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Objective:To investigate the effect of extracellular vesicles derived from lung tissue on intrapulmonary inflammation and the formation of neutrophil extracellular traps (NETs) in sepsis rats.Methods:Sepsis rat model was established by cecal ligation and puncture (CLP). Collagenase D and DNase I were used to dissociate the lung tissue, the impurities were removed by centrifugation, and finally, the extracellular vesicles (Ti-EVs) derived from lung tissue were separated and extracted by differential ultracentrifugation. Eighteen male SD rats were randomly divided into the sham group, sepsis group and Ti-EVs group: in the Ti-sEV group, a sepsis model was established by CLP, and Ti-EVs suspension was instilled through the airway; rats in the CLP group received CLP, and an equal volume of PBS was instilled through the airway; and rats in the sham group was treated with sham operation. The pathological changes of lung tissue were detected by hematoxylin-eosin (HE) staining after 24 h. The content of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the bronchoalveolar lavage fluid (BALF) was measured by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect the NETs content in lung tissue.Results:The isolated extracellular vesicles derived from rat lung tissue were observed by transmission electron microscopy as double-layer circular cystic vesicles with particle diameter mainly distributed at 150 nm. Western blot showed positive expression of EVs markers CD9, CD63, and Tsg101. HE staining of lung tissue showed alveolar integrity damage and a large number of inflammatory cells infiltrated in the lung of sepsis rats. Compared with the CLP group, the degree of lung tissue damage was more serious in the Ti-EVs group and the levels of IL-1β, TNF-α and IL-6 in the BALF of rats were significantly increased ( P<0.01). The formation of NETs in the lungs of the rats in the sepsis group and the Ti-EVs group was observed under the laser confocal microscope. Compared with the sepsis group, the fluorescence intensity of NETs in the lung tissues of the Ti-EVs group increased significantly. Conclusions:After enzymatic digestion, differential ultracentrifugation and other treatments, the extracellular vesicles derived from rat lung tissue with high purity can be successfully isolated and extracted. In the process of septic lung injury, extracellular vesicles in lung tissue can aggravate the inflammatory response in the lung and promote the formation of NETs.
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Objective:To investigate the effect and mechanism of exosomes derived from human-induced pluripotent mesenchymal stem cells (iMSC-Exos) on alveolar macrophages (AM) pyroptosis.Methods:The exosomes in the culture supernatant of human-induced pluripotent mesenchymal stem cells (iMSC) were extracted by rotating ultrafiltration, and the extracted exosomes were identified by transmission electron microscopy, Western blotting and high-resolution adjustable resistance pulse. The rat alveolar macrophage cells (NR8383 cells) were cultured in vitro and the logarithmic growth phase cells were divided into three groups: the control group was added with an equal volume of phosphate buffered saline (PBS) in the AM supernatant; in LPS/ATP group AM cells were stimulated with 500 μg/L LPS for 23 hours and then 5 mmol/L ATP was added for 1 hour to induce pyrolysis; iMSC-Exos group was incubated with AM and 100 mg/L iMSC-Exos for 3 hours before giving LPS and ATP. The cytotoxic activity was detected by cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) analysis, the apoptosis and the expression of caspase-1 were observed by immunofluorescence, the levels of inflammatory factors interleukins (IL-1β and IL-18) released by AM were detected by enzyme linked immunosorbent assay (ELISA), the NOD-like receptor protein 3 (NLRP3) inflammasome pathway and the expression level of pyroptosis related protein gasdermin D (GSDMD) were detected by Western blotting. Results:The extracted exosomes were observed by transmission electron microscopy as round vesicles, expressing exosomal markers CD63 and CD9 showed by Western blotting, high-resolution adjustable resistance pulse showed the average diameter of the particles was 130 nm, and could be uptaken by AM. Compared with the control group, the cell activity decreased [(0.56±0.05)% vs. (1.06±0.07)%, P < 0.01], the release of necrotic substance LDH increased (U/L: 1 218.86±22.73 vs. 188.30±1.61, P < 0.01), the expression levels of inflammatory factors increased [IL-1β (ng/L): 958.91±32.78 vs. 194.63±5.14, IL-18 (ng/L): 870.89±21.86 vs. 288.85±24.48, both P < 0.01], and the apoptosis rate [(55.35±6.19)% vs. (12.01±1.32)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 41.06±3.65 vs. 2.80±0.54, P < 0.01) elevated in the AM after LPS/ATP stimulation, suggesting that LPS combined with ATP successfully induced alveolar pyroptosis. Compared with the LPS/ATP group, AM pretreated with iMSC-Exos showed increased cell viability [(0.81±0.05)% vs. (0.56±0.05)%, P < 0.01], decreased LDH secretion (U/L: 535.05±42.55 vs. 1 218.86±22.73, P < 0.01), decreased expression of inflammatory factors [IL-1β (ng/L): 381.82±19.50 vs. 958.91±32.78, IL-18 (ng/L): 533.77±31.54 vs. 870.89±21.86, both P < 0.01], and decreased apoptosis rate [(19.74±2.96)% vs. (55.35±6.19)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 12.16±1.31 vs. 41.06±3.65, P < 0.01). At the same time, the expression of NLRP3 inflammasome pathway [NLRP3 protein (NLRP3/β-actin): 0.62±0.06 vs. 1.89±0.11; cleaved caspase-1 protein (cleaved caspase-1/β-actin): 0.42±0.07 vs. 1.22±0.17, both P < 0.01] and pyrolysis-related protein was significantly inhibited [GSDMD protein (GSDMD/β-actin): 0.57±0.05 vs. 1.22±0.05, P < 0.01]. Conclusion:iMSC-Exos successfully reversed the AM pyroptosis and inflammatory factor expression induced by LPS/ATP, which may be due to the targeted inhibition of NLRP3 inflammasome pathway, suggesting that iMSC-Exos can exert anti-inflammatory effects by inhibiting the pyrolysis of AM.
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Objective To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients. Methods Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCATM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway. Results The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t =-10.688; REX1 mRNA (2-ΔΔCt):1.419±0.206 vs. 0.103±0.066, t =-14.245; OCT4 mRNA (2-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t =-9.028; Sox2 mRNA (2-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t =-12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC. Conclusion The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.
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Objective@#To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients.@*Methods@#Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCATM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway.@*Results@#The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t = -10.688; REX1 mRNA (2-ΔΔCt): 1.419±0.206 vs. 0.103±0.066, t = -14.245; OCT4 mRNA (2-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t = -9.028; Sox2 mRNA (2-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t = -12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC.@*Conclusion@#The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.
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Objective@#To analyze the specificity and sensitivity of the modified microbubble test in identifying the peripherally inserted central venous catheters (PICC) tip based on the chest X-ray location as the "gold standard", and to find out an accurate and noninvasive PICC tip positioning method that can save time and cost.@*Methods@#Convenient sampling method was conducted. The patients under PICC guided by ultrasound in intensive care unit (ICU) or PICC clinic of the First Affiliated Hospital of Nanchang University from August 2017 to February 2018 were enrolled. All patients were followed up by ultrasound guided PICC catheter placement, modified microbubble test and chest X-ray localization. The relationship between the density of microbubbles in modified microbubble test and the location of PICC tip in chest X-ray localization was analyzed. Using chest X-ray localization as the "gold standard", the diagnostic evaluation indexes such as specificity and sensitivity of PICC tip identification by modified microbubble test were calculated.@*Results@#A total of 120 patients were enrolled during the study period, excluding those who refused to participate in the study, unclear right atrial ultrasound, conscious intolerance, unclear chest X-ray, and finally 108 patients completed the modified microbubble test and chest X-ray tip localization. According to the chest X-ray localization results of 108 patients, 69 patients (63.9%) were in ideal locations, 33 (30.6%) were in dissatisfactory position, and 6 (5.5%) were in malposition. There was no significant difference in gender, age, tube placement, depth of catheterization, placement of catheterization room, and catheterization among the three groups. In the modified microbubble test, there were 74 patients (68.5%) with gradeⅠmicrobubble, 25 (23.2%) with gradeⅡ microbubble, and 9 (8.3%) with grade Ⅲ microbubble. There was a correlation between microbubble density and the tip position of the catheter, showing a moderate intensity correlation, and the contingency coefficient was 0.662. The sensitivity of the modified microbubble test for PICC tip positioning was 95.7% (66/69), the specificity was 89.7% (35/39), the rate of missed diagnosis was 4.4% (3/69), the misdiagnosis rate was 10.3% (4/39), the positive predictive value was 94.3% (66/70), the negative predictive value was 92.1% (35/38), and the Youden index was 0.85. The consistency between the two methods was good, and the Kappa value was 0.86.@*Conclusions@#Compared with the chest X-ray localization method, the modified microbubble test method has high sensitivity and specificity in identifying PICC in the position, and the operation is simple, noninvasive, with less time and low cost. The modified microbubble test can be used as a screening test for PICC tip position, especially in ICU. When there are technical limitations or suspicious patient, further chest X-ray is necessary.
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Objective To analyze the specificity and sensitivity of the modified microbubble test in identifying the peripherally inserted central venous catheters (PICC) tip based on the chest X-ray location as the"gold standard", and to find out an accurate and noninvasive PICC tip positioning method that can save time and cost. Methods Convenient sampling method was conducted. The patients under PICC guided by ultrasound in intensive care unit (ICU) or PICC clinic of the First Affiliated Hospital of Nanchang University from August 2017 to February 2018 were enrolled. All patients were followed up by ultrasound guided PICC catheter placement, modified microbubble test and chest X-ray localization. The relationship between the density of microbubbles in modified microbubble test and the location of PICC tip in chest X-ray localization was analyzed. Using chest X-ray localization as the "gold standard", the diagnostic evaluation indexes such as specificity and sensitivity of PICC tip identification by modified microbubble test were calculated. Results A total of 120 patients were enrolled during the study period, excluding those who refused to participate in the study, unclear right atrial ultrasound, conscious intolerance, unclear chest X-ray, and finally 108 patients completed the modified microbubble test and chest X-ray tip localization. According to the chest X-ray localization results of 108 patients, 69 patients (63.9%) were in ideal locations, 33 (30.6%) were in dissatisfactory position, and 6 (5.5%) were in malposition. There was no significant difference in gender, age, tube placement, depth of catheterization, placement of catheterization room, and catheterization among the three groups. In the modified microbubble test, there were 74 patients (68.5%) with gradeⅠmicrobubble, 25 (23.2%) with gradeⅡmicrobubble, and 9 (8.3%) with grade Ⅲ microbubble. There was a correlation between microbubble density and the tip position of the catheter, showing a moderate intensity correlation, and the contingency coefficient was 0.662. The sensitivity of the modified microbubble test for PICC tip positioning was 95.7% (66/69), the specificity was 89.7% (35/39), the rate of missed diagnosis was 4.4% (3/69), the misdiagnosis rate was 10.3% (4/39), the positive predictive value was 94.3% (66/70), the negative predictive value was 92.1% (35/38), and the Youden index was 0.85. The consistency between the two methods was good, and the Kappa value was 0.86. Conclusions Compared with the chest X-ray localization method, the modified microbubble test method has high sensitivity and specificity in identifying PICC in the position, and the operation is simple, noninvasive, with less time and low cost. The modified microbubble test can be used as a screening test for PICC tip position, especially in ICU. When there are technical limitations or suspicious patient, further chest X-ray is necessary.
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Objective To observe the amplification effect of mitochondrial DNA (mtNDA) on the inflammatory response of rat alveolar macrophage induced by lipopolysaccharide (LPS), and to preliminarily explore its mechanism. Methods mtDNA of Sprague-Dawley (SD) rat liver tissue was harvested, ultra-micro spectrophotometer and 1% agarose gel electrophoresis were used to detect the concentration and quality of mtDNA. The alveolar macrophages of SD rat were isolated and cultured, the macrophages in logarithmic growth phase were divided into phosphatic buffer solution (PBS) group, LPS group, mtDNA group and LPS+mtDNA group. The first three groups were added equal volumes of PBS, LPS 1 mg/L or mtDNA 10 mg/L to the alveolar macrophages medium for 6 hours and 12 hours, respectively; the alveolar macrophage medium of LPS+mtDNA group was stimulated with 1 mg/L LPS for 6 hours and then stimulated with 10 mg/L mtDNA for 6 hours. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell supernatant were detected by enzyme linked immunosorbent assay (ELISA);the expression of the key protein of Pyroptosis-Gasdermin D (GSDMD) was detected by Western Blot. Results ① The mtDNA A260/280 ratios were between 1.8-2.0, and agarose gel electrophoresis showed a single band, with a size of about 16 kb. ② After alveolar macrophages stimulated by LPS or mtDNA for 6 hours or 12 hours, respectively, the levels of IL-1β and TNF-α were higher than those in PBS group. When cells were treated with mtDNA for 6 hours after LPS stimulation 6 hours, the levels of IL-1β were higher than those in LPS 12 hours group (ng/L: 366.27±23.35 vs. 154.70±23.32, 1 < 0.01), but the levels of TNF-α had no significant difference compared with LPS 12 hours group (ng/L: 836.13±25.01 vs. 802.67±30.48, 1 > 0.05). ③ The protein expressions of GSDMD in LPS group, mtDNA group and LPS+mtDNA group were significantly higher than those in PBS group (GSDMD/β-actin: 1.77±0.05, 1.65±0.04,2.40±0.05, 1.00±0.02, all 1 < 0.01), and the protein expression of GSDMD in LPS+mtDNA group was higher than that in LPS group (1 < 0.01). Conclusion mtDNA amplifies LPS-induced alveolar macrophage inflammatory responses,which mechanism may be related to the increase in pyroptosis mediated by mtDNA.
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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.
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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.
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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.
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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.
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Objective To systematically evaluate the comprehensive effect of subglottic secretion drainage (SSD) on patients with mechanical ventilation (MV) in intensive care unit (ICU). Methods The randomized controlled clinical trials (RCTs) comparing SSD (intervention group) versus non-SSD (control group) in adult patients with MV in ICU was collected through the databases such as the PubMed database of the National Library of Medicine, CNKI, Wanfang database and the Chinese journal of science and technology database (VIP). The subjects were ICU patients with MV, and the retrieval time ranged from January 2006 to December 2016. Two reviewers independently screened the studies according to the inclusive and exclusive criteria, extracted the data, and assessed the quality. Then RevMan 5.3 software was used for Meta-analysis. Sensitivity analysis was performed using Stata 11.0 software. Funnel plot was used to analyze publication bias. Results In the 1004 documents obtained from preliminary screening, a total of 13 studies involving 2052 patients were enrolled after excluding duplicated documents and literature did not meet the inclusion criteria, with 1021 patients in intervention group, and 1031 in control group. Meta-analysis showed that compared with control group, the application of SSD in patients with MV could contribute to the reduction of the incidence of ventilator-associated pneumonia [VAP; risk ratio (RR) = 0.54, 95% confidence interval (95% CI) = 0.46-0.64, P < 0.00001], the duration of MV [mean difference (MD) = -3.29, 95%CI = -4.53 to -2.05, P < 0.00001] and length of hospital stay (MD = -4.27, 95% CI = -7.36 to -1.18, P = 0.007) were shortened, while there was no significant difference in ICU or hospital mortality rate between the intervention group and control group (RR = 0.89, 95%CI = 0.73-1.09, P = 0.25). The sensitivity analysis for studies enrolled in Meta-analysis of MV duration showed that individual research results were stable through step remove of the included literatures and combined calculation of the remaining literature value, suggesting that individual research results were stable, and would not have a significant impact on the overall results. The results of the funnel analysis showed that there was a symmetry in the inclusion studies, and no significant publication bias was found. Conclusions SSD did have effect in reducing the incidence of VAP, shortening the duration of MV and length of hospital stay, while there was no significant effect on reducing mortality rate. Effective use of SSD is an important measure to prevent VAP. It is necessary to objectively evaluate the clinical effect of SSD.
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Objective To analyze the relationship between the catheter to vein ratio and the formation of peripheral insertion of central venous catheter (PICC) related upper extremity deep venous thrombosis (PICC-UEDVT) in cases having undergone PICC in patients at intensive care unit (ICU) and further identify the best optimal ratio cut-off point to reduce the incidence of PICC-UEDVT.Methods A retrospective study was conducted, including 69 patients having undergone PICC with complete clinical data admitted to the Department of Critical Care Medicine of the First Affiliated Hospital of Nanchang University from August 2013 to December 2016; their ages were > 18 years old and catheter indwelling times were > 1 week; the patients' basic information, disease related laboratory parameters and catheter insertion situation were collected. According to the occurrence of PICC-UEDVT, they were divided into PICC-UEDVT group and non PICC-UEDVT group; the receiver operating characteristic (ROC) curve of the catheter to vein ratio versus the incidence ofPICC-UEDVT was plotted to assess the optimal ratio to reduce the incidence of PICC-UEDVT.Results In the 69 patients, there were 7 patients in the PICC-UEDVT group and 62 patients in the non PICC-UEDVT group, the incidence of PICC-UEDVT being 10.14%. Four, 5 and 6 French (Fr) catheters were indwelled in 43, 23 and 3 cases respectively, and the range of catheter to vein ratio was 20% - 67%. The comparisons between PICC-UEDVT group and non PICC-UEDVT group in various aspects were as follows: the incidence of DVT in the PICC-UEDVT group was significantly higher than that in non PICC-UEDVT group [42.9% (3/7) vs. 6.5% (4/62)], the rate of using vasopressor drugs [57.14% (4/7) vs. 17.74% (11/62)], D-dimer level [mg/L: 9.0 (3.0, 12.3) vs. 1.8 (1.0, 3.6)], patients of indwelling 5Fr catheter [71.4% (5/7) vs. 29.0% (18/62)] and the percentage of patientsapplying catheter to vein ratio 45%-67% [57.14% (4/7) vs. 17.74% (11/62)] in PICC-UEDVT group were all higher than those in the non PICC-UEDVT group, the differences being statistically significant (allP < 0.05). ROC analysis showed that the catheter to vein ratio 44% was the optimal cut off or critical point, the area under the ROC curve (AUC) at that point was 0.755, 95% confidence interval (95%CI) = 0.554-0.955, sensitivity = 71.4% and specificity = 79.0%; compared with the patients using 45%-67% catheter to vein ratio, the incidence of PICC-UEDVT was 6.182 times higher than those using the ratio 20%-44% [odds ratio (OR) = 6.182, 95%CI = 1.208-31.634,P = 0.036]; however, there was no significant difference in incidence of PICC-UEDVT between 20%-32% and 33%-44% (P = 1.000).Conclusion It is found that the 44% catheter to vein ratio was the optimal critical point to reduce the incidence of PICC-UEDVT, possessing relatively high sensitivity and specificity; applying <44% catheter to vein ratio can decrease the risk of PICC-UEDVT occurrence in patients at ICU.
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AIM:To investigate the role of microRNA-132 (miR-132) on alveolar macrophage inflammation. METHODS: Rat alveolar macrophage cell line NR8383 was transfected with miR-132 mimic, mimic negative control ( NC) , miR-132 inhibitor, or inhibitor NC.The cells were divided into transfection group, transfection +lipopolysaccha-ride ( LPS) group, and transfection +LPS +acetylcholine ( ACh) group.The mRNA expression of acetylcholinesterase ( AChE) was detected by real-time PCR.The protein levels of AChE, signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 (p-STAT3) in the cells, and nuclear factor-κB (NF-κB) in the cytoplasm and nu-cleus were analyzed by Western blot.The activity of AChE in the culture supernatant was measured by AChE activity assay kit.The nuclear translocation of NF-κB was detected by immunofluorescence assay.RESULTS: Up-regulation or down-regulation of miR-132 had no effect on the mRNA expression of AChE.However, up-regulation of miR-132 decreased the protein level of AChE compared with mimic NC group (P<0.05).Transfection with miR-132 inhibitor increased the pro-tein expression of AChE compared with inhibitor NC group ( P<0.05 ) .In the alveolar macrophages treated with LPS+ACh, the inhibition of nuclear translocation of NF-κB p65 in miR-132 mimic group was more effective than that in mimic NC group ( P<0.05) .The inhibitory effect in miR-132 inhibitor group was weaker than that in inhibitor NC group ( P<0.05 ) .The inhibitory effect of miR-132 mimic on the protein levels of STAT3 and p-STAT3 was stronger than that of mimic NC (P<0.05).CONCLUSION:miR-132 in LPS-stimulated alveolar macrophages reinforced ACh-mediated anti-inflam-matory reaction by targeting AChE to suppress ACh hydrolyzation, which was related to the suppression of NF-κB and STAT3 activation.
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ObjectiveTo observe the effect of acetylcholine (ACh) on lipopolysaccharide (LPS) induced inflammatory model of rat alveolar macrophages, and to observe the effect of the acetylcholinesterase inhibitor physostigmine (Phy) on the anti-inflammatory effect of ACh.Methods The rat alveolar macrophages NR8383 were cultured in vitro, which were divided into five groups: blank control group, LPS group (stimulated with 1 mg/L LPS for 12 hours), LPS+ ACh group (0.01, 0.1, 1, 10, 100μmol/L of ACh were added for 5 minutes before LPS stimulation), LPS+ Phy group (1 mmol/L Phy was added for 5 minutes before LPS stimulation), and LPS+ ACh+ Phy group (1 mmol/L Phy and 10μmol/L ACh were added for 5 minutes before LPS stimulation). The supernatants were collected in each group, the enzyme-linked immunosorbent assay (ELISA) was used to assay the contents of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, and IL-6). The activity of acetylcholine esterase (AChE ) in the supernatant was also determined.Results① The contents of TNF-α (ng/L: 605.09±57.13 vs. 34.07±8.62), IL-1β (ng/L: 377.09±28.55 vs. 32.33±10.62) and IL-6 (ng/L: 558.04±77.45 vs. 42.62±11.21) in the LPS group were significantly higher than those in the blank control group (allP 0.05). Nevertheless, 10μmol/L and 100μmol/L ACh notably reduced the production of TNF-α (ng/L: 451.19±30.67, 332.19±32.19 vs. 604.96±22.56), IL-1β(ng/L: 261.08±24.78, 143.98±28.39 vs. 367.06±10.44) and IL-6 (ng/L: 342.75±54.60, 235.48±29.75 vs. 562.69±63.34) in the culture supernatants compared with the LPS group (allP< 0.05).③ The activity of AChE in the LPS group was significantly higher than that in the blank control group (kU/L: 5.21±0.63 vs. 3.09±0.10,P< 0.05). The activity of AChE was successfully inhibited by 1 mmol/L acetylcholinesterase inhibitor Phy pretreatment compared with that in the LPS group (1.51±0.12 vs. 5.21±0.63,P< 0.05).④ The level of TNF-α (ng/L: 183.17±35.44 vs. 451.19±30.67), IL-1β (ng/L: 91.49±12.27 vs. 261.08±24.78) and IL-6 (ng/L: 108.17±22.82 vs. 342.75±54.60) in the culture supernatants of LPS+ ACh+ Phy group was significantly decreased as compared with LPS+ ACh group (allP< 0.05).Conclusions ACh with the final concentrations of 10μmol/L and 100μmol/L can inhibit the LPS induced inflammatory reaction in alveolar macrophages. The acetylcholinesterase inhibitor Phy can reinforce the ACh-mediated anti-inflammatory effect on alveolar macrophages inflammatory model.
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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.
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<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>
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Aged , Female , Humans , Male , Middle Aged , Antiviral Agents , Therapeutic Uses , Fluoroquinolones , Therapeutic Uses , Imipenem , Therapeutic Uses , Influenza A Virus, H10N8 Subtype , Virulence , Influenza, Human , Diagnosis , Drug Therapy , Oseltamivir , Therapeutic UsesABSTRACT
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.
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Objective To evaluate the efficacy of early continuous high-volume-hemofiltration in the treatment of patients with severe acute pancreatitis (SAP).Methods Based on the method of prospective,randomized and controlled clinical trial,60 patients with SAP between January 2005 and July 2011 from the First Affiliated Hospital of Nanchang University were divided into control group and hemofiltration group.The hemofiltration group was treated with early continuous high-volume-hemofiltration and not in the control group.The changes of vital signs,clinical symptoms and laboratory indicators were compared between the two groups before and after the treatment.Results After hemofiltration,the clinical symptoms such as abdominal pain,fever,tachycardia and respiratory distress in hemofiltration group were significantly remitted compared to those in the control group (P ＜0.05).The APACHE Ⅱ score (13.3 ± 1.0 vs 14.1 ± 1.2) and the level of TBil[(20.4±11.3) μmol/L vs (28.1 ±10.9) μmol/L],creatinine[(178.7 ±71.8)μmol/L vs (215.6 ± 51.3) μmol/L],blood urea nitrogen[(10.1 ± 5.6) mmol/L vs (13.2 ± 3.8) mmol/L] and ALT[(51.3 ± 13.2) U/L vs (62.5 ±14.3) U/L] were decreased compared to those in the control group (all P values ＜0.05).The level of PaO2/FiO2(197.3 ±32.4 vs 178.3 ±31.7) was increased (P ＜ 0.05).After hemofiltration,heart rate was decreased gradually (P ＜ 0.05) in the hemofiltration group than in the control group.Mean artery pressure (mAP) increased gradually (P ＜ 0.05) in the hemofiltration group than in the control group.Conclusion Early continuous high-volume-hemofiltration has significant effects on the treatment of SAP including the improvement of clinic symptoms,the blockade of development from systemic inflammatory response syndrome (SIRS) to multiple organ dysfuction syndrome(MODS),improvement of organ function and prevention from the complications.It may become one of the important therapies for SAP.
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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.