OBJECTIVE: To identify the underlying mechanism by which quercetin (Que) alleviates sepsis-related acute respiratory distress syndrome (ARDS). METHODS: In vivo, C57BL/6 mice were assigned to sham, cecal ligation and puncture (CLP), and CLP+Que (50 mg/kg) groups (n=15 per group) by using a random number table. The sepsisrelated ARDS mouse model was established using the CLP method. In vitro, the murine alveolar macrophages (MH-S) cells were classified into control, lipopolysaccharide (LPS), LPS+Que (10 μmol/L), and LPS+Que+acetylcysteine (NAC, 5 mmol/L) groups. The effect of Que on oxidative stress, inflammation, and apoptosis in mice lungs and MH-S cells was determined, and the mechanism with reactive oxygen species (ROS)/p38 mitogen-activated protein kinase (MAPK) pathway was also explored both in vivo and in vitro. RESULTS: Que alleviated lung injury in mice, as reflected by a reversal of pulmonary histopathologic changes as well as a reduction in lung wet/dry weight ratio and neutrophil infiltration (P<0.05 or P<0.01). Additionally, Que improved the survival rate and relieved gas exchange impairment in mice (P<0.01). Que treatment also remarkedly reduced malondialdehyde formation, superoxide dismutase and catalase depletion, and cell apoptosis both in vivo and in vitro (P<0.05 or P<0.01). Moreover, Que treatment diminished the release of inflammatory factors interleukin (IL)-1β, tumor necrosis factor-α, and IL-6 both in vivo and in vitro (P<0.05 or P<0.01). Mechanistic investigation clarifified that Que administration led to a decline in the phosphorylation of p38 MAPK in addition to the suppression of ROS expression (P<0.01). Furthermore, in LPS-induced MH-S cells, ROS inhibitor NAC further inhibited ROS/p38 MAPK pathway, as well as oxidative stress, inflammation, and cell apoptosis on the basis of Que treatment (P<0.05 or P<0.01). CONCLUSION Que was found to exert anti-oxidative, anti-inflammatory, and anti-apoptotic effects by suppressing the ROS/p38 MAPK pathway, thereby conferring protection for mice against sepsis-related ARDS.
Animals ; Sepsis/drug therapy* ; Quercetin/therapeutic use* ; Respiratory Distress Syndrome/enzymology* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; Mice, Inbred C57BL ; Reactive Oxygen Species/metabolism* ; Apoptosis/drug effects* ; Male ; Oxidative Stress/drug effects* ; MAP Kinase Signaling System/drug effects* ; Lung/drug effects* ; Mice ; Lipopolysaccharides ; Macrophages, Alveolar/pathology* ; Inflammation/pathology* ; Protective Agents/therapeutic use*

Objective To investigate the effect of interleukin-6 (IL-6) on the phagocytosis of MH-S alveolar macrophages and its related mechanisms. Methods A mouse acute lung injury (ALI) model was constructed by instilling lipopolysaccharide (LPS) into the airway. ELISA was used to detect the content of IL-6 in bronchoalveolar lavage fluid (BALF). In vitro cultured MH-S cells, in the presence or absence of signal transducer and activator 3 of transcription(STAT3) inhibitor Stattic (5 μmol/L), IL-6 (10 ng/mL~500 ng/mL) was added to stimulate for 6 hours, and then incubated with fluorescent microspheres for 2 hours. The phagocytosis of MH-S cells was detected by flow cytometry. Western blot analysis was used to detect the expression levels of phosphorylated Janus kinase 2 (p-JAK2), phosphorylated STAT3 (p-STAT3), actin-related protein 2 (Arp2) and filamentous actin (F-actin). Results The content of IL-6 in BALF was significantly increased after the mice were injected with LPS through the airway. With the increase of IL-6 stimulation concentration, the phagocytic function of MH-S cells was enhanced, and the expression levels of Arp2 and F-actin proteins in MH-S cells were increased. The expression levels of p-JAK2 and p-STAT3 proteins increased in MH-S cells stimulated with IL-6(100 ng/mL). After blocking STAT3 signaling, the effect of IL-6 in promoting phagocytosis of MH-S cells disappeared completely, and the increased expression of Arp2 and F-actin proteins in MH-S cells induced by IL-6 was also inhibited. Conclusion IL-6 promotes the expression of Arp2 and F-actin proteins by activating the JAK2/STAT3 signaling pathway, thereby enhancing the phagocytic function of MH-S cells.
Animals ; Mice ; Actins ; Disease Models, Animal ; Interleukin-6 ; Janus Kinase 2 ; Lipopolysaccharides ; Macrophages, Alveolar ; Signal Transduction

OBJECTIVES: To investigate the inhibitory effect of FER-1 on methylglyoxal-induced ferroptosis in cultured mouse alveolar macrophages. METHODS: MH-S cells derived from mouse alveolar macrophages treated with 90 μg/mL methylglyoxal, 10 μmol/mL FER-1MG+FER-1, or both were examined for intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and ferrous ion (Fe²⁺) levels and changes in mitochondrial membrane potential. Western blotting was performed to detect the protein expression levels of glutathione peroxidase 4 (GPX4) and long-chain acyl-CoA synthase 4 (ACSL4). RESULTS: Methylglyoxal treatment of MH-S cells for 24 h significantly decreased the protein expression level of GPX4, upregulated the protein expression of ACSL4, increased intracellular concentrations of ferrous ions, ROS and MDA, caused loss of mitochondrial membrane potential, and decreased cell viability. Treatment of the cells with FER-1 effectively attenuated these detrimental effects of methylglyoxal in MH-S cells by increasing GPX4 expression, reducing ACSL4 expression and intracellular ferrous ions, ROS and MDA levels, and restoring the mitochondrial membrane potential. CONCLUSIONS Methylglyoxal can induce ferroptosis in MH-S cells in a dose-dependent manner, and FER-1 can rescue the cells from methylglyoxal-induced ferroptosis.
Animals ; Ferroptosis/drug effects* ; Mice ; Pyruvaldehyde ; Macrophages, Alveolar/drug effects* ; Reactive Oxygen Species/metabolism* ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism* ; Membrane Potential, Mitochondrial/drug effects* ; Coenzyme A Ligases/metabolism* ; Malondialdehyde/metabolism* ; Cell Survival/drug effects*

This work aims to explore the effect of glycolysis on the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in porcine alveolar macrophages (PAMs). The changes of glucose metabolism, PRRSV protein levels, PRRSV titers, and the relative expression levels of genes and proteins in PAMs were analyzed by ELISA, qPCR, virus titration, and Western blotting after PRRSV infection. The effect of hypoxia-inducible factor-1α (HIF-1α) on PRRSV replication was subsequently assessed by specific siRNAs targeting to HIF-1α. The results showed that PRRSV infection enhanced glycolysis, elevated the levels of glucose uptake and lactate in the supernatant (P<0.05 and 0.01, respectively), reduced ATP production (P<0.05), and up-regulated the expression of hexokinase 2 (HK2), 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), and pyruvate kinase isozyme type M2 (PKM2) in PAMs (P<0.05 and 0.01, respectively). Glycolysis inhibitors down-regulated the expression of PRRSV proteins and reduced virus titers (P<0.01). The knockdown of HIF-1α by siRNAs inhibited glycolysis and lowered PRRSV titers (P<0.05). In addition, the interferon pathways inhibited by PRRSV infection were reversed by the inhibition of glycolysis. These findings may facilitate further investigation of the role of glycolysis in PRRSV replication.
Porcine respiratory and reproductive syndrome virus/physiology* ; Glycolysis ; Swine ; Animals ; Virus Replication ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Macrophages, Alveolar/metabolism* ; Porcine Reproductive and Respiratory Syndrome/virology* ; Cells, Cultured ; RNA, Small Interfering/genetics*

Humans ; Nitric Oxide ; Carcinoma, Non-Small-Cell Lung/drug therapy* ; Lung Neoplasms ; Lung ; Nitric Oxide Synthase ; Macrophages, Alveolar ; Pulmonary Alveoli

The aim of this study was to investigate the effects of polarization program on the ability of macrophages to regulate iron metabolism. M1 and M2 macrophages were propagated in vitro from porcine alveolar macrophages 3D4/2 and polarized by cytokines. The 3D4/2 macrophages were treated with 20 ng/mL interferon gamma (IFN-γ) and 10 ng/mL interleukin-4 (IL-4) combined with 10 ng/mL macrophage colony-stimulating factor (M-CSF) to induce polarization to M1 and M2, respectively. After incubation for 24 h, the expression levels of inflammatory factors and iron-metabolism genes were determined using real-time qPCR, Western bot and immunofluorescence. The M1/M2 macrophages culture media supernatant was collected and used to treat porcine intestinal epithelial cells IPEC-J2. The proliferation ability of IPEC-J2 was detected using CCK-8 assay kit. Following exogenous addition of ammonium ferric citrate (FAC) to M1/M2 macrophages, the phagocytic function of macrophages was detected using fluorescein isothiocyanate-dextran (FITC-dextran) and flow cytometry. The results showed that, compared with control, M1 macrophages had higher mRNA levels of iron storage proteins (ferritin heavy and light polypeptide, i.e. FtH and FtL), hepcidin and lipocalin-2, as well as iron content. Moreover, iron enhanced the ability of M1 macrophages to phagocytize FITC-dextran. There was no significant change in these mRNA expression levels in M2 macrophages, but the mRNA expression levels of ferroportin and transferrin receptor were up-regulated. In addition, the conditioned media supernatant from M2 macrophages promoted cell proliferation of IPEC-J2. These findings indicate that M1 macrophages tend to lock iron in the cell and reduce extracellular iron content, thereby inhibiting the proliferation of extracellular bacteria. While M2 macrophages tend to excrete iron, which contributes to the proliferation of surrounding cells and thus promotes tissue repair.
Animals ; Cytokines ; Ferritins ; Iron/metabolism* ; Macrophages/metabolism* ; Macrophages, Alveolar/metabolism* ; Swine

In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.
Animals ; Cells, Cultured ; Circovirus ; Interferon Type I/genetics* ; Macrophages, Alveolar/virology* ; Membrane Proteins/metabolism* ; Nucleotidyltransferases/metabolism* ; Signal Transduction ; Swine

The aim of the present study was to investigate the effect of salidroside (Sal) on inflammatory activation induced by lipopolysaccharide (LPS) in the co-culture of rat alveolar macrophages (AM) NR 8383 and type II alveolar epithelial cells (AEC II) RLE-6TN. CCK-8 colorimetric method was used to detect cell proliferation percentage. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-10 (IL-10) in the supernatant. Western blot was used to examine the expression levels of phosphorylated AKT (p-AKT) and total AKT protein. The results showed that pretreatment of RLE-6TN cells or co-culture of RLE-6TN and NR 8383 cells with 32 and 128 µg/mL Sal for 1 h, followed by continuous culture for 24 h, significantly increased the cell proliferation (P < 0.05). Compared with control group, 32 and 128 µg/mL Sal pretreatment significantly increased the ratio of p-AKT/AKT in RLE-6TN cells (P < 0.05). Pretreatment of 32 µg/mL Sal not only inhibited the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05), but also enhanced the inhibitory effect of RLE-6TN and NR 8383 cells co-culture on the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05). In addition, 32 µg/mL Sal pretreatment promoted LPS-induced IL-10 secretion by NR 8383 cells (P < 0.05), and enhanced the promoting effect of co-culture of RLE-6TN and NR 8383 cells on the IL-10 secretion by LPS-induced NR 8383 cells (P < 0.05). In conclusion, Sal may directly inhibit LPS-induced inflammatory activation of AM (NR 8383), promote the proliferation of AEC II (RLE-6TN) through PI3K/AKT signaling pathway, and enhance the regulatory effect of AEC II on LPS-induced inflammatory activation of AM.
Alveolar Epithelial Cells ; drug effects ; metabolism ; Animals ; Cell Line ; Chemokine CXCL2 ; metabolism ; Coculture Techniques ; Glucosides ; pharmacology ; Interleukin-10 ; metabolism ; Lipopolysaccharides ; Macrophages, Alveolar ; drug effects ; metabolism ; Phenols ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVE: To investigate the effect of sulfur dioxide (SO₂) on the apoptosis of alveolar macrophage (AM) in lung protection of limb ischemia/reperfusion (I/R) induced acute lung injury (ALI), and to find a new target for the control of inflammatory response. METHODS: Twenty pathogen-free, adult male Sprague-Dawley (SD) rats (180-230 g) were used in this study. Five rats were to be used for limb ischemia/reperfusion, then plasma was extracted as ischemia/reperfusion serum stimulation. Fifteen rats were to be used for extracting AM by bronchoalveolar lavage. The AM was isolated and cultured, then the cell count was adjusted to 1×10⁶/mL, and randomly divided into the following 4 groups (n=6): control group, I/R group, SO₂ group, and I/R+SO₂ group. The I/R group was given ischemia/reperfusion serum (500 μg/L) to stimulate 6 h; the SO₂ group was given an SO₂ donor, Na₂SO₃/NaHSO₃ [(0.54 mmol/kg) / (0.18 mmol/kg)]; and the I/R+SO₂ group was given the same ischemia/reperfusion serum and Na₂SO₃/NaHSO₃ at the same time. The level of mitochondrial membrane potential, the state of mitochondrial permeability transition pore (mPTP), the rate of AM apoptosis, the expression of Bcl-2 and Caspase-3 proteins were detected by flow cytometry, microplate reader and Western blotting. RESULTS: Compared with the control group, in the I/R group, the ratio of red to green fluorescence and the absorbance decreased significantly, the percentage of apoptotic cells increased obviously, the apoptotic rate was 43.81%±2.40%, Caspase-3 protein expression increased, Bcl-2 protein expression decreased. While compared with the I/R group, in the I/R+SO₂ group, the ratio of red to green fluorescence and the absorbance increased significantly; the apoptotic rate decreased to 37.01%±1.93%, Caspase-3 protein expression decreased, Bcl-2 protein expression increased. CONCLUSION Exogenous SO₂ has the effect of accelerating AM apoptosis by stimulating mPTP to open and mitochondrial membrane potential to decrease; besides, exogenous SO₂ could stimulate AM to secrete more anti-inflammatory cytokines and less inflammatory cytokines. In conclusion, exogenous SO₂ can reduce macrophage apoptosis by inhibiting mitochondrial pathways.
Acute Lung Injury ; Animals ; Apoptosis ; Ischemia ; Macrophages, Alveolar ; Male ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; Sulfur Dioxide

Hypoxia (low oxygen level) is an important feature during infections and affects the host defence mechanisms. The host has evolved specific responses to address hypoxia, which are strongly dependent on the activation of hypoxia-inducible factor 1 (HIF-1). Hypoxia interferes degradation of HIF-1 alpha subunit (HIF-1α), leading to stabilisation of HIF-1α, heterodimerization with HIF-1 beta subunit (HIF-1β) and subsequent activation of HIF-1 pathway. Apical periodontitis (periapical lesion) is a consequence of endodontic infection and ultimately results in destruction of tooth-supporting tissue, including alveolar bone. Thus far, the role of HIF-1 in periapical lesions has not been systematically examined. In the present study, we determined the role of HIF-1 in a well-characterised mouse periapical lesion model using two HIF-1α-activating strategies, dimethyloxalylglycine (DMOG) and adenovirus-induced constitutively active HIF-1α (CA-HIF1A). Both DMOG and CA-HIF1A attenuated periapical inflammation and tissue destruction. The attenuation in vivo was associated with downregulation of nuclear factor-κappa B (NF-κB) and osteoclastic gene expressions. These two agents also suppressed NF-κB activation and subsequent production of proinflammatory cytokines by macrophages. Furthermore, activation of HIF-1α by DMOG specifically suppressed lipopolysaccharide-stimulated macrophage differentiation into M1 cells, increasing the ratio of M2 macrophages against M1 cells. Taken together, our data indicated that activation of HIF-1 plays a protective role in the development of apical periodontitis via downregulation of NF-κB, proinflammatory cytokines, M1 macrophages and osteoclastogenesis.
Alveolar Bone Loss ; metabolism ; prevention & control ; Amino Acids, Dicarboxylic ; pharmacology ; Animals ; Cytokines ; metabolism ; Down-Regulation ; Gene Expression ; drug effects ; Hypoxia-Inducible Factor 1, alpha Subunit ; physiology ; Macrophages ; physiology ; Mice ; NF-kappa B ; metabolism ; Osteogenesis ; physiology ; Periapical Periodontitis ; metabolism ; prevention & control ; Real-Time Polymerase Chain Reaction ; X-Ray Microtomography