bacteroides, unclassified- Helicobacillae, Prevotella, unclassified- Enterobacteriaceae, Brautia, Parabacteroides, Escherichia coli, etc., and the main dominant microflora of patients in burn group were Bacteroides, Prevotella, unclassified-Enterobacteriaceae, and Parabacteroides. The OTU number, Ace index, Chao1 index, and Shannon index of fecal microflora of patients in burn group were 149±47, 199±45, 190±45, 2.0±0.9, which were significantly lower than 266±57, 323±51, 318±51, 3.8±0.5 of volunteers in healthy group ( t=10.325, 11.972, 12.224, 11.662, P<0.01). The relative abundance of fecal microflora of patients in burn group and volunteers in healthy group was clearly divided into two groups by principal component 1, and the contribution rate of principal component 1 was 32.50%, P<0.01. The fecal microflora of volunteers in healthy group were more concentrated on principal component 2, the fecal microflora of patients in burn group were dispersed in principal component 2, and the contribution rate of principal component 2 was 13.44%, P>0.05. The metabolic levels of alanine-aspartate-glutamate, arginine- proline, cysteine-methionine, glycine-serine-threonine, phenylalanine, tryptophan, and tyrosine in amino acid, tricarboxylic acid cycle, glucose and mannose, galactolipin, glycolysis/gluconiogenesis, starch and sucrose in carbohydrate of fecal microflora of patients in burn group were significantly lower than those of volunteers in healthy group ( Z=-4.75, -4.54, -4.75, -4.62, -3.71, -3.28, -4.19, -3.82, -4.72, -4.35, -4.75, -4.71, P<0.01). The levels of lipoic acid metabolism and coenzyme Q synthesis of fecal microflora of patients in burn group were significantly higher than those of volunteers in healthy group ( Z=-6.07, -4.51, P<0.01). The metabolic level of arachidonic acid of fecal microflora of patients in burn group was similar to that of volunteers in healthy group ( P>0.05). Conclusions:There are significant differences in intestinal microflora between severe burn patients at the early stage and healthy people, and the species and diversity of microflora are decreased, and the nutrient metabolism level is decreased in burn patients by 16S rRNA high-throughput sequencing.

Objective:To investigate the effect of neutrophils on T lymphocyte function in septic mice and the role of CD80/cytotoxic T lymphocyte antigen-4 (CTLA-4) signaling pathway in this modulated effects.Methods:① In vivo experiment: 6-8 weeks old male C57BL/6 mice were divided into sham operation group (Sham group, n = 20), Sham+CTLA-4 antibody treatment group (Sham+aCTLA-4 group, n = 20), cecal ligation and perforation (CLP) induced sepsis model group (CLP group, n = 30) and CLP+CTLA-4 antibody treatment group (CLP+aCTLA-4 group, n = 30) according to the random number table. CLP was used to reproduce mouse sepsis model. The mice in the Sham group were treated identically but their cecums were neither punctured nor ligated. In CTLA-4 antibody treatment groups, 50 μg CTLA-4 antibody was injected intraperitoneally 6 hours and 24 hours after the operation. Forty-eight hours after operation, 6 mice in Sham group and Sham+aCTLA-4 group, 14 mice in CLP group and CLP+aCTLA-4 group were randomly selected to detect the expression of CD69 in spleen. At the same time, spleen, bone marrow and peripheral blood were collected, and the expression of CD80 on neutrophils was detected by flow cytometry. The expression of CTLA-4 on the surface of T lymphocytes in spleen was detected by immunofluorescence and flow cytometry. The remaining mice in each group were used to observe the 96-hour survival after operation.② In vitro experiment 1: neutrophils were extracted from bone marrow of healthy mice and stimulated with LPS (1 mg/L) for 4, 8 and 12 hours respectively. The control group was added with the same amount of phosphate buffered saline (PBS) at each time point, and the expression of CD80 was detected at each time point.③ In vitro experiment 2: splenic T lymphocytes of healthy mice were extracted and divided into PBS control group, LPS group (final concentration of LPS 1 mg/L), neutrophil group and neutrophil+LPS group. In the latter two groups, the co-culture model of neutrophils and T lymphocytes was established, and then the corresponding treatment was given to detect the expression of CTLA-4 on the surface of T lymphocytes. With the above four groups as controls, CTLA-4 antibody treatment groups (final concentration of CTLA-4 antibody 50 mg/L) were set up respectively. After 48 hours, the level of interleukin-2 (IL-2) in the cell supernatant was detected by enzyme linked immunosorbent assay (ELISA). Results:① Results of in vivo experiment: compared with Sham group, the expression of CD80 on neutrophils in spleen, bone marrow and peripheral blood was significantly up-regulated, while the expression of CTLA-4 on the surface of T lymphocytes was significantly increased [(9.98±0.84)% vs. (3.48±0.64)%, P < 0.05]. It suggested that neutrophils may affect T lymphocytes function through CD80/CTLA-4 pathway in sepsis. Compared with CLP group, CTLA-4 antibody could significantly improve the 96-hour cumulative survival rate of CLP mice (56.25% vs. 18.75%, P < 0.05), and increase the expression of CD69 on the surface of T lymphocytes. It suggested that CTLA-4 antibodies might increase T lymphocytes activation in sepsis and improve survival. ② Results of in vitro experiment: with the prolongation of LPS stimulation, the expression of CD80 on neutrophils gradually increased in time-dependent manner as compared with PBS control group [4 hours: (6.35±0.40)% vs. (3.41±0.40)%, 8 hours: (8.57±0.64)% vs. (3.09±0.27)%, 12 hours: (19.83±1.06)% vs. (5.16±0.36)%, all P < 0.05]. Compared with PBS control group, the expression of CTLA-4 on CD4 +/CD8 + T lymphocytes was not significantly affected by LPS stimulation alone, but CTLA-4 was increased after co-culture with neutrophils [CD4 +: (4.92±0.30)% vs. (3.33±0.25)%, CD8 +: (4.26±0.21)% vs. (2.53±0.66)%, both P < 0.05], and the increased trend of CTLA-4 was more obvious after co-culture with LPS-stimulated neutrophils [CD4 +: (6.34±0.50)% vs. (3.33±0.25)%, CD8 +: (6.21±0.41)% vs. (2.53±0.66)%, both P < 0.05]. In the PBS control group and LPS group, CTLA-4 antibody had no significant effect on IL-2 secretion of T lymphocytes. Compared with PBS control group, co-culture with neutrophils could inhibit the secretion of IL-2 by T lymphocytes (ng/L: 1 938.00±68.45 vs. 2 547.00±218.00, P < 0.05), and the inhibitory effect of neutrophils stimulated by LPS was more obvious (ng/L: 1 073.00±34.39 vs. 2 547.00±218.00, P < 0.05). CTLA-4 antibodies could partially restore IL-2 secretion. In conclusion, after promoting the expression of CTLA-4 on the surface of T lymphocytes, neutrophils might mediate the inhibition of T lymphocytes function by reducing the production of IL-2. Conclusions:Neutrophils mediate T lymphocytes dysfunction in sepsis, and the CD80/CTLA-4 pathway plays an important role. The CTLA-4 antibody improves survival and T lymphocytes function in sepsis mice, which may be a new method of immunotherapy for sepsis.

Objective:To investigate the effect of heparin-binding protein (HBP) on the damage of vascular permeability in early burn.Methods:① Clinical research: 12 patients with severe burns admitted to Suzhou Hospital of Nanjing Medical University from January 1st to August 30th in 2019 were enrolled. Eight patients with severe trauma admitted to the same hospital during the same period were also enrolled as controls to explain the specificity of burn injury. Whole blood samples were obtained within 0.5 hour after admission. The white blood cell count (WBC), absolute value and ratio of neutrophils, and serum HBP levels were measured. Serum samples of 12 patients with severe burn were collected within 9 days after admission, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of metabolism products of glycocalyx including syndecan-1 and hyaluronic acid (HA). The correlation between HBP and neutrophils ratio, syndecan-1 and HA were analyzed by linear correlation. ② Basic research: a 30% total body surface area (TBSA) Ⅲ° burn model of Sprague-Dawley (SD) rat aged 6-8 weeks was prepared. In low molecular weight heparin (LMWH) intervention group ( n = 5), 200 U/kg LMWH was injected subcutaneously immediately and every 2 hours after injury for 4 times in total; the burn group ( n = 5) was given the same amount of normal saline. No intervention was given to the normal control group ( n = 5). The peripheral venous blood was collected at 0, 2, 4, and 8 hours after injury, and the serum levels of HBP, syndecan-1 and HA were measured; the injury of glycocalyx on pulmonary vascular endothelial cells was observed under transmission electron microscope. Results:① Clinical research results: the WBC, neutrophils absolute value and ratio, and HBP levels were increased in 12 patients with severe burn and 8 patients with severe trauma. There was no significant difference in the WBC, absolute value and ratio of neutrophils between severe burn and severe trauma patients [WBC (×10 9/L): 14.5±6.1 vs. 10.8±3.6, the absolute value of neutrophils (×10 9/L): 12.0±5.9 vs. 9.0±4.0, the ratio of neutrophils: 0.81±0.10 vs. 0.79±0.14, all P > 0.05], but the HBP levels in the burn patients were significantly higher than those in the trauma patients (μg/L: 192.92±61.73 vs. 51.17±23.05, P < 0.01). Twelve patients with severe burns had a sharp increase in serum syndecan-1 and HA levels after burns, which continued to maintain high levels and peaked at the 9th day [syndecan-1 (μg/L): 16.02±0.39, HA (μg/L): 106.83±4.90]. The analysis showed that HBP was positively correlated with neutrophils ratio, syndecan-1 and HA in severe burn patients at the 1st day after admission ( r values were 0.805, 0.732 and 0.900, respectively, all P < 0.01). It indicated that the sharp increase of neutrophils after the burn released a lot of HBP, and the glycocalyx of the vascular endothelium was severely damaged. ② Basic research results: the levels of serum HBP, syndecan-1 and HA in the burn group were increased sharply as compared with the normal control group, and continued to increase with time, reaching a peak at 8 hours after burn. In the LMWH intervention group, the serum levels of HBP, syndecan-1 and HA were significantly lower than those in the burn group, and the difference was still statistically significant after 8 hours [HBP (μg/L): 6.47±0.25 vs. 12.48±0.08, syndecan-1 (μg/L): 19.06±1.48 vs. 25.92±3.34, HA (μg/L): 35.76±2.10 vs. 54.91±2.64, all P < 0.01]. The results of transmission electron microscopy showed that in the normal control group, the glycocalyx pulmonary vascular endothelial cells was continuous, evenly distributed and dense. The glycocalyx on pulmonary vascular endothelial cells of rats were significantly damaged and shed 2 hours after burn in the burn group, and no glycocalyx was observed at 8 hours. In the LMWH intervention group, the glycocalyx on pulmonary vascular endothelial cells was damaged and the phenomenon of shedding was significantly relieved, and the glycocalyx could be observed 8 hours after the injury. Conclusion:The massive exudation of body fluids and the significant increase of vascular permeability in patients in early burns may be related to the destruction of the glycocalyx on endothelial cells by HBP released from increased neutrophils.

Objective:To observe the early changes of chemotactic function of peripheral blood neutrophil of patients with severe burns and the influence factor.Methods:Seven severe burn patients who met the inclusion criteria and were admitted to Suzhou Hospital Affiliated to Nanjing Medical University in 6 hours post burns from January to May 2019 were selected and included in burn group (4 males and 3 females, aged (36±10) years). Seven healthy volunteers with normal physical examination results in the Physical Examination Center of the same hospital in the same period of time were included in healthy control group (5 males and 2 females, aged (35±8) years). A prospective and controlled study was performed. (1) The venous blood of 2 mL was taken from each patient in burn group on post admission day (PAD) 1, 3, 5 and venous blood of 2 mL was taken from each volunteer in healthy control group for routine detection of white blood cell count, platelet count, neutrophil count, serum procalcitonin level, and C-reactive protein level. (2) The venous blood of patients and healthy volunteers was taken as before for measuring interleukin-6 (IL-6), IL-10, and tumor necrosis factor α (TNF-α) by enzyme-linked immunosorbent assay. (3) The venous blood of patients and healthy volunteers was taken as before, and peripheral blood neutrophils were isolated by Ficoll density gradient centrifugation. The chemotactic distance of neutrophil was detected by agarose chemotaxis test, and the positive expression rates of chemokine receptor CXCR1 and CXCR2 of patients in burn group on PAD 3 and volunteers in healthy control group were detected by flow cytometer. Data were statistically analysed with analysis of variance for repeated measurement, t test, and Bonferroni correction. Results:(1) The platelet count of patients in burn group on PAD 1, 3, 5 was close to that of volunteers in healthy control group respectively ( t=0.55, 0.44, 0.12, P>0.05). The counts of neutrophil and white blood cell and the expression levels of serum procalcitonin and C-reactive protein of patients in burn group on PAD 1, 3, 5 were significantly higher than those of volunteers in healthy control group ( t=196.96, 273.31, 45.22, 3.46, 4.18, 5.55, 4.36, 5.26, 11.13, 64.94, 89.97, 84.31, P<0.01). (2) The level of IL-6 of patients in burn group on PAD 1, 3, 5 was significantly higher than that of volunteers in healthy control group respectively ( t=187.43, 213.54, 195.74, P<0.01), the level of IL-10 of patients in burn group on PAD 1, 3, 5 was significantly higher than that of volunteers in healthy control group respectively ( t=21.47, 11.13, 6.23, P<0.01), and the level of TNF-α of patients in burn group on PAD 1, 3, 5 was significantly higher than that of volunteers in healthy control group respectively ( t=5.27, 7.89, 15.58, P<0.01). (3) The chemotactic distances of neutrophil of patients in burn group were (1 479±102), (1 395±82), and (1 017±91) μm respectively on PAD 1, 3, 5, which were significantly shorter than (1 902±120) μm of volunteers in healthy control group ( t=7.11, 9.23, 15.55, P<0.01). (4) The CXCR1 and CXCR2 positive expression rates of neutrophil of patients in burn group on PAD 3 were (48.3±1.6)% and (79.0±1.8)%, respectively, which were significantly lower than (95.4±4.5)% and (97.8±2.1)% of volunteers in healthy control group ( t=27.13, 23.10, P<0.01). Conclusions:The chemotactic dysfunction of peripheral blood neutrophil was detected in the early stage of severe burn patients, which may be related to the decreases of CXCR1 and CXCR2.

OBJECTIVE: To investigate the effect and mechanism of autophagy on the expression of neutrophil programmed death ligand-1 (PD-L1) in mice with sepsis. METHODS: (1) In vivo experiment: male C57BL/6 mice aged 6-8 weeks were divided into sham operation group (Sham group), cecum ligation and perforation (CLP) group, and rapamycin (RAP)+CLP group by random number table with 10 mice in each group. The sepsis model was reproduced by CLP, and the cecum and perforation were not ligated in Sham group, and other operations were the same as CLP group. The mice in RAP+CLP group were intraperitoneally injected with autophagy agonist RAP 4 mg×kg^-1×d^-1 7 days before modeling, while the mice in Sham group and CLP group were not treated. Lung, liver, spleen and pancreas tissues were harvested for immunohistochemical staining 4 days after the operation, and the infiltration of neutrophils in various organs was observed under light microscope. Meanwhile, the expressions of immunosuppressive molecule PD-L1 and autophagy marker microtubule-associated protein 1 light chain 3 (LC3) in lung neutrophils were determined by immunofluorescence staining. (2) In vitro experiment: mouse bone marrow neutrophils were extracted and re-suspended to 1×10¹⁰/L, and they were divided into blank control group (without any treatment), RAP control group (RAP 100 μmol/L), autophagy inhibitor Bafilomycin A1 (Baf) control group (Baf 10 μmol/L), lipopolysaccharide (LPS) stimulation group (LPS 1 mg /L), RAP+LPS group, and Baf+LPS group. The latter two groups were pretreated with 100 μmol/L RAP or 10 μmol/L Baf 30 minutes before LPS stimulation, respectively. The expression of PD-L1 mRNA of neutrophils was determined by reverse transcription-polymerase chain reaction (RT-PCR) at 0, 4, 12 hours after LPS stimulation. At the same time, the expressions of PD-L1, LC3 and p62 at the protein level were determined by Western Blot. RESULTS: (1) In vivo experiment: according to immunohistochemical experiments, a large amount of infiltration of neutrophils in lung, liver, spleen and pancreas was found at 4 hours after CLP. In the immunofluorescence, with the time extension after CLP, the positive expression of LC3 in the lung tissue showed a decreased tendency, and PD-L1 expression was significantly increased. RAP pretreatment could promote the expression of LC3 and reduce the expression of PD-L1 in CLP mice. (2) In vitro experiment: in terms of mRNA levels, with the extension of LPS stimulation time, the expression of PD-L1 mRNA in mouse neutrophils was increased continuously, and peaked at 12 hours, it was significantly higher than that in the blank control group (2^-ΔΔCT: 72.2±10.0 vs. 13.0±0.8, P < 0.01). Compared with LPS stimulation group, the expression of PD-L1 mRNA in RAP+LPS group was significantly down-regulated [12-hour PD-L1 mRNA (2^-ΔΔCT): 47.4±7.3 vs. 72.2±10.0, P < 0.01]. In Baf+LPS group, PD-L1 mRNA expression was significantly up-regulated as compared with that in LPS stimulation group [12-hour PD-L1 mRNA (2^-ΔΔCT): 109.1±7.4 vs. 72.2±10.0, P < 0.01]. At the protein levels, at 4 hours after LPS stimulation, the positive expressions of PD-L1, LC3 and p62 were increased significantly as compared with those in the blank control group, and PD-L1 and p62 were increased continuously with time. Compared with the LPS stimulation group, the expressions of PD-L1 and p62 in the RAP+LPS group were significantly down-regulated, while the expression of LC3 was continually increased, indicating that the level of autophagy was increased, and autophagy was circulated smoothly. On the contrary, the expressions of PD-L1, LC3 and p62 in the Baf+LPS group were significantly up-regulated, indicating that the binding of autophagy and lysosome was blocked, and autophagy was not smooth. CONCLUSIONS In sepsis, the infiltration of neutrophils in all organs increased, and the expression of PD-L1 of neutrophils in lungs was increased significantly, while the expression level of autophagy was decreased. The expression of PD-L1 stimulated by LPS can be inhibited by autophagy agonists, and promoted by autophagy inhibitors. PD-L1 has a negative regulatory effect on sepsis. It can reduce the expression of PD-L1 molecule in sepsis by targeting autophagy, so as to improve sepsis.
Animals ; Autophagy ; B7-H1 Antigen/metabolism* ; Male ; Mice ; Mice, Inbred C57BL ; Models, Animal ; Neutrophils ; Sepsis

OBJECTIVE: To observe the changes of peripheral blood neutrophil apoptosis rate and the effect of p38 mitogen-activated protein kinase (p38MAPK) inhibitor on peripheral blood neutrophil apoptosis in severely burned patients. METHODS: Severe burn patients [burn area ≥ 30% total body surface area (TBSA), deep II to III degrees, burn index ≥ 20%, age > 20 years old] admitted to the department of burn and plastic surgery of Affiliated Suzhou Hospital of Nanjing Medical University from January to May in 2019 and health examination volunteers during the same period were enrolled. The peripheral blood neutrophils were isolated by Ficoll gradient centrifugation. The neutrophils of severely burned patients were divided into burn group and p38MAPK inhibitor SB203580 group (after 30 minutes incubation at room temperature and 24 hours incubation in incubator); the neutrophils of healthy volunteers were used as control group. The apoptosis of neutrophils was detected by flow cytometry; the level of reactive oxygen species (ROS) in neutrophils was detected by fluorescence probe; the expression of p38MAPK protein and its phosphorylation (p-p38MAPK) level were detected by Western Blot. RESULTS: Compared with the control group, the apoptosis rate of neutrophils in burn group was significantly decreased [(37.42±3.14)% vs. (50.20±9.87)%, P < 0.05], and the ROS production level in neutrophils was significantly increased (fluorescence intensity: 83.28±0.29 vs. 75.23±0.34, P < 0.05). The apoptosis rate of neutrophils in SB203580 group was further lower than that in burn group [(25.51±1.56)% vs. (37.42±3.14)%, P < 0.05], and the level of ROS production was further higher than that in burn group (fluorescence intensity: 95.56±3.67 vs. 83.28±0.29, P < 0.05). There was no significant difference in the protein expression of p38MAPK among the three groups. The p-p38MAPK protein level in burn group was significantly lower than that in control group (p-p38MAPK/p38MAPK: 0.45±0.06 vs. 0.91±0.09, P < 0.05), while the expression level of p-p38MAPK in SB203580 group was further lower than that in burn group (p-p38MAPK/p38MAPK: 0.22±0.04 vs. 0.45±0.06, P < 0.05). CONCLUSIONS Peripheral blood neutrophil apoptosis delayed and ROS production were increased in severely burned patients. The mechanism may be related to p38MAPK pathway inhibitor.
Apoptosis ; Burns ; Humans ; Neutrophils ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases