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En Cao,1,* Jun Xu,1,* Yuanqi Gong,2,* Jingjing Yuan,3 Anbang Chen,1 Jiayi Liu,4 Yunfei Fan,4 Xiangyang Fan,4 Xiaodong Kuang1 1Department of Pathology, Basic Medical College of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Critical Care Medicine/ICU (Intensive Care Unit), Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 3Department of Physiology, School of Basic Medicine, Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 4The Basic Medical School of Nanchang University, Nanchang, Jiangxi, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaodong Kuang, Department of Pathology, Basic Medical College of Nanchang University, Nanchang, Jiangxi, People’s Republic of China, Email xdssmu2005@163.comBackground: Macrophages are known to play a crucial role in the chronic inflammation associated with Chronic Obstructive Pulmonary Disease (COPD). BML-111, acting as a lipoxin A4 (LXA4) receptor agonist, has shown to be effective in protecting against COPD. However, the precise mechanism by which BML-111 exerts its protective effect remains unclear.Methods: In order to establish a cell model of inflammation, cigarette smoke extract (CSE) was used on the RAW264.7 cell line. Afterwards, an Enzyme-linked immunosorbent assay (ELISA) kit was employed to measure concentrations of tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1β), interleukin-18 (IL-18), and interleukin-10 (IL-10) in the cell supernatants of the RAW264.7 cells.In this study, we examined the markers of macrophage polarization using two methods: quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Additionally, we detected the expression of Notch-1 and Hes-1 through Western blotting.Results: BML-111 effectively suppressed the expression of pro-inflammatory cytokines TNF-α, IL-1β, and IL-18, as well as inflammasome factors NLRP3 and Caspase-1, while simultaneously up-regulating the expression of the anti-inflammatory cytokine IL-10 induced by CSE. Moreover, BML-111 reduced the expression of iNOS, which is associated with M1 macrophage polarization, and increased the expression of Arg-1, which is associated with M2 phenotype. Additionally, BML-111 downregulated the expression of Hes-1 and the ratio of activated Notch-1 to Notch-1 induced by CSE. The effect of BML-111 on inflammation and macrophage polarization was reversed upon administration of the Notch-1 signaling pathway agonist Jagged1.Conclusion: BML-111 has the potential to suppress inflammation and modulate M1/M2 macrophage polarization in RAW264.7 cells. The underlying mechanism may involve the Notch-1 signaling pathway.Keywords: BML-111, macrophage polarization, Notch-1 signaling pathway, COPD

Background: Equipoise exists regarding the implementation of evidence-based guidelines for nutritional therapy in critical illness because some previous cluster-randomised controlled trials (cRCTs) demonstrate patient benefits whilst others do not. We aimed to resolve this issue by initiating an appropriately powered cRCT to assess whether active implementation of an evidence-based guideline for nutrition therapy in critical illness could reduce mortality. Methods: We did a multicentre, cluster-randomised, parallel-controlled trial in 97 intensive care units (ICUs) throughout China. We developed an up-to-date, evidence-based nutrition guideline. ICUs randomly allocated to implement the guideline formed a local "intervention team", which actively implemented the guideline using standardized educational materials. ICUs assigned to the control group remained unaware of the guideline. All ICUs enroled critically ill patients who were expected to stay longer than seven days. The primary outcome was all-cause mortality within 28 days of enrolment . Findings Between March, 2018 and July, 2019, we enrolled 1,373 patients at 48 guideline ICUs and 1,399 patients at 49 control ICUs. Implementation of the nutrition guideline resulted in significantly earlier enteral nutrition (EN) initiation (1.20 vs. 1.55 mean days to initiation of EN; difference -0.40 [95% CI, -0.71 to -0.09]; P=0.01) and delayed parenteral nutrition (PN) initiation (1.29 vs. 0.80 mean days to start of PN; difference 1.06 [95% CI, 0.44 to 1.67]; P=0.001). There was no significant difference in 28-day mortality (14.2% vs. 15.2%; difference -1.6% [95% CI -4.3% to 1.2%]; p=0.42) between study groups. Interpretation: Active implementation of an evidence-based guideline can change practice. Our active implementation strategy improved the provision of nutrition therapy to critically ill patients. However, in our cRCT, this improvement in care did not translate to a reduction in mortality. We did not identify any safety concerns associated with the active implementation of our evidence-based guideline for nutrition therapy. Trial Registration: The study was registered in the ISRCTN registry before enrolment commenced (No. ISRCTN12233792). Funding Statement: The study was funded by the Key Research and Development Program Foundation of Jiangsu Province of China (no. BE2015685) and Nutricia, Wuxi, China. Declaration of Interests: Dr. GSD reported receiving academic research grants related to nutrition in critical illness from the Australian National Health and Medical Research Council, Fresenius Kabi Deutschland GmbH and Baxter Healthcare Pty Ltd and speakers honoraria from Fresenius Kabi Deutschland GmbH, Baxter Healthcare Australia, Pty Ltd, Nestle Healthcare, Vevy, Switzerland and Nutricia Pharmaceutical (Wuxi) Co., Ltd. China. Dr. AvZ reports personal fees from Baxter, personal fees from Nestle, personal fees from Fresenius Kabi, grants and personal fees from Nutricia, grants from Cardinal Health grants from Mermaid grants from Lyric, outside the submitted work. Dr. WqL reported receiving speakers honoraria from Nutricia Pharmaceutical (Wuxi) Co., Ltd. China. No other disclosures were reported. Ethics Approval Statement: The study was approved by the local hospital ethics committees of all the participating ICUs. At each site, informed consent was obtained from the patients or their next of kin before enrolment.

Background There is a lack of large-scale epidemiological data on the clinical practice of enteral nutrition (EN) feeding in China. This study aimed to provide such data on Chinese hospitals and to investigate factors associated with EN delivery. Methods This cross-sectional study was launched in 118 intensive care units (ICUs) of 116 mainland hospitals and conducted on April 26, 2017. At 00:00 on April 26, all patients in these ICUs were included. Demographic and clinical variables of patients on April 25 were obtained. The dates of hospitalization, ICU admission and nutrition initiation were reviewed. The outcome status 28 days after the day of investigation was obtained. Results A total of 1953 patients were included for analysis, including 1483 survivors and 312 nonsurvivors. The median study day was day 7 (IQR 2–19 days) after ICU entry. The proportions of subjects starting EN within 24, 48 and 72 h after ICU entry was 24.8% (84/352), 32.7% (150/459) and 40.0% (200/541), respectively. The proportion of subjects receiving > 80% estimated energy target within 24, 48, 72 h and 7 days after ICU entry was 10.5% (37/352), 10.9% (50/459), 11.8% (64/541) and 17.8% (162/910), respectively. Using acute gastrointestinal injury (AGI) 1 as the reference in a Cox model, patients with AGI 2–3 were associated with reduced likelihood of EN initiation (HR 0.46, 95% CI 0.353–0.599; p

Acute lung injury (ALI) is a severe disease with high morbidity and mortality, and is characterized by devastating inflammation of the lung and increased production of reactive oxygen species (ROS). Recent studies have indicated that fatty acid binding protein (FABP4) is important in the regulation of inflammation. However, the role of FABP4 in sepsis-related ALI, and the specific mechanism of action have not been examined. In vitro, the exposure of human alveolar epithelial A549 cells to lipopolysaccharide (LPS) and recombinant FABP4 (hrFABP4) resulted in the production of pro-inflammatory cytokines, inflammatory cytokines, and ROS, while these changes were ameliorated by pretreatment with the FABP4 inhibitor BMS309403 and FABP4 siRNA. Sequentially, treatment of A549 cells with N-acetylcysteine (NAC) significantly attenuated LPS and hrFABP4-induced the generation of ROS and the release of inflammatory cytokines. In vivo, a cecal ligation and puncture (CLP)-induced ALI murine model was successfully established. Then, the mice were treated with FABP4 inhibitor BMS309403. The results showed treatment with BMS309403 improved the survival rate of CLP-induced ALI mice, and prevented lung inflammation, histopathological changes, and increase of FABP4 induced by CLP. These data indicate that FABP4 plays an important role in lung inflammation of sepsis-induced ALI. Blockade of FABP4 signaling exhibits a protective effect in a CLP-induced ALI mouse model, and in A549 cell LPS specifically induces enhanced expression of FABP4, which then causes inflammatory cytokine production by elevating the ROS level.

Macrophage migration inhibitory factor (MIF) is an inflammatory mediator that contributes to asthmatic airway remodeling; however, little is known regarding the effects of MIF on airway smooth muscle cells (ASMCs). In the present study, we found that an enhanced expression of MIF promoted ASMC proliferation, increased the population of cells in the S/G2 phase, downregulated P21 expression, and upregulated cyclin D1, cyclin D3, and Cdk6 expression. In addition, the apoptosis of ASMCs was significantly decreased in response to MIF overexpression, compared with the negative control. Moreover, MIF facilitated the migration of ASMCs by upregulating the expression of matrix metalloproteinase (MMP)-2. Finally, we showed that MIF increased the phosphorylation of extracellular regulated protein kinases (ERK) 1/2 and focal adhesion kinase (FAK), which are associated with proliferation and migration. In conclusion, this study demonstrated that MIF overexpression promotes the proliferation and migration of ASMCs by upregulating the activity of the ERK1/2 and FAK signaling pathways in these cells, further indicating that inhibition of MIF may prove to be an effective strategy for treating asthma patients with airway remodeling.

Recent studies have indicated that Macrophage migration inhibitory factor (MIF) plays an important role in the prevention and treatment of asthma. However the role of MIF in airway inflammation and airway epithelial barrier disruption in house dust mite (HDM)-induced asthma has not been addressed. We hypothesized that MIF contributed to HDM-induced the production of Th2-associated cytokines and E-cadherin dysfunction in asthmatic mice and 16HBE cells. In vivo, a HDM-induced asthma mouse model was set up and mice treated with MIF antagonist ISO-1 after HDM. The mice treated with the ISO-1 ameliorated airway hyper-reactivity, airway inflammation, increased serum IgE levels, the aberrant arrangement of E-cadherin as well as the release of Th2 cytokines induced by HDM. In vitro, the exposure of 16HBE cells to HDM and rhMIF resulted in airway epithelial barrier disruption, inflammatory cytokine production and enhanced glycolytic flux. While these changes were attenuated by MIF siRNA treatment. Sequentially, treatment of 16HBE cells with PFKFB3 antagonist PFK15 significantly lowered rhMIF-induced these changes in 16HBE cells. Therefore, these results indicate that MIF may be an important contributor in airway inflammation and airway epithelial barrier disruption of HDM-induced asthma. Moreover, HDM specifically induces airway inflammation and airway epithelial barrier disruption of 16HBE cells through MIF-mediated enhancement of aerobic glycolysis.

Soluble TGF-β1 type II receptor (sTβRII) via TGF-β1 inhibition could inhibit hepatic fibrosis, but over-dosage triggers autoimmune responses. To test whether the use of a TGF-β1-responsive collagen I promoter COL1A1, via generating a feedback loop to TGF-β1 level, could offer accurate control on sTβRII expression. Recombinant adenoviruses with COL1A1 (Ad-COL-sTβRII/Luc) or CMV promoter (Ad-CMV-sTβRII/Luc) were constructed and characterized. Inhibition of TGF-β activity was determined both in vitro and in vivo. Total and bioactive TGF-β, hepatic fibrosis scale, α-SMA, collagen levels, and liver function were determined. COL1A1, but not CMV, responded to TGF-β1 in vitro. Both in vitro and in vivo, Ad-COL-sTβRII could significantly, but not completely inhibit TGF-β1 activity while Ad-CMV-sTβRII almost completely inhibited TGF-β1 activity. As evidenced by fibrosis scale, α-SMA, and collagen levels in liver tissue, Ad-COL-sTβRII and Ad-CMV-sTβRII had comparable efficacies in treating hepatic fibrosis. Ad-COL-sTβRII was better than Ad-CMV-sTβRII in liver function restore. Ad-CMV-sTβRII, but not Ad-COL-sTβRII, induced high level of anti-dsDNA and anti-Sm antibodies in rats. COL1A1 can precisely control sTβRII expression to inhibit excessive bioactive TGF-β level and thus inhibit hepatic fibrosis but without inducing autoimmune responses.

Sepsis-related acute lung injury (ALI) is characterized by excessive lung inflammation and apoptosis of alveolar epithelial cells resulting in acute hypoxemic respiratory failure. Recent studies indicated that anaerobic glycolysis play an important role in sepsis. However, whether inhibition of aerobic glycolysis exhibits beneficial effect on sepsis-induced ALI is not known. In vivo, a cecal ligation and puncture (CLP)-induced ALI mouse model was set up and mice treated with glycolytic inhibitor 3PO after CLP. The mice treated with the 3PO ameliorated the survival rate, histopathological changes, lung inflammation, lactate increased and lung apoptosis of mice with CLP-induced sepsis. In vitro, the exposure of human alveolar epithelial A549 cells to lipopolysaccharide (LPS) resulted in cell apoptosis, inflammatory cytokine production, enhanced glycolytic flux and reactive oxygen species (ROS) increased. While these changes were attenuated by 3PO treatment. Sequentially, treatment of A549 cells with lactate caused cell apoptosis and enhancement of ROS. Pretreatment with N-acetylcysteine (NAC) significantly lowered LPS and lactate-induced the generation of ROS and cell apoptosis in A549 cells. Therefore, these results indicate that anaerobic glycolysis may be an important contributor in cell apoptosis of sepsis-related ALI. Moreover, LPS specifically induces apoptotic insults to A549 cell through lactate-mediated enhancement of ROS.

Macrophage migration inhibitory factor (MIF) is an inflammatory mediator that contributes to asthmatic airway remodeling; however, little is known regarding the effects of MIF on airway smooth muscle cells (ASMCs). In the present study, we found that an enhanced expression of MIF promoted ASMC proliferation, increased the population of cells in the S/G2 phase, downregulated P21 expression, and upregulated cyclin D1, cyclin D3, and Cdk6 expression. In addition, the apoptosis of ASMCs was significantly decreased in response to MIF overexpression, compared with the negative control. Moreover, MIF facilitated the migration of ASMCs by upregulating the expression of matrix metalloproteinase (MMP)-2. Finally, we showed that MIF increased the phosphorylation of extracellular regulated protein kinases (ERK) 1/2 and focal adhesion kinase (FAK), which are associated with proliferation and migration. In conclusion, this study demonstrated that MIF overexpression promotes the proliferation and migration of ASMCs by upregulating the activity of the ERK1/2 and FAK signaling pathways in these cells, further indicating that inhibition of MIF may prove to be an effective strategy for treating asthma patients with airway remodeling.

To investigate the role of miR-181c in glycolysis of cancer-associated fibroblasts (CAFs) and explore the mechanism.Human lung CAFs and normal fibroblasts (NFs), isolated from fresh human lung adenocarcinoma tissue specimens by primary culture of tissue explants, were transfected with a miR -181c mimics, a miR-181c inhibitor, a siRNA siRNA-HK2 or the vector HK2-vector via Lipofectamine(TM) 2000. Quantitative real-time PCR was used to analyze the changes in miR-125b expression in the transfected cells; hexokinase-2 (HK2) protein expression in the cells was detected using Western blotting, and the cellular glucose uptake was assessed with 2-NBDG. Lactate production in the cells was examined and expression of HK2 mRNA was detected with dual luciferase reporter gene assay.No obvious difference was found in the cell morphology between CAFs and NFs. Compared with the NFs, the CAFs showed obviously increased glucose uptake, lactate production and HK2 protein expression with decreased expressions of the miR-181 family (P0.05). Transfection with the miR-181 inhibito- rsignificantly increased glucose uptake, lactate production and HK2 protein expression in the NFs. In CAFs, transfection with the miR-181 mimics caused significantly lowered glucose uptake, lactate production and HK2 protein expression of. Knockdown of endogenous HK2 by siRNA abolished miR-181 mimics-mediated decrease of glucose uptake and lactate production in CAFs, while transfection with miR-181 mimics suppressed HK2 overexpression-induced enhancement of glucose uptake and lactate production in NFs.Transfection with miR-181 mimics can suppress glycolysis in CAFs by inhibiting HK2 expression.