Chromosome evolution of Escherichia coli Nissle 1917 for high-level production of heparosan.

Heparosan is a crucial-polysaccharide precursor for the chemoenzymatic synthesis of heparin, a widely used anticoagulant drug. Presently, heparosan is mainly extracted with the potential risk of contamination from Escherichia coli strain K5, a pathogenic bacterium causing urinary tract infection. Here, a nonpathogenic probiotic, E. coli strain Nissle 1917 (EcN), was metabolically engineered to carry multiple copies of the 19-kb kps locus and produce heparosan to 9.1 g/L in fed-batch fermentation. Chromosome evolution driven by antibiotics was employed to amplify the kps locus, which governed the synthesis and export of heparosan from EcN at 21 mg L-1 OD-1 . The average copy number of kps locus increased from 1 to 24 copies per cell, which produced up to 104 mg L-1 OD-1 of heparosan in the shaking flask cultures of engineered strains. The following in-frame deletion of recA stabilized the recombinant duplicates of chromosomal kps locus and the productivity of heparosan in continuous culture for at least 56 generations. Fed-batch fermentation of the engineered strain EcN8 was carried out to bring the yield of heparosan up to 9.1 g/L. Heparosan from the fermentation culture was further purified at a 75% overall recovery. The structure of purified heparosan was characterized and further modified by N-sulfotransferase with 3'-phosphoadenosine-5'-phosphosulfate as the sulfo-donor. The analysis of element composition showed that heparosan was N-sulfated by over 80%. These results indicated that duplicating large DNA cassettes up to 19-kb, followed by high-cell-density fermentation, was promising in the large-scale preparation of chemicals and could be adapted to engineer other industrial-interest bacteria metabolically.

Inhibition of bacterial swimming by heparin binding of flagellin FliC from Escherichia coli strain Nissle 1917.

Escherichia coli Nissle1917 (EcN) is a non-pathogenic probiotic strain widely used to maintain gut health, treat gastrointestinal disorders, and modulate the gut microbiome due to its anti-inflammatory and competitive exclusion effects against pathogenic bacteria. Heparin, abundant on intestinal mucosal surfaces, is a highly sulfated glycosaminoglycan primarily produced by mast cells. Currently, the interaction between EcN surface protein and heparin has remained elusive. In this study, the flagellin FliC responsible for EcN's movement was separated and characterized as a heparin binding protein by mass spectrometry (MS) analysis. The recombinant FliC protein, expressed by plasmid pET28a( +)-fliC, was further prepared to confirm the interaction between FliC and heparin. The results showed that heparin-Sepharose's ability to bind FliC was 48-fold higher than its ability to bind the negative control, bovine serum albumin (BSA). Neither the knockout of gene fliC nor the addition of heparin affects the growth of EcN, but both significantly inhibit the swimming of EcN. Adding 10 mg/ml heparin reduced the swimming diameter of the wild type and the complemented strain to 29-41% of the original, but that did not affect the swimming ability of the knockout strains. These results demonstrate that heparin interacts with EcN flagellin FliC and inhibits bacteria swimming. Exploring this interaction could improve our understanding of the relationship between hosts and microorganisms and provide a potential basis for disease treatment.

SN50 attenuates alveolar hypercoagulation and fibrinolysis inhibition in acute respiratory distress syndrome mice through inhibiting NF-κB p65 translocation.

BACKGROUND: It has been confirmed that NF-κB p65 signaling pathway is involved in the regulation of alveolar hypercoagulation and fibrinolysis inhibition in acute respiratory distress syndrome (ARDS). Whether SN50, a NF-κB cell permeable inhibitor, could attenuate alveolar hypercoagulation and fibrinolysis inhibition in ARDS remains to be elucidated. PURPOSE: We explored the efficacy and potential mechanism of SN50 on alveolar hypercoagulation and fibrinolysis inhibition in ARDS in mice. MATERIALS AND METHODS: Mouse ARDS was made by 50 µl of lipopolysaccharide (LPS) (4 mg/ml) inhalation. Male BALB/c mice were intraperitoneally injected with different does of SN50 1 h before LPS inhalation. Lung tissues were collected for hematoxylin-eosin (HE) staining, wet/dry ratio. Pulmonary expressions of tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), collagen III, as well as phosphorylated p65 (p-p65), p65 in nucleus (p'-p65), IκBα and IKKα/ß were measured. Bronchoalveolar lavage fluid (BALF) was gathered to test the concentrations of TF, PAI-1, activated protein C (APC) and thrombinantithrombin complex (TAT). DNA binding activity of NF-κB p65 was also determined. RESULTS: After LPS stimulation, pulmonary edema and exudation and alveolar collapse occured. LPS also stimulated higher expressions of TF and PAI-1 in lung tissues, and higher secretions of TF, PAI-1, TAT and low level of APC in BALF. Pulmonary collagen III expression was obviously enhanced after LPS inhalation. At same time, NF-κB signaling pathway was activated with LPS injury, shown by higher expressions of p-p65, p'-p65, p-IKKα/ß, p-Iκα in pulmonary tissue and higher level p65 DNA binding activity. SN50 dose-dependently inhibited TF, PAI-1 and collagen IIIexpressions, and decreased TF, PAI-1, TAT but increased APC in BALF. SN50 treatment attenuated pulmonary edema, exudation and reduced lung tissue damage as well. SN50 application significantly reduced p'-p65 expression and weakened p65 DNA binding activity, but expressions of p-p65, p-IKKα/ß, p-Iκα in cytoplasm of pulmonary tissue were not affected. CONCLUSIONS: SN 50 attenuates alveolar hypercoagulation and fibrinolysis inhibition in ARDS via inhibition of NF-κB p65 translocation. Our data demonstrates that NF-κB p65 pathway is a viable new therapeutic target for ARDS treatment.

Managed grassland alters soil N dynamics and N2O emissions in temperate steppe.

Reclamation of degraded grasslands as managed grasslands has been increasingly accelerated in recent years in China. Land use change affects soil nitrogen (N) dynamics and nitrous oxide (N2O) emissions. However, it remains unclear how large-scale grassland reclamation will impact the grassland ecosystem as a whole. Here, we investigated the effects of the conversion from native to managed grasslands on soil N dynamics and N2O emissions by field experiments in Hulunber in northern China. Soil (0-10cm), nitrate (NO3-), ammonium (NH4+), and microbial N were measured in plots in a temperate steppe (Leymus chinensis grassland) and two managed grasslands (Medicago sativa and Bromus inermis grasslands) in 2011 and 2012. The results showed conversion of L. chinensis grassland to M. sativa or B. inermis grasslands decreased concentrations of NO3--N, but did not change NH4+-N. Soil microbial N was slightly decreased by the conversion of L. chinensis grassland to M. sativa, but increased by the conversion to B. inermis. The conversion of L. chinensis grassland to M. sativa (i.e., a legume grass) increased N2O emissions by 26.2%, while the conversion to the B. inermis (i.e., a non-legume grass) reduced N2O emissions by 33.1%. The conversion from native to managed grasslands caused large created variations in soil NO3--N and NH4+-N concentrations. Net N mineralization rates did not change significantly in growing season or vegetation type, but to net nitrification rate. These results provide evidence on how reclamation may impact the grassland ecosystem in terms of N dynamics and N2O emissions.

Evaluation and intercomparison of MODIS and GEOV1 global leaf area index products over four sites in North China.

This study investigated the performances of the Moderate Resolution Imaging Spectroradiometer (MODIS) and GEOLAND2 Version 1 (GEOV1) Leaf Area Index (LAI) products using ground measurements and LAI reference maps over four sites in North China for 2011-2013. The Terra + Aqua MODIS and Terra MODIS LAI retrieved by the main algorithm and GEOV1 LAI within the valid range were evaluated and intercompared using LAI reference maps to assess their uncertainty and seasonal variability The results showed that GEOV1 LAI is the most similar product with the LAI reference maps (R2 = 0.78 and RMSE = 0.59). The MODIS products performed well for biomes with low LAI values, but considerable uncertainty arose when the LAI was larger than 3. Terra + Aqua MODIS (R2 = 0.72 and RMSE = 0.68) was slightly more accurate than Terra MODIS (R2 = 0.57 and RMSE = 0.90) for producing slightly more successful observations. Both MODIS and GEOV1 products effectively followed the seasonal trajectory of the reference maps, and GEOV1 exhibited a smoother seasonal trajectory than MODIS. MODIS anomalies mainly occurred during summer and likely occurred because of surface reflectance uncertainty, shorter temporal resolutions and inconsistency between simulated and MODIS surface reflectances. This study suggests that further improvements of the MODIS LAI products should focus on finer algorithm inputs and improved seasonal variation modeling of MODIS observations. Future field work considering finer biome maps and better generation of LAI reference maps is still needed.

Effects of different grassland utilization methods on the germinable soil seed bank of the Hulunbuir meadow steppe.

Seed banks are crucial regenerative resources for aboveground vegetation. The pattern of their changes holds immense significance in understanding alterations in the belowground seed bank. This understanding is pivotal for uncovering both short-term and long-term shifts in plant communities. Additionally, it contributes to the restoration of grassland ecosystems. To better protect grassland biodiversity and provide a theoretical basis for the restoration of degraded grasslands, in this study, the germination characteristics of soil seed banks in free-grazed, enclosed and mown areas were compared, and the results were combined with those of previous studies for a comprehensive analysis. The density of soil seed bank and perennial forage soil seed bank were significantly affected by different grassland utilization and soil depths. Grazing and enclosure grassland utilization methods increased the content of the soil seed bank, and mowing reduced the content of the seed bank. The soil seed bank density of perennial grasses accounted for the highest proportion under grazing, followed by mowing, and its lowest proportion was observed in the enclosures. Grazing not only facilitated the germination of the perennial grass seed bank but also substantially augmented its content. Mowing inhibited the germination of the upper growth grasses seed bank, which was particularly significant in the 0-2 cm soil layer under grazing. The content of the upper growth grasses seed bank affected the total seed bank to a certain extent, mainly in the 5-10 cm layer. The general correlations among the perennial grasses, upper growth grasses and soil germination seed bank resulted in 84.58% information extraction, and this information has practical significance for grassland ecological restoration.

Sown alfalfa pasture decreases grazing intensity while increasing soil carbon: Experimental observations and DNDC model predictions.

Introduction: Grasslands are the most important land use in China and have experienced extensive degradation in the past few decades due to overgrazing. However, regionally viable solutions to grazing intensity alleviation remained elusive to date. Methods: Here, we evaluated the grazing intensity effects of sown alfalfa pastures in northern China using an experiment-modeling combined approach that involved six sites in field experiments and five provinces in DNDC modeling of sown alfalfa pasture's forage production and carbon sequestration potentials in marginal lands. Results: Our results showed that the sown alfalfa pasture's dry-matter yield varied between 4.5 and 9.0 Mg ha-1 under rainfed and irrigated conditions, respectively, from 2025 to 2035. If half of the available marginal lands were mobilized for alfalfa forage production, these yield levels meant that livestock grazing intensity on natural grasslands may drop 8-13% under rainfed and 20-33% under irrigated conditions. Our results also showed that marginal land's soil organic carbon contents were systematically higher under sown alfalfa pasture than under fallow management by a big margin of 8.5 and 9.9 g kg-1 (i.e., +79 and +95%), under rainfed and irrigated conditions, respectively, during 2025-2035. Discussion: Overall, these results demonstrated that sown alfalfa pasture on marginal lands represents an effective grassland conservation pathway over the short- to medium-term time horizon based on current technologies.

Andrographolide Suppresses Expressions of Coagulation and Fibrinolytic Inhibition-Related Factors in LPS-Induced Alveolar Epithelial Cell Type II via NF-κB Signal Pathway In Vitro.

Background: Andrographolide (Andro) has been confirmed to ameliorate alveolar hypercoagulation and fibrinolysis inhibition via NF-κB pathway in acute respiratory distress syndrome (ARDS), but the specific target of Andro is unknown. Purpose: Our aim is to explore the specific target of Andro through which the drug exerted its effects on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS. Methods: AECII was treated with different doses of Andro for 1 h, and then stimulated with LPS for 24 h. Expressions of tissue factor (TF), plasminogen activator inhibitor (PAI)-1 and tissue factor pathway inhibitor (TFPI) were detected. Concentrations of thrombin-antithrombin complex (TAT), pro-collagen type III peptide (PIIIP), antithrombin III (ATIII) and activated protein C (APC) in cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). NF-κB signaling pathways activation was simultaneously determined. AECII with p65 down-/over-expression were used as control. Results: Andro effectively inhibited TF and PAI-1 and promoted TFPI expressions on AECII induced by LPS stimulation. Andro also significantly suppressed the productions of TAT and PIIIP but promoted ATIII and APC secretions from the LPS-treated cell. Furthermore, Andro application obviously inhibited NF-κB signaling pathway activation provoked by LPS, as shown by decreased level of phosphorylation (p­)-IKKß/IKKß, p-p65/p65 and p65 DNA binding activity. The effects of Andro on those factors were obviously strengthened by down- but were weakened by up-regulation of p65 gene in AECII cell. Conclusions: Our data demonstrates that targeting AECII is the mechanism by which Andro ameliorates alveolar hypercoagulaiton and fibrinolytic inhibition via NF-κB pathway in ARDS. Andro is worth to be clinically further studied in ARDS treatment.

Novel botanical active component derivatives containing carboxamide and 1,3,4-Thiadiazole thioether moieties: Design, synthesis, and inhibitory activity.

In this study, using the botanical active components of carvacrol, thymol, guaiacol, and sesamol as the lead structures, 19 novel botanical active component derivatives containing carboxamide and 1,3,4-thiadiazole thioether moieties (5a-5s) were synthesized and structurally characterized by 1H NMR, 13C NMR, and HRMS. The antibacterial bioassay results in vitro showed that compound 2-(2-methoxyphenoxy)-N-(5-(methylthio)-1,3,4-thiadiazol-2-yl)acetamide (5k) revealed excellent inhibitory activities against Xanthomonas axonopodis pv. citri (Xac) and Xanthomonas oryzae pv. oryzicolaby (Xoc), with the median effective concentration (EC50) values of 22 and 15 µg/ml, respectively, which were even better than those of thiodiazole copper and bismerthiazol. Meanwhile, all the target compounds revealed lower in vitro inhibitory effects on Mucor bainieri (M. bainieri), Mucor fragilis (M. fragilis), and Trichoderma atroviride (T. atroviride), than carbendazim.

Sandwich structure Aloin-PVP/Aloin-PVP-PLA/PLA as a wound dressing to accelerate wound healing.

We have prepared a new type of Aloin/Polyvinylpyrrolidone (PVP)-Aloin/PVP/polylactic acid (PLA)-PLA sandwich nanofiber membrane (APP), to achieve a time-regulated biphasic drug release behavior, used for hemostasis, antibacterial activity and accelerated wound healing. We tested the water absorption capacity, water contact angle, tensile strength, thermogravimetric analysis, Fourier transform infrared spectroscopy and in vitro drug release of the prepared material, as well as analyzed the morphology of the nanofiber membrane with a scanning electron microscope. In the wound healing experiment, the wound healing rate of APP on the 15th day was 96.67%, and it demonstrated excellent antibacterial activity by the disc diffusion method, showing superior antibacterial activity against Gram-negative bacteria. The skin defect model on the back of mice showed that APP nanofibers significantly induced granulation tissue growth, collagen deposition and epithelial tissue remodeling. Current research shows that the prepared composite nanofibers can quickly stop bleeding and can effectively promote wound healing.

Binding domain peptide ameliorates alveolar hypercoagulation and fibrinolytic inhibition in mice with lipopolysaccharide-induced acute respiratory distress syndrome Via NF-κB signaling pathway.

BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition are shown to be associated with refractory hypoxemia in acute respiratory distress syndrome (ARDS), and the NF-κB pathway is involved in this process. The purpose of this study is to explore the role of NEMO-binding domain peptide (NBDP) in alleviating alveolar hypercoagulation and fibrinolytic inhibition induced by lipopolysaccharide (LPS) in ARDS mice and its related mechanisms. MATERIALS AND METHODS: ARDS was induced by inhalation of LPS (mg/L) in adult male BALB/c mice. Mice were treated with intratracheal inhalation of NBDP or saline aerosol at increased concentrations 30 minutes before LPS administration. Six hours after LPS treatment, bronchoalveolar lavage fluids (BALF) were collected and then all mice were euthanized. In addition, coagulation and fibrinolysis associated factors in lung tissues and BALF were detected, and the activation of NF-κB signaling pathway was observed. RESULTS: NBDP pretreatment dose-dependently inhibited the expression of tissue factor (TF) and plasminogen activator inhibitor (PAI) 1 in lung tissues, reduced the secretions of TF, PAI-1, thrombin-antithrombin (TAT) complex, and promoted activated protein C (APC) secretion in BALF induced by LPS. LPS-induced high expression of pulmonary procollagen peptide type lll (PIIIP) was also reduced in a dose-dependent manner under NBDP pretreatment. Western blotting showed that NBDP pretreatment significantly attenuated LPS-induced activation of IKKα/ß, Iκα and NF-κB p65. NBDP pretreatment also inhibited the DNA binding activity of p65 induced by LPS. We also noticed that NBDP protected mice against LPS-induced lung injury in a dose-dependent manner. CONCLUSIONS: The experimental findings demonstrate that through inhibiting the NF-κB signaling pathway, NBDP dose-dependently ameliorates LPS-induced alveolar hypercoagulation and fibrinolytic inhibition, which is expected to be a new therapeutic target to correct the abnormalities of alveolar coagulation and fibrinolytic pathways in ARDS.

Andrographolide sulfonate attenuates alveolar hypercoagulation and fibrinolytic inhibition partly via NF-κB pathway in LPS-induced acute respiratory distress syndrome in mice.

BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition are important characteristics during acute respiratory distress syndrome (ARDS), and NF-κB p65 signaling pathway is involved to regulate these pathophysiologies. We hypothesize that targeting NF-κB signal pathway could ameliorate alveolar hypercoagulation and fibrinolyitc inhibition, thus attenuating lung injury in ARDS. PURPOSE: We explore the efficacy and the potential mechanism of andrographolide sulfonate (Andro-S) on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS in mice. METHODS: ARDS was made by lipopolysaccharide (LPS) inhalation in C57BLmice. Andrographolide sulfonate (2.5, 5 and 10 mg/kg) was intraperitoneally given to the mice (once a day for three consecutive days) before LPS administration. NEMO binding domain peptide (NBD), an inhibitor of NF-κB, was used as the positive control and it replaced Andro-S in mice of NBD group. Mice in normal control received saline instead of LPS. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis of alveolar coagulation, fibrinolytic inhibition as well as of pulmonary inflammatory response after 8 h of LPS inhalation. NF-κB signal pathway in lung tissue was simultaneously determined. RESULTS: Andro-S dose-dependently inhibited tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 expressions either in mRNA or in protein in lung tissue of ARDS mice, and it also decreased the concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) while promoting the production of activated protein C (APC) in BALF. Meanwhile, Andro-S effectively inhibited inflammatory response (interleukin 1ß and myeloperoxidase) induced by LPS. LPS stimulation dramatically activated NF-κB signal pathway, indicated by increased expressions of phosphorylation of p65 (p-p65), p-IKKα/ß and p-IκBα and the higher p65-DNA binding activity, which were all dose-dependently reversed by Andro-S. Andro-S and NBD presented similar efficacies. CONCLUSIONS: Andro-S treatment improves alveolar hypercoagulation and fibrinolytic inhibition and attenuates pulmonary inflammation in LPS-induced ARDS in mice partly through NF-κB pathway inactivation. The drug is expected to be an effective choice for ARDS.

Triptolide dose-dependently improves LPS-induced alveolar hypercoagulation and fibrinolysis inhibition through NF-κB inactivation in ARDS mice.

BACKGROUND: Alveolar hypercoagulation and fibrinolysis inhibition were associated with the refractory hypoxemia and the high mortality in patient with acute respiratory distress syndrome (ARDS), and NF-κB pathway was confirmed to contribute to the process. Triptolide (TP) significantly inhibited NF-κB pathway and thus depressed accessive inflammatory response in ARDS. We speculate that TP could improve alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS via NF-κB inactivation. PURPOSE: The aim of this experiment was to explore the efficacy and potential mechanism of TP on alveolar hypercoagulation and fibrinolysis inhibition in LPS-induced ARDS in mice. METHODS: 50 µl of LPS (5 mg/ml) was inhalationally given to C57BL/6 mice to set up ARDS model. Male mice were randomly accepted with LPS, LPS + TP (1 µg/kg, 10 µg/kg, 50 µg/kg respectively), or with NEMO Binding domain peptide (NBD), an inhibitor of NF-κB. TP (1 µg/kg, 10 µg/kg, 50 µg/kg) were intraperitoneally injected or 10 µg/50 µl of NBD solution were inhaled 30 min before LPS inhalation. A same volume of normal saline (NS) substituted for TP in mice in control. The endpoint of experiment was at 8 hours after LPS stimulation. Pulmonary tissues were taken for hematoxylin-eosin (HE) staining, wet / dry ratio and for lung injury scores (LIS). Tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue were detected by Western-blotting and by quantitative Real-time PCR(qPCR) respectively. Concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) and activated protein C (APC) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. NF-κB activation and p65-DNA binding activity in pulmonary tissue were simultaneously determined. RESULTS: LPS stimulation resulted in pulmonary edema, neutrophils infiltration, obvious alveolar collapse, interstitial congestion, with high LIS, which were all dose-dependently ameliorated by Triptolide. LPS also dramatically promoted the expressions of TF and PAI-1 either in mRNA or in protein in lung tissue, and significantly stimulated the secretions of TF, PAI-1, TAT, PⅢP but inhibited APC production in BALF, which were all reversed by triptolide treatment in dose-dependent manner. TP dose-dependently inhibited the activation of NF-κB pathway induced by LPS, indicated by the changes of phosphorylations of p65 (p-p65), p-IKKα/ß and p-IκBα, and weakened p65-DNA binding activity. TP and NBD had same efficacies either on alveolar hypercoagulation and fibrinolysis inhibition or on NF-κB signalling pathway in ARDS mice. CONCLUSIONS: TP dose-dependently improves alveolar hypercoagulation and fibrinolysis inhibition in ARDS mice through inhibiting NF-κB signaling pathway. Our data demonstrate that TP is expected to be an effective selection in ARDS.

[Berberine dose-dependently inhibits the expression of procoagulant and fibrinolytic inhibitory factors in lipopolysaccharide-induced rat type II alveolar epithelial cells].

OBJECTIVE: To observe the effects of berberine on procoagulant and fibrinolytic inhibitory factors produced by rat type II alveolar epithelial cell (AEC II) induced by lipopolysaccharide (LPS). METHODS: AEC II cells (RLE-6TN cells) were cultured in vitro, and the cells in logarithmic growth phase were collected. The cytotoxicity text of berberine was detected by cell counting kit-8 (CCK-8) to determine the drug concentration range according to inhibition concentration of half cells (IC50). The RLE-6TN cells were divided into five groups, the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 5 mg/L LPS; and the cells in berberine pretreatment groups were pretreated with 20, 50 and 80 µmol/L berberine for 1 hour, and then were co-cultured with 5 mg/L LPS. The cells were collected after LPS induced for 24 hours. The protein and mRNA expression levels of tissue factor (TF), tissue factor pathway inhibitor (TFPI) and plasminogen activator inhibitor-1 (PAI-1) in the cells were detected by Western blotting and real-time fluorescence quantification reverse transcription-polymerase chain reaction (RT-qPCR). The levels of activated protein C (APC), precollagen III peptide (PIIIP), thrombin-antithrombin complex (TAT) and antithrombin III (AT III) in the cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). RESULTS: According to the inhibition rate curve, the IC50 of berberine on RLE-6TN cells was 81.16 µmol/L. Therefore, 20, 50 and 80 µmol/L were selected as the intervention concentration of berberine. Compared with the blank control group, the expression and secretion of procoagulant and fibrinolytic inhibitory factors were abnormal in RLE-6TN cells after LPS induced for 24 hours. The protein and mRNA expression levels of TF and PAI-1 in the LPS group were significantly increased, but the protein and mRNA expression levels of TFPI were significantly decreased. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly decreased, while the levels of PIIIP and TAT were significantly increased. After pretreatment with berberine, the abnormal expression and secretion of procoagulant and fibrinolytic inhibitory factors induced by LPS were corrected in a dose-dependent manner, especially in 80 µmol/L. Compared with the LPS group, the protein and mRNA expression levels of TF and PAI-1 in the berberine 80 µmol/L group were significantly decreased [TF protein (TF/GAPDH): 0.45±0.02 vs. 0.55±0.03, TF mRNA (2-ΔΔCt): 0.39±0.08 vs. 1.48±0.11, PAI-1 protein (PAI-1/GAPDH): 0.37±0.02 vs. 0.64±0.04, PAI-1 mRNA (2-ΔΔCt): 1.14±0.29 vs. 4.18±0.44, all P < 0.01] and those of TFPI were significantly increased [TFPI protein (TFPI/GAPDH): 0.53±0.02 vs. 0.45±0.02, TFPI mRNA (2-ΔΔCt): 0.94±0.08 vs. 0.40±0.05, both P < 0.01]. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly increased [APC (µg/L): 1 358.5±26.0 vs. 994.2±23.1, AT III (µg/L): 118.0±7.4 vs. 84.4±2.7, both P < 0.01], while those of PIIIP and TAT were significantly decreased [PIIIP (µg/L): 11.2±0.4 vs. 18.6±0.9, TAT (ng/L): 222.1±2.8 vs. 287.6±7.0, both P < 0.01]. CONCLUSIONS: Berberine could inhibit the LPS-induced expressions of procoagulant and fibrinolytic inhibitory factors in rat AEC II cells and promote the expressions of anticoagulant factors in a dose-dependent manner. Berberine may be a new therapeutic target for alveolar hypercoagulability and fibrinolysis inhibition in acute respiratory distress syndrome (ARDS).

[Effects of andrographolide on the expression of procoagulant and fibrinolytic inhibition factors in rat type II alveolar epithelial cells stimulated by lipopolysaccharide].

OBJECTIVE: To determine the effect of andrographolide (AD) on the expression of procoagulant and fibrinolytic inhibitory factors in rat type II alveolar epithelial cells (AEC II) stimulated by lipopolysaccharide (LPS). METHODS: The AEC II cells RLE-6TN in the logarithmic growth phase were divided into 5 groups: the normal control (NC) group, the LPS group, and the 6.25, 12.5, and 25 mg/L AD groups (AD 6.25 group, AD 12.5 group, AD 25 group). The NC group was cultured with RPMI 1640 conventional medium. In the LPS group, 5 mg/L LPS was added to the RPMI 1640 conventional medium for stimulation. Cells in the AD groups were treated with 6.25, 12.5, and 25 mg/L AD in advance for 1 hour and then given LPS to stimulate the culture. The cells and cell culture supernatant were collected 24 hours after LPS stimulation. The protein and mRNA expressions of tissue factor (TF), tissue factor pathway inhibitor (TFPI), and plasminogen activator inhibition-1 (PAI-1) in cells were detected by Western blotting and real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The levels of procollagen III peptide (PIIIP), thrombin-antithrombin complex (TAT), antithrombin III (AT-III) and activated protein C (APC) in the cell supernatant were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Compared with the NC group, the protein and mRNA expressions of TF and PAI-1 in the LPS group were significantly increased, and the protein and mRNA expressions of TFPI were significantly reduced. At the same time, the levels of PIIIP and TAT in the cell supernatant were significantly increased, the levels of AT-III, APC were significantly reduced. Compared with the LPS group, the protein and mRNA expressions of TF and PAI-1 in AD 6.25 group, AD 12.5 group, AD 25 group were significantly reduced [TF/GAPDH: 0.86±0.08, 0.45±0.04, 0.44±0.04 vs. 1.32±0.10, TF mRNA (2-ΔΔCt): 2.59±0.25, 2.27±0.05, 1.95±0.04 vs. 4.60±0.26, PAI-1/GAPDH: 2.11±0.07, 1.45±0.04, 0.86±0.09 vs. 2.56±0.09, PAI-1 mRNA (2-ΔΔCt): 3.50±0.22, 2.23±0.29, 1.84±0.09 vs. 6.60±0.27, all P < 0.05], while the protein and mRNA expressions of TFPI were significantly increased [TFPI/GAPDH: 0.78±0.05, 0.81±0.03, 0.84±0.07 vs. 0.36±0.02, TFPI mRNA (2-ΔΔCt): 0.46±0.09, 0.69±0.07, 0.91±0.08 vs. 0.44±0.06, all P < 0.05]. Also the levels of PIIIP and TAT in the cell supernatant were significantly reduced, and the levels of AT-III and APC were significantly increased [PIIIP (µg/L): 13.59±0.23, 12.66±0.23, 10.59±0.30 vs. 15.82±0.29, TAT (ng/L): 211.57±6.41, 205.69±4.04, 200.56±9.85 vs. 288.67±9.84, AT-III (µg/L): 102.95±3.86, 123.92±2.63, 128.67±1.67 vs. 92.93±3.36, APC (µg/L): 1 188.95±14.99, 1 366.12±39.93, 1 451.15±29.69 vs. 1 145.55±21.07, all P < 0.05]. With the increase of the dose of AD, the above-mentioned promotion and inhibition effects became more obvious. In the AD 25 group, TF, PAI-1 protein and mRNA expressions decreased, TFPI mRNA expression increased, PIIIP level in the supernatant decreased and AT-III, APC levels increased compared with AD 6.25 group, the difference was statistically significant, and the decrease of PAI-1 protein expression and PIIIP level in the supernatant were also statistically significant compared with AD 12.5 group. CONCLUSIONS: Andrographolide in the dose range of 6.25-25 mg/L can dose-dependently inhibit the expression and secretion of procoagulant and fibrinolytic inhibitor-related factors in AEC II cells RLE-6TN stimulated by LPS, and promote the secretion of anticoagulant factors. 25 mg/L has the most obvious effect.

Preparation of gelatin/poly (γ-glutamic acid) hydrogels with stimulated response by hot-pressing preassembly and radiation crosslinking.

The conventional preparation method of natural polymers is low in strength, which limits the application of multi stimuli responsive hydrogels as intelligent wound dressings. In this work, interpenetrating gelatin and γ-polyglutamic acid (γ-PGA) hydrogels with good biocompatibility, biodegradability and mechanical properties were prepared by hot-pressing pre-gelation of gelatin/γ-PGA and synergistic with gamma irradiation. The effects of radiation crosslinking dosage on gel fraction, swelling ratio, tensile strength and surface morphology were investigated. The biocompatibility of radiation cross-linked hydrogel was evaluated by aseptic test, hemolysis test, cytotoxicity test, delayed hypersensitivity reaction as well as in vivo degradation studies from in vitro to in vivo. The optimized hydrogel irradiated by 25 kGy has good water retention and biodegradability, especially the stimulation of temperature, pH value, salt species and concentration. The mechanical strength, biocompatibility and responsive properties of the hydrogel indicate that the intelligent hydrogel prepared by this method is a good hydrogel biomaterial for developing interactive wound dressing.

Enhanced Low-Neutron-Flux Sensitivity Effect in Boron-Doped Silicon.

Space particle irradiation produces ionization damage and displacement damage in semiconductor devices. The enhanced low dose rate sensitivity (ELDRS) effect caused by ionization damage has attracted wide attention. However, the enhanced low-particle-flux sensitivity effect and its induction mechanism by displacement damage are controversial. In this paper, the enhanced low-neutron-flux sensitivity (ELNFS) effect in Boron-doped silicon and the relationship between the ELNFS effect and doping concentration are further explored. Boron-doped silicon is sensitive to neutron flux and ELNFS effect could be greatly reduced by increasing the doping concentration in the flux range of 5 × 109-5 × 1010 n cm-2 s-1. The simulation based on the theory of diffusion-limited reactions indicated that the ELNFS in boron-doped silicon might be caused by the difference in the concentration of remaining vacancy-related defects (Vr) under different neutron fluxes. The ELNFS effect in silicon becomes obvious when the (Vr) is close to the boron doping concentration and decreased with the increase in boron doping concentration due to the remaining vacancy-related defects being covered. These conclusions are confirmed by the p+-n-p Si-based bipolar transistors since the ELNFS effect in the low doping silicon increased the reverse leakage of the bipolar transistors and the common-emitter current gain (ß) dominated by highly doped silicon remained unchanged with the decrease in the neutron flux. Our work demonstrates that the ELNFS effect in boron-doped silicon can be well explained by noise diagnostic analysis together with electrical methods and simulation, which thus provide the basis for detecting the enhanced low-particle-flux damage effect in other semiconductor materials.

[Meta-analysis of the impact of continuous anticoagulation therapy with low-dose heparin or low molecular weight heparin on the prognosis of patients with acute respiratory distress syndrome].

OBJECTIVE: The effects of low-dose heparin and low molecular weight heparin on the efficacy and prognosis of patients with acute respiratory distress syndrome (ARDS) were systematically evaluated. METHODS: Chinese and English databases such as Wanfang data, VIP database, China National Knowledge Infrastructure (CNKI), China Biomedical Literature Service system (SinoMed), American National Medical Library database (PubMed), Netherlands Medical Abstracts database (Embase) and Cochrane library database were searched for the randomized controlled trials (RCTs) of heparin or low molecular weight heparin in the treatment of ARDS. The search time is from July 1999 to June 2020. The control group was given routine treatment, while the experimental group was given heparin 5-10 U×kg-1×h-1, or low molecular heparin subcutaneous injected 2 500-5 000 U once every 12 hours for 7 days on the basis of the routine treatment. The main outcome were 28-day mortality, arterial oxygen saturation (SaO2), acute physiology and chronic health evaluation II (APACHE II), length of stay in (intensive care unit) ICU and mechanical ventilation time, and the secondary outcome indexes were C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), lung injury score, respiratory frequency, activated partial thromboplastin time (APTT) and prothrombin time (PT) after 7 days' treatment. The two researchers collected data and evaluated the quality of the literature according to the Cochrane 5.1. The stability of the Meta-analysis results was tested by sensitivity analysis, and the publication bias was included in the funnel chart analysis. RESULTS: Fourteen Chinese literatures were included, with a total of 894 cases, including 454 cases in the experimental group and 440 cases in the control group. The results of Meta-analysis showed that the 28-day fatality rate in the experimental group was significantly lower than that in the control group [relative risk (RR) = 0.54, 95% confidence interval (95%CI) was 0.38-0.76, P = 0.000 4], and 7-day SaO2 significantly increased [heparin: mean difference (MD) = 48.27, 95%CI was 29.95-66.59, P < 0.000 01]. Low molecular weight heparin: MD = 56.67, 95%CI was 41.22-71.13, P < 0.000 01]. Compared with the control group, the APACHE II score (MD = -4.28, 95%CI was -5.15 to -3.42, P < 0.000 01), the lung injury score (MD = -1.19, 95%CI was -1.35 to -1.03, P < 0.000 01) and the respiratory rate (MD = -4.76, 95%CI was -6.26 to -3.26, P < 0.000 01) in the experimental group were significantly lower than those in the control group after 7 days' treatment. The time of staying in ICU (MD = -4.85, 95%CI was -6.94 to -2.76, P < 0.000 01) and the time of mechanical ventilation (MD = -2.93, 95%CI was -3.34 to -2.52, P < 0.000 01) in the experimental group were lower than those in the control group. After 7 days' treatment, the levels of IL-6 (MD = -38.50, 95%CI was -59.01 to -17.99, P < 0.000 01), TNF-α (MD = -16.24, 95%CI was -31.11 to -1.38, P < 0.000 01) and CRP (MD = -5.5, 95%CI was -6.47 to -4.27, P < 0.000 01) were significantly lower in the experimental group than those in the control group. However, after 7 days' treatment, there were no significant differences in APTT (MD = -0.55, 95%CI was -1.61 to 0.51, P = 0.27) or PT (MD = -0.41, 95%CI was -1.48 to 0.66, P = 0.45). For the indicators with high heterogeneity, Meta-analysis was carried out again by excluding any study, and the overall effect value did not change significantly, suggesting that the results were relatively robust. Funnel chart analysis was carried out on the indexes with more than 10 articles included in the literature. The results showed that the literature had publication bias, but the bias was small. CONCLUSIONS: Continuous anticoagulation therapy with low dose heparin and low molecular weight heparin can improve the prognosis and reduce the mortality of patients with ARDS.

Emodin improves alveolar hypercoagulation and inhibits pulmonary inflammation in LPS-provoked ARDS in mice via NF-κB inactivation.

BACKGROUND: Alveolar hypercoagulation and pulmonary inflammation are important characteristics and they regulate each other in acute respiratory distress syndrome (ARDS). NF-κB pathway has been confirmed to be involved in regulation of this crosstalk. Emodin, a traditional Chinese herb, shows potent inhibitory effect on NF-κB pathway, but whether it is effective in alveolar hypercoagulation and pulmonary inflammation in ARDS remains to be elucidated. PURPOSE: The aim of this experiment was to evaluate the efficacy of emodin on LPS-provoked alveolar hypercoagulation and excessive pulmonary inflammation in ARDS, and its potential mechanism. METHODS: Mice ARDS was set up through LPS (40 µl, 4 mg/ml) inhalation. Male mice were randomly received with BPS, LPS only, LPS+ emodin (5 mg/kg, 10 mg/kg, 20 mg/kg, respectively) and BAY65-1942, an inhibitor of IKKß. After 48 h of LPS stimulation, pulmonary pathological injury, expressions of Tissue factor (TF), plasminogen activator inhibitor (PAI)-1, activated protein C (APC), collagen Ⅰ, collagen III, interleukin (IL) 8, IL-1ß and tumor necrosis factor (TNF)-α in lung tissues, as well as concentrations of antithrombin III (AT III), procollagen peptide type III (PIIIP), soluble thrombomodulin (sTM), thrombin antithrombin complex (TAT), myeloperoxidase (MPO) and the percentage of inflammatory cells in bronchoalveolar lavage fluid (BALF) were all determined. NF-κB pathway activation as well as NF-κB DNA binding activity in pulmonary tissue were simultaneously checked. RESULTS: LPS stimulation resulted in obvious lung injury, excessive inflammatory cells infiltration, which all were dose-dependently ameliorated by emodin. Expressions of TF, PAI-1, collagen Ⅰ and collagen III as well as IL-8, IL-1ß and TNF-α in pulmonary tissue were all elevated while APC decreased under LPS provocation, which were all reversed by emodin treatment in dose-dependent manner. LPS promoted the secretions of PIIIP, sTM, TAT and inhibited AT III production in BALF, and resulted in high levels of MPO and the percentage of inflammatory cells in BALF, all of which were significantly and dose-dependently attenuated while AT III production was increased by emodin. Meanwhile, emodin effectively inhibited NF-κB pathway activation and attenuated p65 DNA binding activity induced by LPS inhalation. Emodin and BAY-65-1942 had similar impacts in this experiment. CONCLUSIONS: Emodin improves alveolar hypercoagulation and fibrinolytic inhibition and depresses excessive pulmonary inflammation in ARDS mice in dose-dependent manner via NF-κB inactivation. Our data demonstrate that emodin is expected to be an effective drug in alveolar hypercoagulation and pulmonary inflammation in ARDS.

[Small dose of low molecular weight heparin improves the prognosis of elderly patients with severe pneumonia: a Meta-analysis of 1 173 patients].

OBJECTIVE: To explore the effect of small dose of low molecular weight heparin on the prognosis of elderly patients with severe pneumonia using systematic evaluation method. METHODS: Databases including Wanfang data, VIP, CNKI, SinoMed, PubMed, Embase and Cochrane Library were searched for randomized controlled trial (RCT) studies about the comparison of conventional therapy and low molecular weight heparin on prognosis of elderly patients with severe pneumonia from the time of database establishment to August 2019. The patients in conventional treatment group were treated by improving ventilation, anti-infection, eliminating phlegm, relieving asthma and maintaining homeostasis while those in low molecular weight heparin group were subcutaneously injected with low molecular weight heparin of 4 000 U, once a day for 7 days. The patients' main outcomes included the oxygenation index (PaO2/FiO2) after 7 days of treatment, duration of mechanical ventilation, mortality in hospital, and secondary outcomes included acute physiology and chronic health evaluation II (APACHE II) score and coagulation function after 7 days of treatment, the length of intensive care unit (ICU) stay, and incidence of bleeding. Data extraction and quality evaluation were conducted. The Meta-analysis of included studies that met the quality standards was performed using RevMan 5.3 software. Funnel diagram analysis was used to analyze the parameters with no less than 10 studies enrolled. RESULTS: A total of 14 RCT studies were enrolled involving 1 173 elderly patients with severe pneumonia, among whom 590 received low molecular weight heparin while the other 583 received conventional therapy. All the included studies were well designed and of high quality. The results of Meta-analysis showed that compared with conventional therapy, small dose of low molecular weight heparin significantly elevated PaO2/FiO2 after 7 days of treatment [mean difference (MD) = 19.25, 95% confidence interval (95%CI) was 16.88 to 21.61, P < 0.000 01], shortened the duration of mechanical ventilation (MD = -48.88, 95%CI was -67.42 to -30.33, P < 0.000 01), and decreased mortality in hospital [odds ratio (OR) = 0.40, 95%CI was 0.22 to 0.73, P = 0.003] and APACHE II score after 7 days of treatment (MD = -3.38, 95%CI was -3.94 to -2.83, P < 0.000 01), and shortened the length of ICU stay (MD = -4.51, 95%CI was -5.75 to -3.27, P < 0.000 01). There was no significant difference in the changes of coagulation parameters after 7 days of treatment or the incidence of bleeding between low molecular weight heparin group and conventional therapy group [7-day thrombin time (TT): MD = 0.57, 95%CI was -0.15 to 1.28, P = 0.12; 7-day prothrombin time (PT): MD = 0.32, 95%CI was -0.35 to 0.98, P = 0.35; 7-day fibrinogen (FIB): MD = -0.17, 95%CI was -0.45 to 0.10, P = 0.22; incidence of bleeding: OR = 0.86, 95%CI was 0.36 to 2.07, P = 0.74]. The funnel diagram showed that there was publication bias of included 10 studies about APACHE II score after 7 days of treatment. CONCLUSIONS: Small dose of low molecular weight heparin can improve the prognosis of elderly patients with severe pneumonia and it has no obvious side-effect on coagulation function.