MicroRNA-155 reduces inflammatory response induced by lipopolysaccharide in alveolar macrophages / 中华危重病急救医学

Objective To observe the effect of microRNA-155 (miR-155) on the inflammatory response of rat alveolar macrophages induced by lipopolysaccharide (LPS). Methods The alveolar macrophages NR8383 of rat were cultured in vitro, the macrophages in logarithmic growth phase were harvested to conduct experiment. ① The 1 mg/L LPS was used to stimulate the rat alveolar macrophages for 3, 6, 12, and 24 hours, a phosphate buffer solution (PBS) control group was also set up. Enzyme linked immunosorbent assay (ELISA) was used to detect the dynamic changes of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the supernatant, and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the dynamics expression of miR-155 in the cells, which confirmed the optimal time for LPS stimulation was 12 hours. ② Carboxyfluorescein (FAM) labeled mimic (FAM mimic) and inhibitor (FAM inhibitor) were used to transfect the alveolar macrophage, and the transfection effect was observed under inverted fluorescence microscope 6 hours later to confirm the optimal transfection concentration of mimic was 20 nmol/L, and the optimal transfection concentration of inhibitor was 100 nmol/L. miR-155 mimic and miR-155 inhibitor were transfected to alveolar macrophages respectively at the optimal transfection concentration for 24 hours, and 1 mg/L LPS was used to stimulate the cells for 12 hours. A mimic negative control + LPS group and an inhibitor negative control + LPS group were set up. The expressions of IL-1β and TNF-α in the supernatant were determined by ELISA to observe the regulation of miR-155 on inflammatory response of alveolar macrophages. Results ① After stimulation of 1 mg/L LPS on alveolar macrophages, the contents of IL-1β and TNF-α in the supernatant and the expression of miR-155 in the cells were increased gradually with time prolongation, IL-1β and TNF-α contents peaked at 12 hours, and the expression of miR-155 peaked at 24 hours [as compared with PBS control group, IL-1β (ng/L): 910.43±36.09 vs. 22.66±7.84, TNF-α (ng/L): 3 138.39±394.10 vs. 233.92±8.84, miR-155 (2-ΔΔCt): 7.82±0.30 vs. 1, all P < 0.05]. ② Under inverted fluorescence microscope, after 20 nmol/L FAM mimic or 100 nmol/L FAM inhibitor transfected alveolar macrophages for 6 hours, a large number of cells showed green fluorescence, indicating that the transfection was successful. The expression of miR-155 in the cells transfected with 20 nmol/L miR-155 mimic was up-regulated by (236.73±46.49) times as much as that in the negative control group (P < 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly lower than those in the negative control group [IL-1β (ng/L): 324.37±36.59 vs. 799.31±39.44, TNF-α (ng/L): 1 554.01±342.48 vs. 3 020.49±418.30, both P < 0.05]. The miR-155 activity was significantly inhibited in the cells transfected with 100 nmol/L miR-155 inhibitor, and the expression of miR-155 was decreased by (4.00±3.26)% as compared with the negative control group, but the difference was not statistically significant (P > 0.05), and the levels of IL-1β and TNF-α in the supernatant of the cells stimulated by 1 mg/L LPS for 12 hours were significantly higher than those in the negative control group [IL-1β (ng/L): 1 358.98±212.04 vs. 878.68±53.42, TNF-α (ng/L): 4 225.57±281.11 vs. 2 881.32±286.08, both P < 0.05]. Conclusion In LPS induced inflammatory response of alveolar macrophages, miR-155 plays an obvious inhibitory role.

The inflammatory effect of exosomes derived from alveolar epithelial cells stimulated by lipopolysaccharide on the alveolar macrophages / 中华急诊医学杂志

Objective To explore the inflammatory effect of exosomes derived from alveolar epithelial cells stimulated by lipopolysaccharide (LPS) on the alveolar macrophages (NR8383). Methods The alveolar epithelial cells disposed with different treatments were co-cultured with alveolar macrophages by using a Transwell system separately. Alveolar epithelial cells (RLE-6TN) were randomly divided into 4 groups: normal group, LPS-stimulated group, exosome inhibitor group, and exosome inhibitor pretreatment + LPS stimulation group. NR8383 cultured alone was considered as a blank control. After the 12-h co-culture, the real-time PCR (qPCR) was performed to examine the mRNA relative expression of IL-6, TNF-α, and IL-1β in NR8383 cells. To further explore the role of exosomes derived from RLE-6TN on alveolar macrophages mediated inflammationary response, the experimental exosomes (exosomes derived from LPS-induced RLE-6TN) and control exosomes exosomes derived from normal RLE-6TN were extracted by gradient ultracentrifugation. Transmission electron microscopy and Western blotting analyses was performed to identify the exosomes, and qNano particle diameter analyzer was conducted to measure the particle diameter of exosomes. In vitro, NR8383 cells were divided into 3 groups which were cultured with exosomes derived from LPS-stimulated RLE-6TN at a concentration of 10 μg/mL (experimental group), exosomes derived from untreated RLE-6TN at the same concentration of 10 μg/mL (control group), and the PBS at the same volume with experimental group (PBS group), respectively for 12 h. After the treatment, the phagocytosis of NR8383 cells was observed by laser confocal microscope and the release of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in supernatants of NR8383 was detected by enzyme-linked immunosorbent assay ELISA Results (1)In the co-culture experiment, the mRNA relative expression of pro-inflammatory cytokine in the LPS group was significantly increased compared with the blank control group (P<0.01), however comparing the exosome inhibitor pretreatment+LPS group with the LPS group, the expression of pro-inflammatory cytokine was decreased (P<0.01). (2) The extracted exosomes were observed as circular or elliptical vesicles with a diameter of 40-100 nm under the transmission electron microscopy. Western blotting analyses showed that the extracted exosomes express the protein marker, such as CD63 and CD9; After incubation with NR8383 cells for 5 h, laser scanning confocal microscope showed that the exosomes labeled with red fluorescent were uptaken by NR8383 cells. (3)After the exosomes derived from the LPS-disposed RLE-6TN and the normal RLE-6TN cells were incubated with NR8383 cells respectively. The ELISA test showed that treated the alveolar macrophages with LPS induced alveolar epithelial secreted exosomes led to a robustly increased release of pro-inflammatory cytokine (P<0.01), but there was no significant difference between the control group and PBS group (P>0.05). Conclusions Exosomes derived from LPS-disposed alveolar epithelial cells activate the alveolar macrophage-mediated inflammatory response.

Simultaneous esophagectomy and off-pump coronary artery bypass grafting for esophageal cancer:a report of five cases with literature review / 中华胸心血管外科杂志

Objective To explore the possibility and safety of the simultaneous esophagectomy and off-pump coronary artery bypass grafting(OPCABG) in patients suffering from esophageal cancer combined with coronary artery and summarize the clinical experiences.Methods We retrospectively analyzed the clinical dates of 5 patients performed at the Henan province Chest Hospital from May 2009 to December 2014.The related literature was reviewed.Results All patients were performed the simultaneous esophagectomy and OPCABG through the single left posterolateral thoracotomy 4cases and through median sternotomy and left thoracotomy 1 case.Instrument anastomosis under aortic arch 2 cases and above aortic arch 1 case , left neck anastomosis by hand 2 cases.Coronary artery three ressel disease 3cases, double-vessel 1 case, left main single vessel 1 case.There was no hospital death in this series.Postoperative complications included arrhythmia,anastomotic fistula and pneumonia.Only one patient was still alive, the other patients died of tumor recurrence or metastasis and median survival time was 20.6 months.Conclusion Simultaneous esophagectomy and OPCABG is a safe and feasible treatment modality in patients with severe CAD and esophageal cancer and it may be more beneficial for the patient with early esophageal cancer.

A clinical study of lung protective ventilation and sequential lung recruitment maneuver for treatment of severe acute pancreatitis complicated with acute respiratory distress syndrome / 中国中西医结合急救杂志

Objective To investigate the clinical therapeutic effects of lung protective ventilation and sequential recruitment maneuver (RM) on treatment of patients with severe acute pancreatitis (SAP) complicated with acute respiratory distress syndrome (ARDS). Methods Sixty patients with SAP complicated with ARDS admitted to the Department of Critical Care Medicine of the First Affiliated Hospital of Nanchang University from April 2014 to March 2016 were enrolled. They were divided into control group and experimental group by random number table, 30 patients in each group. On the basis of comprehensive treatment, the patients in control group were treated with lung protective ventilation mode: low tidal volume ventilation (6 mL/kg) + optimal end-expiratory positive pressure (PEEP) ventilation mode, when the intra-abdominal pressure (IAP) was essentially returned to a normal level (Ⅰ grade intra-abdominal hypertension), the patients in experimental group were treated by the combination with RM therapy, and the rest treatment was the same as the control group. Under the two types of ventilation strategies, the clinical effects, respiratory mechanics, hemodynamics and arterial blood gas indexes were compared between the two groups. Results The mechanical ventilation time (days: 13.82±4.40 vs. 19.87±7.40), the length of ICU stay (days:22.67±4.40 vs. 26.43±5.39) and incidence of ventilator associated pneumonia [VAP: 16.67% (5/30) vs. 26.67% (8/30)] of the experimental group were lower than those of the control group (all P < 0.05), the mortality rate of the experimental group was slightly lower than that of the control group [26.67% (8/30) vs. 30.00% (9/30)] without statistical significance (P > 0.05). Plateau pressure (Pplat) and the peak airway pressure (PIP) at each time point were decreased after treatment in both groups, while the static lung compliance (Cst), the arterial partial pressure of oxygen (PaO2) and oxygenation index (PaO2/FiO2) were increased compared with those before treatment, especially the changes at 72 hours after recruitment in the experimental group were more significant than those in the control group [Pplat (cmH2O, 1 cmH2O = 0.098 kPa):15.6±4.0 vs. 21.2±5.6, PIP (cmH2O): 18.3±5.0 vs. 25.1±5.4, Cst (mL/cmH2O): 41.2±4.8 vs. 31.2±6.0, PaO2 (mmHg, 1 mmHg = 0.133 kPa): 90.93±6.45 vs. 80.27±4.51, PaO2/FiO2 (mmHg): 238.33±18.31 vs. 185.83±11.14]. Heart rate [HR (bpm): 110.23±7.92 vs. 98.23±8.44] and the central venous pressure [CVP (mmHg): 8.62±1.52 vs. 6.32±1.42] were significantly higher than those before treatment, the mean arterial pressure [MAP (mmHg): 86.74±7.65 vs. 94.92±10.93] and cardiac output [CO (L/min): 5.32±1.36 vs. 6.42±1.32] were significantly reduced compared with those before treatment (all P < 0.05). The values of HR, MAP, CVP, CO at 5 minutes after recruitment were (97.87±5.77) bpm, (94.54±6.87) mmHg, (6.33±1.44) mmHg, (6.32±1.41) L/min, respectively. The changes of these parameters were not significant when compared with those of the basal conditions (P > 0.05) Conclusions Based on the lung protective ventilation in the early stage, sequential RM is applied in treatment of patients with SAP complicated with ARDS, after the IAP is essentially returned to a normal, which is beneficial to improving lung compliance, promoting oxygenation, shortening the time of mechanical ventilation, reducing the length of ICU stay, and decreasing the incidence of VAP without any obvious hemodynamic influence.

A clinical study of lung protective ventilation and sequential lung recruitment maneuver for treatment of severe acute pancreatitis complicated with acute respiratory distress syndrome / 中国中西医结合急救杂志

Objective To investigate the clinical therapeutic effects of lung protective ventilation and sequential recruitment maneuver (RM) on treatment of patients with severe acute pancreatitis (SAP) complicated with acute respiratory distress syndrome (ARDS). Methods Sixty patients with SAP complicated with ARDS admitted to the Department of Critical Care Medicine of the First Affiliated Hospital of Nanchang University from April 2014 to March 2016 were enrolled. They were divided into control group and experimental group by random number table, 30 patients in each group. On the basis of comprehensive treatment, the patients in control group were treated with lung protective ventilation mode: low tidal volume ventilation (6 mL/kg) + optimal end-expiratory positive pressure (PEEP) ventilation mode, when the intra-abdominal pressure (IAP) was essentially returned to a normal level (Ⅰ grade intra-abdominal hypertension), the patients in experimental group were treated by the combination with RM therapy, and the rest treatment was the same as the control group. Under the two types of ventilation strategies, the clinical effects, respiratory mechanics, hemodynamics and arterial blood gas indexes were compared between the two groups. Results The mechanical ventilation time (days: 13.82±4.40 vs. 19.87±7.40), the length of ICU stay (days:22.67±4.40 vs. 26.43±5.39) and incidence of ventilator associated pneumonia [VAP: 16.67% (5/30) vs. 26.67% (8/30)] of the experimental group were lower than those of the control group (all P < 0.05), the mortality rate of the experimental group was slightly lower than that of the control group [26.67% (8/30) vs. 30.00% (9/30)] without statistical significance (P > 0.05). Plateau pressure (Pplat) and the peak airway pressure (PIP) at each time point were decreased after treatment in both groups, while the static lung compliance (Cst), the arterial partial pressure of oxygen (PaO2) and oxygenation index (PaO2/FiO2) were increased compared with those before treatment, especially the changes at 72 hours after recruitment in the experimental group were more significant than those in the control group [Pplat (cmH2O, 1 cmH2O = 0.098 kPa):15.6±4.0 vs. 21.2±5.6, PIP (cmH2O): 18.3±5.0 vs. 25.1±5.4, Cst (mL/cmH2O): 41.2±4.8 vs. 31.2±6.0, PaO2 (mmHg, 1 mmHg = 0.133 kPa): 90.93±6.45 vs. 80.27±4.51, PaO2/FiO2 (mmHg): 238.33±18.31 vs. 185.83±11.14]. Heart rate [HR (bpm): 110.23±7.92 vs. 98.23±8.44] and the central venous pressure [CVP (mmHg): 8.62±1.52 vs. 6.32±1.42] were significantly higher than those before treatment, the mean arterial pressure [MAP (mmHg): 86.74±7.65 vs. 94.92±10.93] and cardiac output [CO (L/min): 5.32±1.36 vs. 6.42±1.32] were significantly reduced compared with those before treatment (all P < 0.05). The values of HR, MAP, CVP, CO at 5 minutes after recruitment were (97.87±5.77) bpm, (94.54±6.87) mmHg, (6.33±1.44) mmHg, (6.32±1.41) L/min, respectively. The changes of these parameters were not significant when compared with those of the basal conditions (P > 0.05) Conclusions Based on the lung protective ventilation in the early stage, sequential RM is applied in treatment of patients with SAP complicated with ARDS, after the IAP is essentially returned to a normal, which is beneficial to improving lung compliance, promoting oxygenation, shortening the time of mechanical ventilation, reducing the length of ICU stay, and decreasing the incidence of VAP without any obvious hemodynamic influence.

The protective effects of transfected microRNA-146a on mice with sepsis-induced acute lung injury in vivo / 中华危重病急救医学

ObjectiveTo investigate the protective effect of transfected microRNA-146a (miR-146a) on mice with sepsis-induced acute lung injury (ALI) in vivo.Methods Twenty-four healthy male BALB/C mice were randomly divided into sham group, sepsis group, transfection group and transfection control group, eachn = 6. Mice in transfection group were given miR-146a agomir loaded by in vivo-jetPEITM via airway before reproduction of model, and mice in transfection control group were given negative control loaded by in vivo-jetPEITM only via airway. The septic model was reproduced by cecal ligation and puncture (CLP) 12 hours after transfection , and the mice in the sham group underwent laparotomy and closure only without ligation and puncture of the cecum. The mice of each group were sacrificed at 24 hours post-operation. The expression of miR-146a in lung tissue was determined by real time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the quantity of tumor necrosis factor-α (TNF-α) in the bronchial alveolar lavage fluid (BALF) was determined with enzyme-linked immunosorbent assay (ELISA). The wet/dry ratio of lung (W/D) was determined. The pathohistological changes in the lung were observed and scored. Results The expression of miR-146a showed a significant increase in sepsis group, transfection group and transfection control group, which were (3.56±0.43), (27.64±3.46) and (3.72±0.54) folds of that in sham group, respectively (P 0.05). Compared with the sham group, higher level of TNF-αin the BALF was found in the sepsis group, transfection group and transfection control group (ng/L: 511.65±43.47, 305.74±34.76, 492.27±42.21 vs. 50.72±7.23, allP< 0.01). The level of TNF-α in transfection group was significantly lower than that in sepsis group and transfection control group (bothP< 0.01). Compared with the sham group, the W/D ratio of lung in sepsis group, transfection group and transfection control group showed a significant increase (6.11±0.32, 5.02±0.29, 6.05±0.43 vs. 4.18±0.10, allP< 0.01). The W/D ratio of lung in transfection group was significantly lower than that of sepsis group and transfection control group (bothP< 0.01). The lung injury score of transfection group was significantly lower than that of sepsis group and transfection control group (6.12±0.75 vs. 10.53±1.52, 9.73±1.08, bothP< 0.01).Conclusions miR-146a agomir loaded by in vivo-jetPEITM instillation into airway was able to increase the expression of miR-146a in the lung tissue of septic mice. Up-regulation of miR-146a inhibit the release of the inflammatory cytokine TNF-α stimulated by sepsis, and alleviate inflammatory reaction and lung tissue injury in mice with sepsis-induced ALI.

Clinical characteristics of human infection with a novel avian-origin influenza A(H10N8) virus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Novel influenza A viruses of avian-origin may be the precursors of pandemic strains. This descriptive study aims to introduce a novel avian-origin influenza A (H10N8) virus which can infect humans and cause severe diseases.</p><p><b>METHODS</b>Collecting clinical data of three cases of human infection with a novel reassortment avian influenza A (H10N8) virus in Nanchang, Jiangxi Province, China.</p><p><b>RESULTS</b>Three cases of human infection with a new reassortment avian influenza A(H10N8) virus were described, of which two were fatal cases, and one was severe case. These cases presented with severe pneumonia that progressed to acute respiratory distress syndrome (ARDS) and intractable respiratory failure.</p><p><b>CONCLUSION</b>This novel reassortment avian influenza A (H10N8) virus in China resulted in fatal human infections, and should be added to concerns in clinical practice.</p>

The relationship between microRNA-146a and TNF-α in lipopolysaccharide-stimulated alveolar macrophages of rats / 中华急诊医学杂志

Objective To determine kinetics of TNF-α and miR-146a (microRNA-146a)expressions in lipopolysaccharide (LPS)-induced NR8383 alveolar macrophages (AM) at different intervals and their relationships in order to explore regulatory effect and mechanism of miR-146a on alveolar macrophages inflammatory responses.Methods NR8383 alveolar macrophages were seeded in a 6-well plate,and stimulated with 1 μg/ml of LPS for 0 h,3 h,6 h and 12 h separately after 90 min.Cells were harvested and supernatant were collected 0 h,3 h,6 h and 12 h after incubation.The expressions of miR146a and TNF-α mRNA in cells were detected by real-time qPCR and the levels of TNF-α protein in the supematant of cells were assayed by enzyme-linked immunosorbent assay ( ELISA ).Pearson correlation analysis was used to analyze the correlation between miR-146a and TNF-α mRNA.Results ①The level of TNF-α protein in the supernatant of cell was significantly increased 3 h after LPS challenge ( 359.80 ±57.54) pg/ml (P ＜0.01 ),and peaked 12 h later (729.22 ±50.40) pg/ml (P＜0.01 ) ; ②the expression of TNF-α mRNA peaked 3 h after LPS challenge (67.48 ±24.52) fold,P ＜0.01 ),and then decreased gradually; ③the expression of miR-146a mRNA increased continuously until 6 h or 12 h after LPS challenge 6 h:(5.33 ±0.81) fold,12 h:(8.21 ±1.19) fold,(P＜0.01),and it showed an upward tendency;④ the expression of miR-146a mRNA was negatively correlated with TNF-α mRNA ( r =-0.895,P ＜0.01).Conclusions The miR-146a mRNA showed a negative correlation with TNF-α mRNA present in lipopolysaccharide-stimulated alveolar macrophages,suggesting miR-146a mRNA involved in regulating the inflammatory response of alveolar macrophages.

The expression changes of miR-146a in lipopolysaccharide-induced alveolar macrophages / 中华急诊医学杂志

Objective To explore the mechanism and effect of miR-146a on alveolar macrophages and to observe the changes of miR-146a expression in the LPS-induced alveolar macrophages. Method NR8383 alveolar macrophages were divided into LPS-stimulated group and control group, and the cells of former group were treated with LPS ( 1 μg/mL) and then incubated for 3 h, 6 h and 12 h, respectively. The level of TNF-α in the supernatant of cells was assayed by using enzyme-linked immunosorbent assay (ELISA), and the expression of miR-146a of cells was detected by using Real-Time PCR (TaqMan probe).Statistical analysis carried out by using SPSS 13.0 software package in which One-way ANOVA and Student's t-test were used. Results Compared with control group, the levels of TNF-α in the supernatant of cells were significantly increased 3 h, 6 h and 12 h after LPS challenge (P ＜ 0.01 ). The expression of miR-146a increased 6 h and 12 h after LPS stimulation in NR8383 cells( P ＜0.01 ), and it had an upward tendency.Conclusions The expression of miR-146a in alveolar macrophages increases after LPS-stimulation. It hints miR-146a may be involved in the regulation of the inflammatory responses produced by alveolar macrophages.