Inhibition of phosphoinositide 3-kinase activity attenuates neutrophilic airway inflammation and inhibits pyrin domain-containing 3 inflammasome activation in an ovalbumin-lipopolysaccharide-induced asthma murine model.

BACKGROUND: Existing investigations suggest that the blockade of phosphoinositide 3-kinase (PI3K) activity contributes to inflammatory solution in allergic asthma, but whether this inhibition directly attenuates neutrophilic airway inflammation in vivo is still unclear. We explored the pharmacological effects of LY294002, a specific inhibitor of PI3K on the progression of neutrophilic airway inflammation and investigated the underlying mechanism. METHODS AND RESULTS: Female C57BL/6 mice were intranasally sensitized with ovalbumin (OVA) together with lipopolysaccharide (LPS) on days 0 and 6, and challenged with OVA on days 14-17 to establish a neutrophilic airway disease model. In the challenge phase, a subset of mice was treated intratracheally with LY294002. We found that treatment of LY294002 attenuates clinic symptoms of inflammatory mice. Histological studies showed that LY294002 significantly inhibited inflammatory cell infiltration and mucus production. The treatment also significantly inhibited OVA-LPS induced increases in inflammatory cell counts, especially neutrophil counts, and IL-17 levels in bronchoalveolar lavage fluid (BALF). LY294002 treated mice exhibited significantly increased IL-10 levels in BALF compared to the untreated mice. In addition, LY294002 reduced the plasma concentrations of IL-6 and IL-17. The anti-inflammatory effects of LY29402 were correlated with the downregulation of NLRP3 inflammasome. CONCLUSIONS: Our findings suggested that LY294002 as a potential pharmacological target for neutrophilic airway inflammation.

TLR2-ERK signaling pathway regulates expression of galectin-3 in a murine model of OVA-induced allergic airway inflammation.

Toll-like receptor 2 (TLR2) and galectin-3 (Gal-3) are involved in the pathological process of asthma, but the underlying mechanism is not fully understood. We hypothesized that TLR2 pathway may regulate expression of Gal-3 in allergic airway inflammation. Wild-type (WT) and TLR2-/- mice were sensitized on day 0 and challenged with ovalbumin (OVA) on days 14-21 to establish a model of allergic airway inflammation, and were treated with a specific ERK inhibitor U0126. Histological changes in the lungs were analyzed by hematoxylin-eosin (HE) and Periodic Acid-Schiff (PAS) staining; cytokines and anti-OVA immunoglobulin E (IgE) were tested by ELISA; and related protein expression in lung tissues was measured by western blot. We found that the expression levels of TLR2 and Gal-3 markedly increased concomitantly with airway inflammation after OVA induction, while TLR2 deficiency significantly alleviated airway inflammation and reduced Gal-3 expression. Moreover, the expression levels of phosphorylated mitogen-activated protein kinases (p-MAPKs) were significantly elevated in OVA-challenged WT mice, while TLR2 deficiency only significantly decreased phosphorylated extracellular signal-regulated kinase (p-ERK) levels. Furthermore, we found that U0126 treatment significantly alleviated allergic airway inflammation and decreased Gal-3 levels in OVA-challenged WT mice, but had no further effect in OVA-challenged TLR2-/- mice. These above results suggested that TLR2 is an upstream signal molecule of ERK. We further demonstrated that TLR2 regulates Gal-3 expression through the ERK pathway in LTA-stimulated macrophages in vitro. Our findings showed that the TLR2-ERK signaling pathway regulates Gal-3 expression in a murine model of allergic airway inflammation.

Particulate matters induce acute exacerbation of allergic airway inflammation via the TLR2/NF-κB/NLRP3 signaling pathway.

BACKGROUND: Exposure to particulate matters (PMs) can lead to an acute exacerbation of allergic airway diseases, increasing the severity of symptoms and mortality. However, little is known about the underlying molecular mechanism. This study aimed to investigate the effects of PMs on acute exacerbation of allergic airway inflammation and seek potential therapeutic targets. METHODS: Non-allergic control and ovalbumin (OVA)-allergic wide-type (WT) and Toll-like receptor 2 knockout (Tlr2-/-) mice were exposed to 100 µg of PM (diameter 5.85 µm) or saline by the oropharyngeal instillation. The responses were examined three days after exposure. In the RAW264.7 macrophage cell line, Tlr2 was knocked down by small-interfering RNA or the NF-κB inhibitor JSH-23 was used, and then the cells were stimulated with PMs for 12 h before comparison of the inflammatory responses. RESULTS: PM exposure led to increased inflammatory cell recruitment and airway intensity of PAS + staining in OVA-allergic WT mice, accompanied with an accumulation of inflammatory cells and elevated inflammatory cytokines, such as IL-6 and IL-18, in the bronchoalveolar lavage fluid (BALF). Furthermore, the protein levels of TLR2 and the NLRP3 inflammasome were elevated concomitantly with the airway inflammation post-OVA/PMs challenge. Tlr2 deficiency effectively inhibited the airway inflammation, including pulmonary inflammatory cell recruitment, mucus secretion, serum OVA-specific immunoglobulin E (IgE), and BALF inflammatory cytokine production. Additionally, the P-induced NLRP3 activation in the RAW 264.7 cell line was diminished by the knockdown of Tlr2 or JSH-23 treatment in vitro. CONCLUSION: Our results indicated that PMs exacerbate the allergic airway inflammation mediated by the TLR2/ NF-κB/NLRP3 signaling pathway. Inhibition of NF-κB seems to be a possible treatment.

15-Lipoxygenase-1 in osteoblasts promotes TGF-ß1 expression via inhibiting autophagy in human osteoarthritis.

BACKGROUND: 15-Lipoxygenase-1 (15-LOX-1) belongs to the lipoxygenase family involved in the inflammatory response and pathological process of various diseases, including osteoarthritis (OA). The overexpression of TGF-ß1 in osteoblasts leads to abnormal changes in subchondral bone structure, eventually causing OA. However, the pathogenesis of the disease is poorly defined, and the interaction between 15-LOX-1 and TGF-ß1 in osteoblasts has not been evaluated in OA. In this study, the role of 15-LOX-1 in subchondral bone osteoblasts in OA was evaluated. METHOD: 15-LOX-1 expression in osteoblasts of the subchondral bone of patients with OA was measured by immunohistochemistry, qRT-PCR, and western blotting. Osteoblasts extracted from the subchondral bone of OA were transfected with 15-LOX-1 siRNA and an overexpression vector. The eff ;ect of 15-LOX-1 on the expression of TGF-ß1 in OA osteoblasts was assessed by qRT-PCR and western blotting. The effect of 15-LOX-1 on autophagy via AMPK pathway in OA osteoblasts was evaluated by qRT-PCR, western blotting, and transmission electron microscopy. RESULTS: The expression levels of 15-LOX-1 and TGF-ß1 were higher in OA subchondral bone osteoblast than that in non-OA subchondral bone. 15-LOX-1, which downregulated autophagy by inhibiting AMPK following the activation of mTORC1, upregulated the osteoblast expression of TGF-ß1. Treatment with autophagy inhibitors significantly increased the expression levels of TGF-ß1 in osteoblasts. CONCLUSION: In the present study, our findings suggested that 15-Lipoxygenase-1 in Osteoblasts Promotes TGF-ß1 expression via inhibiting autophagy in human Osteoarthritis. These novel results suggested that 15-Lipoxygenase-1 expressed by subchondral bone osteoblasts might be a promising therapeutic target in human OA.

Elevated levels of 15-lipoxygenase-1 contribute to the abnormal phenotypes of osteoblasts in human osteoarthritis.

AIMS: 15-lipoxygenase-1 (15-LOX-1) plays a vital role in aggravating the inflammatory response in various pathological processes, including osteoarthritis (OA). Abnormal osteoblast phenotypes including elevated runt-related transcription factor 2 (RUNX2), collagen type 1 alpha 1 (COL1), and osteocalcin (OCN) lead to osteosclerosis of the subchondral bone, which eventually causes OA. However, the pathogenesis of OA is poorly defined, and it is unclear if 15-LOX-1 induces osteoblast abnormal phenotypes in OA. Therefore, this study aimed to determine the roles of 15-LOX-1 on the abnormal phenotypes present in osteoblasts of the subchondral bone in OA. MAIN METHODS: The expression levels of 15-LOX-1 were measured by Immunohistochemistry, qRT-PCR and western blotting from the OA subchondral bone osteoblasts. To further investigate the roles of 15-LOX-1 in abnormal phenotypes of osteoblasts and its mechanisms in OA, 15-LOX-1 siRNA or overexpressing lv-15-lox-1 were transfected into osteoblasts, respectively. The effects of 15-LOX-1 on abnormal phenotypes of osteoblasts in OA were assessed by qRT-PCR, and western blotting. We also examined the role of 15-LOX-1-inhibited autophagy in OA osteoblasts by qRT-PCR, and western blotting, transmission electron microscopy. KEY FINDINGS: The expression levels of 15-LOX-1 along with osteoblast phenotype markers such as RUNX2, COL1, and OCN were significantly increased in OA subchondral bone. Furthermore, 15-LOX-1 inhibited autophagy significantly upregulated the expression levels of RUNX2, COL1 and OCN through activated mTORC1. Similarly, treatment with autophagy inhibitors alleviated osteoblast abnormal phenotypes of osteoblasts in OA. SIGNIFICANCE: In conclusion, our results suggested that the expression of 15-LOX-1 on osteoblasts from the subchondral bone increased in OA. 15-LOX-1 inhibited autophagy by activated mTORC1, which in turn upregulated the markers of abnormal osteoblast phenotypes RUNX2, COL1, and OCN.

Anti-inflammatory Property of Galectin-1 in a Murine Model of Allergic Airway Inflammation.

Galectin-1 (Gal-1) has immunomodulatory activities in various allergic inflammatory disorders, but its potential anti-inflammatory properties on allergic airway diseases have not been confirmed. We explored the pharmacological effects of Gal-1 on the progression of allergic airway inflammation and investigated the underlying mechanism. Female C57BL/6 mice were sensitized on day 0 and challenged with ovalbumin (OVA) on days 14-17 to establish an allergic airway inflammation model. In the challenge phase, a subset of mice was treated intraperitoneally with recombinant Gal-1 (rGal-1) or dexamethasone (Dex). We found that rGal-1 inhibited pulmonary inflammatory cell recruitment, mucus secretion, bronchoalveolar lavage fluid (BALF) inflammatory cell infiltration, and cytokine production. The treatment also suppressed the infiltration of eosinophils into the allergic lung as indicated by decreased expression levels of eotaxin and eosinophil peroxidase (EPX). However, only the expression levels of IL-25, neither IL-33 nor TSLP, were significantly decreased in the lung by rGal-1 treatment. These immunomodulatory effects in the allergic lung were correlated with the activation of extracellular signal-regulated kinase (ERK) signaling pathway and downregulation of endogenous Gal-1. In addition, rGal-1 reduced the plasma concentrations of anti-OVA immunoglobulin E (IgE) and IL-17. Our findings suggest that rGal-1 is an effective therapy for allergic airway inflammation in a murine model and may be a potential pharmacological target for allergic airway inflammatory diseases.

PI3K/Akt-Beclin1 signaling pathway positively regulates phagocytosis and negatively mediates NF-κB-dependent inflammation in Staphylococcus aureus-infected macrophages.

Although autophagy and phagocytosis are involved in the regulation of host inflammatory response to bacterial infection in macrophages, the underlying mechanisms have not been completely elucidated. In the present study, we found that infecting RAW264.7 macrophages with Staphylococcus aureus (S. aureus) activated multiple signaling pathways including phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt), nuclear factor-κB (NF-κB), and Rac1, as well as triggered autophagy. LY294002, a specific PI3K activity inhibitor, significantly decreased autophagy and phagocytosis of macrophages upon S. aureus infection. Similarly, knockdown of Beclin1 by specific siRNA significantly inhibited autophagy and phagocytosis of S. aureus-infected macrophages. Additionally, we showed that although administration of Beclin1 siRNA had no effects on phosphorylation of Akt (p-Akt), inhibition of PI3K activity by LY294002 significantly decreased the expression of Beclin1, suggesting that Beclin1 is a downstream molecular of PI3K. Furthermore, inhibition of autophagy significantly increased the production of NF-κB-dependent TNFα/IL-1ß in S. aureus-infected macrophages. Collectively, these findings demonstrated, for the first time, that the PI3K/Akt-Beclin1 signaling pathway positively regulates phagocytosis and negatively mediates NF-κB-dependent inflammation in S. aureus-infected macrophages.

Short-term treatment of irbesartan and hydrochlorothiazide decreases plasma N-terminal pro-brain natriuretic peptide levels in subjects with acute exacerbations of COPD.

BACKGROUND: Plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP) are elevated in subjects with COPD, and high plasma NT-proBNP levels are correlated with a poor prognosis. Thus, it is crucial to decrease the plasma NT-proBNP levels at the early stage of disease. We aimed to assess the effects of short-term treatment of irbesartan and hydrochlorothiazide on plasma NT-proBNP levels and health-related quality of life (HRQOL) in subjects with acute exacerbations of COPD (AECOPD). SUBJECTS AND METHODS: Eighty subjects with AECOPD and high plasma NT-proBNP levels, without any clinical evidence of cor pulmonale, were enrolled. The subjects were randomly allocated into two groups of 40 subjects. In addition to standard treatment for AECOPD, the subjects in group I were treated with irbesartan alone, and those in group II were treated with irbesartan and hydrochlorothiazide for a week. Forty subjects with stable COPD were enrolled as a control group. Plasma NT-proBNP concentrations were measured on admission and on the first, fourth, and seventh days. The subjects' health-related quality of life was evaluated applying the 36-item short-form questionnaire on the first day before treatment and on the seventh day after treatment. RESULTS: Treatment of irbesartan and hydrochlorothiazide significantly decreased plasma NT-proBNP levels in subjects with AECOPD, and this reduction was more significant in group II than that in group I. There were no significant differences in 36-item short-form domain scores between subjects with stable COPD and those with AECOPD who were treated with irbesartan and hydrochlorothiazide. CONCLUSION: Treatment of irbesartan and hydrochlorothiazide rapidly decreased plasma NT-proBNP levels in subjects with AECOPD, and the treatment did not impair their physical status.