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

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

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.

Genistein Inhibits the Proliferation of Human Lung Cancer PC14 Cells through the ERK and JNK Pathways / 中国医科大学学报

Objective To investigate the effect of genistein on the proliferation of human lung cancer PC14 cells and the underlying mechanisms. Methods Cell proliferation was examined using the MTT and colony formation assays. Western blotting was used to analyze protein expression levels. Results Genistein significantly inhibited the proliferation of PC14 cells in a concentration and time dependent manner. PD98059, SB203580, and SP100625, three specific inhibitors of the MAPK pathway, significantly inhibited the proliferation of PC14 cells. Moreover, genistein inhibited the phosphorylation of ERK and JNK in a dose dependent manner. Conclusion Genistein can inhibit the proliferation of PC14 cells, which may be related to its inhibitory effect on ERK and JNK activation.

Distribution of methamphetamine concentration in saliva and urine samples and the analysis of the initial screening / 中国法医学杂志

Objective To obtain methamphetamine concentration profiles in saliva and urine samples of drug addicts and to screen the colloidal gold strip. Methods Methamphetamine concentration in saliva and urine samples of drug addicts was determined by liquid chromatography tandem mass spectrometry. The initial screening was obtained by colloidal gold strip test. The results were compared and analyzed. Results using the method of protein and fluid MRM scan method to detect direct precipitation, saliva is linear in the range of 1~100ng/mL, the linear correlation coefficient is 0.9987, the detection limit is 0.1ng/mL, the limit of quantification was 1ng/mL, the urine is linear in the range of 1~100ng/mL, the linear correlation coefficient is 0.9943, the detection limit is 0.5ng/mL, the limit of quantification was 1ng/mL. Saliva and urine samples diluted, the concentration in the linear range. Saliva and urine samples of four types of methamphetamine colloidal gold reagent strip were screened directly, and the results were judged visually. Conclusion the detection rate of colloidal gold strip is about 79%, the detection rate of saliva is about 81%, and the detection rate can be increased to more than 93% by using two reagent strips. Combined with the initial screening results and the instrument confirmation concentration, it can be found that the gray zone setting and sensitivity setting have certain influence on the detection rate, and it is suggested to improve the sensitivity to meet the needs of screening.

Low ankle-brachial index predicts cerebral microbleeds in patients with ischemic stroke / 医学研究生学报

Objective The abnormal ankle-brachial index ( ABI) is associated with the incidence of cardiocerebral vascular diseases, but little is known about its relationship with cerebral microbleeds (CMB).This study aimed to investigate the correlation be-tween ABI≤0.9 and different distribution patterns of CMB . Methods We enrolled 187 patients with acute lacunar infarction , inclu-ding 115 non-CMB cases and 72 CMB cases (20 strictly lobar, 24 strictly deep, and 28 lobar and deep).We analyzed the differences between the two groups and the association of abnormal ABI with the occurrence and distribution of CMB by logistic regression analysis . Results ABI≤0.9 was found in 57 (30.5%) of the patients, with a significantly higher incidence rate in the CMB group than in the non-CMB group (43.1%vs 22.6%, P=0.003).The level of ABI was negatively correlated with the number of CMBs (r=-0.211, P=0.006).Multivariate logistic regression analysis after adjusted for confounders indicated that ABI ≤0.9 was significantly associated with the presence of CMB (OR=2.363;95%CI:1.181-4.729), deep CMB (OR=3.434;95%CI:1.283-9.187), and lobar and deep CMB ( OR=2.837;95%CI:1.098-7.333) in patients with ischemic cerebrovascular disease . Conclusion Decreased ABI is a risk factor of CMB, particularly deep CMB, in patients with ischemic stroke.

To investigate the immunity characteristics of a conserved sequence on the C terminal region of the human papillomavirus major protein L 1 / 重庆医学

Objective To investigate and detect the immunity characteristics of a conserved sequence including 30 amino residues which is located in the C terminal of HPVL1 .Methods The immune model of mouse was establish with a polypeptide synthetized based on this sequence .The amount of CD3+ ,CD3+CD4+ or CD3+CD8+ lymphocyte of the mouse spleen was detected by Flow cy-tometry ,then the value of CD4+ /CD8+ were calculated .The cell proliferation was detected by MTT assay .Sample was took from the cell supernatant to detect the content of IL-4 and IFN-γby double antibody sandwich ELISA .Results (1)The amount of CD3+CD8+ lymphocytes and the value of CD4+ /CD8+ were significantly increased than control group(P0 .05) .(3)The level of IL 4 were significantly higher than that of control group (P0 .05) .Conclusion The results showed that it was affirmative that the polypeptides induced humoral immunity ,which was worth to be furtherly studied as a preventive vaccine .But its cell immunogenicity is weak ,and the attenmpt to induce the response related to the cell immunogenicity was failed ,and the immunogenicity of the peptide remains to be improved .

The association of hepatitis B virus DNA levels in the peripheral blood mononuclear cell with antiviral efficacy in chronic hepatitis B / 中华传染病杂志

Objective To explore the relationship between the antiviral effect and peripheral blood mononuclear cell (PBMC) hepatitis B virus (HBV) DNA when the patients reach the standard of withdrawal of antiviral therapy in chronic hepatitis B (CHB).Methods Ninety CHB patients treated with interferon(n=44) or nucleot (s) ide(n=46) who reached the standard of withdrawal of antiviral therapy were recruited.HBV DNA levels in PBMCs were tested at the end of treatment,and its relationship with serum HBV DNA level before treatment in PBMC HBV DNA positive group and negative group were compared.The correlation between HBV DNA in PBMCs at the end of treatment and relapse were explored.Measurement data were analyzed by student t test and enumeration data were analyzed by X2 test.Results Among 90 patients,67(74.4%) were PBMC HBV DNA negative at the end of treatment,and 23(25.6%) were positive.The serum HBV DNA positive conversion rate in PBMC HBV DNA negative patients was 13.4%,which were significantly lower than that in positive group (73.9%) (X2=30. 4873, P<0.01 ). There were no significant differences of alanine aminotransferase (ALT) levels when hepatitis flare (t=0. 8729, P=0. 3913) and relapse time (t=1. 9222, P=0. 0665) between PBMC HBV DNA negative group and positive group after withdrawal of therapy, while the serum HBV DNA rebound was greater in positive group than that in negative group (t=2. 7493, P=0. 0112). There were five patients who achieved hepatitis B surface antigen (HBsAg) seroconversion, whose PBMC HBV DNA were all undetectable, and none relapsed during follow-up for 6-12 months. The pretreatment HBV DNA as level in PBMC HBV DNA positive was (7.2±1.1) lg copy/mL, which was much higher than that in negative group[(5.2±2.1) lg copy/mL] (t=4. 3557, P<0.01). Conclusions In patients who reach the standard of drug withdrawal,PBMC HBV DNA at the end of treatment is an important predictor for durability of antiviral therapy in CHB.

The relationship between the changes of peripheral blood natural killer cells and HLA-Cw alleles in liver cirrhotic patients with chronic HBV infection / 中华微生物学和免疫学杂志

Objective To investigate the relationship between the frequency of peripheral blood natural killer cells (NK) and HLA-Cw alleles in liver cirrhotic patients with chronic HBV infection and a-cute hepatitis B patients. Methods Thirty liver cirrhotic patients and 30 patients with acute hepatitis B were included in our study, and 41 healthy individuals were enrolled as controls. The numbers of circulating NK cells and activated NK ceils were analyzed by flow cytometry. HLA-Cw genotyping was conducted with polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO). Results The numbers of circu-lating NK cells and activated NK cells in liver cirrhotic patients were 13.22% ± 4.61% and 45.68% ± 14.64%, which was lower than that in healthy subjects (P < 0.05). The numbers of circulating NK cells and activated NK cells in acute hepatitis B patients were 22.62% ± 3.70% and 65.28%± 14.45%, which was higher than that in healthy subjects(P < 0. 05). There were statistically significant differences between the two groups(P < 0.01). The allele frequency of HLA-Cw * 15 in the patients with cirrhosis was signifi-cantly higher than that in the healthy (P < 0.05), and there was a significant negative correlation between the frequency of HLA-Cw * 15 and the numbers of activated NK cells in liver cirrhosis(r =4). 862, P < 0.05). No statistically significance was found between the group of acute hepatitis B and healthy subjects a- bout HLA-Cw(P > 0. 05). Conclusion The function of NK cells in liver cirrhotic patients is low, HLA-Cw * 15 gene may be one of the causes of effecting the antiviral function of NK ceils to induce the persistence of hepatitis B virus(HBV) infection.

Elastase-induced intracranial aneurysm by means of endovascular balloon occlusion:preparation of animal model and progress in its applications / 介入放射学杂志

It is of capital importance to establish a reliable experimental animal model of intracranial aneurysm which should be extremely similar to human intracranial aneurysm in histological and hemodynamic aspects.For recent years,the elastase-induced aneurysm model by means of endovascular balloon occlusion has been widely used abroad.This paper aims to review the preparation of animal mode,the advantages and disadvantages of such a model as well as the recent progress in its applications.

Influnce of oral clonidine and ketorolac premedication on cytokine response to abdominal surgery / 中华麻醉学杂志

Objective The aim of the study was to examine the influence of clonidine and ketorolac pretreatment on cytokine response to abdominal surgery. Methods Twenty patients scheduled for upper-abdominal surgery were randomly allocated to two equal groups: (1) pre-emptive analgesia group: patients received oral clonidine 4?g/kg and ketorolac 10mg 1h before surgery and (2) control group: patients received placebo. Blood samples were taken 24h before surgery , 20min after induction of anesthesia (before surgery), 90min after induction (during surgery), and 0,1,2,4,6 and 24h after surgery for determination of serum tumor necrosis factor-? (TNF-?), interleukin-1?(IL-1?),interleukin-6(IL-6) and interleukin-10 (IL-10) concentrations.Results (1) In the control group, serum IL-6 concentration increased significantly during surgery and peaked at 2h after skin closure. In contrast serum IL-6 concentration in pre-emptive analgesia group increased significantly when the skin was closed and peaked at 4h after skin closure. IL-6 production in the pre-emptive analgesia group was less than that in control group at 0,1 and 2h after surgery(P

Intra-cellular Signal Pathway and Synthesis of Prostaglandin E_2 During Invasion of Macrophage by Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

Objective To investigate the signal transduction pathway of arachidonic acid(AA) and prostaglandin E-2(PGE-2) synthesis in macrophage invaded by Toxoplasma gondii. Methods Synthesis of AA and PGE-2, expression of COX_2 mRNA and protein following stimulation infection by Toxoplasma gondii were evaluated in RAW264