Retinitis pigmentosa (RP) is a genetic disorder of photoreceptor cell apoptosis and retinal pigment epithelium (RPE) cell atrophy caused by gene mutation. The clinical manifestations are night blindness, peripheral visual field loss and progressive vision loss. RPE cell apoptosis plays an important role in the progression of RP, and exogenous implantation of RPE cells as an alternative therapy has shown certain efficacy in animal experiments and clinical trials. With the diversification of cell sources, the update of surgical techniques and the continuous emergence of biological materials, more possibilities and hopes are provided for cell therapy. To further promote the development of this field in the future, it is still necessary to strengthen the cooperation between medicine, bioengineering and other disciplines in the future to jointly promote the innovation and development of therapeutic methods. It is believed that RPE cell transplantation therapy will show a brighter prospect in the future

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 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.

Clostridioides difficile is a key pathogen of antibiotic related diarrhea and hospital associated infection, causing several outbreaks in Europe and North Americans and resulting in severe disease burden. However, the standardized diagnostic principle and detection specifications in C. difficile infection (CDI) survey are limited in China, and the infection rate and disease burden of CDI in China are unclear. Therefore, National Institute for Communicable Disease Control and Prevention,National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, together with another 11 institutions, draft the group standard entitled "Diagnosis of Clostridium difficile infection (T/CPMA 008-2020)" of Chinese Preventive Medicine Association. Based on the principle of "legality, scientificity, advancement, and feasibility", this standard clarifies risk factors, diagnosis principles, diagnoses and differential diagnoses in order to improve the accuracy of CDI diagnosis in clinical practice, guide the surveillance for CDI, and understand the infection rate and disease burden of CDI in China .

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 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 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.

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). Conclusion Peripheral blood neutrophil apoptosis delayed and ROS production were increased in severely burned patients. The mechanism may be related to p38MAPK pathway inhibitor.

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