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*

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*

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

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

We investigated whether Nd2O3 treatment results in cytotoxicity and other underlying effects in rat NR8383 alveolar macrophages. Cell viability assessed by the MTT assay revealed that Nd2O3 was toxic in a dose-dependent manner, but not in a time-dependent manner. An ELISA analysis indicated that exposure to Nd2O3 caused cell damage and enhanced synthesis and release of inflammatory chemokines. A Western blot analysis showed that protein expression levels of caspase-3, nuclear factor-κB (NF-κB) and its inhibitor IκB increased significantly in response to Nd2O3 treatment. Both NF-κB and caspase-3 signaling were activated, suggesting that both pathways are involved in Nd2O3 cytotoxicity.
Animals ; Caspase 3 ; metabolism ; Cell Line ; Macrophages, Alveolar ; drug effects ; enzymology ; NF-kappa B ; metabolism ; Neodymium ; toxicity ; Oxides ; toxicity ; Rats ; Toxicity Tests

OBJECTIVETo investigate the effect of silicon dioxide (SiO₂) on the expression of E-cadherin, α-smooth muscle actin (α-SMA), and transforming growth factor β₁(TGF-β₁) in human pulmonary epithelial cells (A549) with epithelial-mesenchymal transition (EMT), and to study the roles of epidermal growth factor receptor (EGFR) signaling pathway in SiO₂-induced EMT in A549 cells in vitro.

METHODSAlveolar macrophages (AMs) were stimulated with 50 µg/ml SiO₂for 3, 6, 12, 18, 24, or 36 h, and the supernatants were collected to measure the expression of TGF-β₁protein by ELISA. The AM supernatant in which TGF-β₁reached the highest expression (T=18 h) was used as AM-conditioned supernatant. A549 cells were cultured in AM-conditioned supernatant and stimulated with indicated doses of SiO₂(0, 50, 100, and 200 µg/ml) for 48 h. The cell morphological changes were observed using an inverted microscope. The cells were collected at different times, and the mRNA and protein expression levels of E-cadherin, α-SMA, and EGFR were measured by RT-PCR and immunocytofluorescence, respectively.

RESULTSAfter stimulation by SiO₂, the expression level of TGF-β₁protein at each time point was significantly higher in the presence of AM supernatants than in the absence of AM supernatants (P<0.05). With the action time, the expression level of TGF-β₁protein increased at first and then decreased, and the highest level was reached at 18 h. After exposure to SiO₂, A549 cells exhibited mesenchymal characteristics, such as a spindle shape, pseudopodia change, and fibroblast-like morphology, as observed by inverted microscope, especially in the 200 µg/ml group. With increased concentration of SiO₂, the mRNA and protein expression of E-cadherin was down-regulated gradually, especially in the 200 µg/ml group, whereas the mRNA and protein expression of α-SMA and EGFR was up-regulated gradually, especially in the 200 µg/m1 group. There were significant differences between the SiO₂-treated groups (50, 100, and 200 µg/ml SiO₂) and the control group (P<0.05).

CONCLUSIONAfter being stimulated by SiO₂in vitro, AMs have significantly increased expression level of TGF-β₁protein. The AM supernatant together with SiO₂can induce the transition of pulmonary epithelial cells to mesenchymal cells, and its mechanism may be related to the EGFR signaling pathway.

Actins ; metabolism ; Cadherins ; metabolism ; Cell Line, Tumor ; Epithelial Cells ; cytology ; metabolism ; Epithelial-Mesenchymal Transition ; drug effects ; Humans ; Lung ; cytology ; Macrophages, Alveolar ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; Silicon Dioxide ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the role of oxidative stress in the endoplasmic reticulum stress-induced apoptosis of alveolar macrophages triggered by quartz dust.

METHODSSeventy-two healthy adult Wistar rats were randomly divided into control group, quartz dust group, quartz dust plus N-acetyl cysteine (NAC) group, and NAC group, with 18 rats in each group. One milliliter of sterile saline (for the control and NAC groups) or 1 ml of saline with 5%ultrafine quartz dust (for dust group and dust plus NAC group) was given to each rat by non-exposed endotracheal infusion. From the second day after dust infusion, rats in dust plus NAC group and NAC group received intragastric administration of NAC (100 mg/kg). In each week, the treatment with NAC lasted for 5 consecutive days, followed by 2 days' interval. For each group, 6 rats were randomly selected on the 14th, 28th, or 56th day after dust exposure; they were sacrificed by bloodletting from the femoral artery, and the lungs were collected. Bronchoalveolar lavage fluid was collected to separate macrophages. The protein expression of caspase-12 in alveolar macrophages, the apoptosis rate and reactive oxygen species (ROS) content of alveolar macrophages, and the protein carbonyl content of alveolar macrophages were determined by Western blot, flow cytometry, and colorimetry, respectively.

RESULTSIncreased protein expression of caspase-12, apoptosis rate, and content of ROS and protein carbonyl were discovered on the 14th day in the dust group, in comparison with the control group (P < 0.05), and the increase lasted till the 28th and 56th days. (P < 0.05). Compared with the dust group, the dust plus NAC group showed significant decreases in the content of ROS on the 14th, 28th, and 56th days (P < 0.05), significant decreases in the content of protein carbonyl on the 28th and 56th days (P < 0.05), and significant decreases in the protein expression of caspase-12 and apoptosis rate (P < 0.05).

CONCLUSIONOxidative stress is potentially involved in the endoplasmic reticulum stress-induced apoptosis of alveolar macrophages triggered by quartz dust. Oxidative damage of protein in the endoplasmic reticulum may play an important role in the process.

Animals ; Caspase 12 ; metabolism ; Dust ; Endoplasmic Reticulum Stress ; drug effects ; Macrophages, Alveolar ; drug effects ; pathology ; Male ; Oxidative Stress ; drug effects ; Protein Carbonylation ; Quartz ; toxicity ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate the anti-inflammatory and immunoregulatory effects of Yupingfeng (, YPF) Powder and its components in rats.

METHODSA rat chronic bronchitis (CB) model was developed using lipopolysaccharide (LPS) combined with bacillus Calmette Guerin (BCG). YPF, simple recipe Astragalus membranaceus (Fisch.) Bge (AM) and Astragalus membranaceus (Fisch.) Bge plus rhizome of Atractylodes macrocephala Koidz (AM+RA) decoction were administered (intragastric administration, once a day for 21 days) to rats, to prevent and treat CB. Immunoregulatory and anti-inflammatory effects of YPF, AM and AM+RA were tested by serum pharmacology in vitro on splenic lymphocytes of normal rats and alveolar macrophages of CB rats.

RESULTSInflammation in the pulmonary tissue and the bronchus of CB rats was significantly reduced in the YPF-treatment groups, AM and AM+RA groups demonstrating the efficacy of YPF. Serum samples collected at different times from rats after administration of YPF, AM and AM+RA demonstrated increased proliferation of splenic lymphocytes with area under the effect curve (AUE) of 552.6%, 336.3% and 452.0%, respectively. Treatment of alveolar macrophages with serum samples in YPF, AM or AM+RA group inhibited interleukin-8 (IL-8) in the cell culture media, and the effect was much better in the YPF group compared with AM or AM+RA group, with a higher maximal effect (Emax, P<0.05) and larger AUE (P <0.01 and P<0.05). Moreover, serum from rats treated with AM or AM+RA had similar efficacy, while the efficiency was lower than that treated with YPF.

CONCLUSIONYPF demonstrated anti-inflammatory and immunoregulatory effects in a rat model of CB, and timedependent relationships were demonstrated in vitro.

Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Body Weight ; drug effects ; Bronchitis, Chronic ; drug therapy ; pathology ; Cell Proliferation ; drug effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Immunologic Factors ; pharmacology ; therapeutic use ; Interleukin-8 ; metabolism ; Lung ; drug effects ; pathology ; ultrastructure ; Lymphocytes ; drug effects ; Macrophages, Alveolar ; drug effects ; metabolism ; Powders ; Rats ; Rats, Sprague-Dawley ; Spleen ; pathology ; Time Factors

OBJECTIVE: To evaluate whether dexmedetomidine hydrochloride, an α2-adrenergic receptor agonist, can prevent oxidative damage to alveolar macrophages induced by H2O2. METHODS: We used methyl thiazolyl tetrazolium (MTT) colorimetry to test the effect of different concentrations and action time of H2O2 on the survival rate of alveolar macrophages, and then we chose the appropriate H2O2 concentration and action time to build NR8383 cell oxidative damage model. After pre-conditioning of 0.01, 0.10, and 1.00 μmol/L dexmedetomidine hydrochloride for 24 hours, MTT colorimetry was used to demonstrate the survival rate of NR8383 cells damaged by H2O2, and the release of lactate dehydrogenase (LDH) and TNF-α by H2O2-damaged NR8383 cells was detected by corresponding kit. RESULTS: At 50-300 μmol/L, H2O2 caused concentration-dependent oxidative damage in the alveolar macrophages, decreased the cell survival rate, and increased LDH and TNF-α release. At 0.01-1.00 μmol/L dexmedetomidine hydrochloride concentration-dependently protected NR8383 cells from oxidative damage induced by H2O2, significantly increased the cell survival rate, decreased LDH and TNF-α release, and this effect of dexmedetomidine hydrochloride was dose-dependent. Yohimbine, an α2 - adrenergic receptor antagonist, completely neutralized the protective effect of dexmedetomidine hydrochloride on NR8383 cells without affecting the oxidative damage of NR8383 cells. CONCLUSION Dexmedetomidine hydrochloride can prevent alveolar macrophages from oxidative damage induced by H2O2, which may play a protective role through α2 - adrenergic receptors.
Animals ; Cell Line ; Cell Survival ; Dexmedetomidine ; pharmacology ; Hydrogen Peroxide ; L-Lactate Dehydrogenase ; metabolism ; Macrophages, Alveolar ; drug effects ; Oxidative Stress ; Rats ; Receptors, Adrenergic, alpha-2 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the effect of L-Arginine (L-Arg) on pulmonary surfactant (PS) expression and alveolar macrophage (AM) in rats with pulmonary injury induced by lipopolysaccharide (LPS).

METHODSModel of acute lung injury (ALI) was made by injection (iv) with LPS 5 mg/kg in rats. Fourty-eight male SD rats were randomly divided into 3 groups(n = 16): control, model (LPS) and L-Arg groups. L-Arg (500 mg/kg ip ,L-Arg group) or saline (control and LPS group) was administrated at 3 h or 6 h after LPS injection respectively for 3 h. The expression of surfactant protein A (SP-A) mRNA in the lung tissue was detected by ISH. The total protein (TP) in the bronchoalveolar lavage fluid (BALF) was detected. Rat AM were isolated from the bronchial alveolar lavage fluid of SD rats and harvested by selective plating technique. LPS and L-Arg were added to the culture medium. The concentration of nitric oxide (NO),the activity of lactate dehydrogenase (LDH), the contents of tumor necrosis factor alpha (TNF-alpha) and interleukin- 6 (IL-6) in the culture supernatants were respectively measured.

RESULTSCompared with the control group, the expression of SP-A mRNA was significantly decreased, the TP concentration was significantly increased in LPS group. Compared with LPS group at the same time points, treatment with L-Arg at 3 h after LPS, the expression of SP-A mRNA in lung tissue was increased markedly, whereas TP concentration was decreased significantly. In cultured rat AM, LDH activity, NO, TNF-alpha and IL-6 contents in culture medium were significantly increased in LPS group to compared with those of control group. LDH activity, TNF-alpha and IL-6 contents were decreased in L-Arg group compared with those of LPS group.

CONCLUSIONL-Arg can protect the lung against LPS-induced pulmonary injury by up-regulating the expression of PS and inhibiting inflammatory transmitters from AM.

Acute Lung Injury ; chemically induced ; drug therapy ; metabolism ; Animals ; Arginine ; pharmacology ; therapeutic use ; Lipopolysaccharides ; adverse effects ; Macrophages, Alveolar ; metabolism ; Male ; Pulmonary Surfactants ; metabolism ; Rats ; Rats, Sprague-Dawley