Project description:RationaleAirway hyperresponsiveness (AHR) is classically found in asthma, and persistent AHR is associated with poor asthma control. Although airway smooth muscle (ASM) cells play a critical pathophysiologic role in AHR, the paracrine contributions of surrounding cells such as fibroblasts to the contractile phenotype of ASM cells have not been examined fully. This study addresses the hypothesis that nicotine promotes a contractile ASM cell phenotype by stimulating fibroblasts to increase nerve growth factor (NGF) secretion into the environment.MethodsPrimary lung fibroblasts isolated from wild type and α7 nicotinic acetylcholine receptor (α7 nAChR) deficient mice were treated with nicotine (50 µg/ml) in vitro for 72 hours. NGF levels were measured in culture media and in bronchoalveolar lavage (BAL) fluid from asthmatic, smoking and non-smoking subjects by ELISA. The role of the NFκB pathway in nicotine-induced NGF expression was investigated by measuring NFκB nuclear translocation, transcriptional activity, chromatin immunoprecipitation assays, and si-p65 NFκB knockdown. The ability of nicotine to stimulate a fibroblast-mediated, contractile ASM cell phenotype was confirmed by examining expression of contractile proteins in ASM cells cultured with fibroblast-conditioned media or BAL fluid.ResultsNGF levels were elevated in the bronchoalveolar lavage fluid of nicotine-exposed mice, current smokers, and asthmatic children. Nicotine increased NGF secretion in lung fibroblasts in vitro in a dose-dependent manner and stimulated NFκB nuclear translocation, p65 binding to the NGF promoter, and NFκB transcriptional activity. These responses were attenuated in α7 nAChR deficient fibroblasts and in wild type fibroblasts following NFκB inhibition. Nicotine-treated, fibroblast-conditioned media increased expression of contractile proteins in ASM cells.ConclusionNicotine stimulates NGF release by lung fibroblasts through α7 nAChR and NFκB dependent pathways. These novel findings suggest that the nicotine-α7 nAChR-NFκB- NGF axis may provide novel therapeutic targets to attenuate tobacco smoke-induced AHR.

Project description:BackgroundCigarette smoking (CS) is a strong risk factor for idiopathic pulmonary fibrosis (IPF). It can activate lung fibroblasts (LF) by inducing redox imbalance. We previously showed that clearing mitochondrial reactive oxygen species (mtROS) protects against CS-induced pulmonary fibrosis. However, the precise mechanisms of mtROS in LF need further investigation. Here we focused on mtROS to elucidate how it was regulated by CS in LF and how it contributed to LF activation.MethodsWe treated cells with 1% cigarette smoking extract (CSE) and examined mtROS level by MitoSOX™ indicator. And the effect of CSE on expression of SIRT1, SOD2, mitochondrial NOX4 (mtNOX4), fatty acid oxidation (FAO)-related protein PPARα and CPT1a and LF activation marker Collagen I and α-SMA were detected. Nile Red staining was performed to show cellular lipid content. Then, lipid droplets, autophagosome and lysosome were marked by Bodipy 493/503, LC3 and LAMP1, respectively. And lipophagy was evaluated by the colocalization of lipid droplets with LC3 and LAMP1. The role of autophagy on lipid metabolism and LF activation were explored. Additionally, the effect of mitochondria-targeted ROS scavenger mitoquinone and SIRT1 activator SRT1720 on mitochondrial oxidative stress, autophagy flux, lipid metabolism and LF activation were investigated in vitro and in vivo.ResultsWe found that CS promoted mtROS production by increasing mtNOX4 and decreasing SOD2. Next, we proved mtROS inhibited the expression of PPARα and CPT1a. It also reduced lipophagy and upregulated cellular lipid content, suggesting lipid metabolism was disturbed by CS. In addition, we showed both insufficient FAO and lipophagy resulted from blocked autophagy flux caused by mtROS. Moreover, we uncovered decreased SIRT1 was responsible for mitochondrial redox imbalance. Furthermore, we proved that both SRT1720 and mitoquinone counteracted the effect of CS on NOX4, SOD2, PPARα and CPT1a in vivo.ConclusionsWe demonstrated that CS decreased SIRT1 to activate LF through dysregulating lipid metabolism, which was due to increased mtROS and impaired autophagy flux. These events may serve as therapeutic targets for IPF patients.

Project description:Ceftazidime-avibactam is a "second-generation" β-lactam-β-lactamase inhibitor combination that is effective against Enterobacteriaceae expressing class A extended-spectrum β-lactamases, class A carbapenemases, and/or class C cephalosporinases. Knowledge of the interactions of avibactam, a diazabicyclooctane with different β-lactamases, is required to anticipate future resistance threats. FOX family β-lactamases possess unique hydrolytic properties with a broadened substrate profile to include cephamycins, partly as a result of an isoleucine at position 346, instead of the conserved asparagine found in most AmpCs. Interestingly, a single amino acid substitution at N346 in the Citrobacter AmpC is implicated in resistance to the aztreonam-avibactam combination. In order to understand how diverse active-site topologies affect avibactam inhibition, we tested a panel of clinical Enterobacteriaceae isolates producing blaFOX using ceftazidime-avibactam, determined the biochemical parameters for inhibition using the FOX-4 variant, and probed the atomic structure of avibactam with FOX-4. Avibactam restored susceptibility to ceftazidime for most isolates producing blaFOX; two isolates, one expressing blaFOX-4 and the other producing blaFOX-5, displayed an MIC of 16 μg/ml for the combination. FOX-4 possessed a k2/K value of 1,800 ± 100 M-1 · s-1 and an off rate (koff) of 0.0013 ± 0.0003 s-1 Mass spectrometry showed that the FOX-4-avibactam complex did not undergo chemical modification for 24 h. Analysis of the crystal structure of FOX-4 with avibactam at a 1.5-Å resolution revealed a unique characteristic of this AmpC β-lactamase. Unlike in the Pseudomonas-derived cephalosporinase 1 (PDC-1)-avibactam crystal structure, interactions (e.g., hydrogen bonding) between avibactam and position I346 in FOX-4 are not evident. Furthermore, another residue is not observed to be close enough to compensate for the loss of these critical hydrogen-bonding interactions. This observation supports findings from the inhibition analysis of FOX-4; FOX-4 possessed the highest Kd (dissociation constant) value (1,600 nM) for avibactam compared to other AmpCs (7 to 660 nM). Medicinal chemists must consider the properties of extended-spectrum AmpCs, such as the FOX β-lactamases, for the design of future diazabicyclooctanes.

Project description:Aerobic glycolysis has been shown to contribute to the abnormal activation of lung fibroblasts with excessive collagen deposition in lipopolysaccharide (LPS)-induced pulmonary fibrosis. Targeting aerobic glycolysis in lung fibroblasts might therefore be considered as a promising therapeutic approach for LPS-induced pulmonary fibrosis. In the present study, the aim was to investigate whether metformin, a widely used agent for treating type 2 diabetes, could alleviate LPS-induced lung fibroblast collagen synthesis and its potential underlying mechanisms. Different concentrations of metformin were used to treat the human lung fibroblast MRC-5 cells after LPS challenge. Indicators of aerobic glycolysis in MRC-5 cells were detected by measuring glucose consumption and lactate levels in culture medium in addition to lactate dehydrogenase activity in cellular lysates. The glucose consumption, lactate levels and the lactate dehydrogenase activity were measured respectively using colorimetric/fluorometric and ELISA kits. The effects of metformin in AMP-activated protein kinase (AMPK) activation was assessed by mitochondrial complex I activity kits. Collagen I, α-smooth muscle actin (α-SMA) and collagen III were used as markers of collagen synthesis, which was measured using western blotting, whereas phosphorylated (p-) AMPK, AMPK, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and mTOR were detected by western blotting. Metformin significantly decreased mitochondrial complex I activity and upregulated the expression of p-AMPK/AMPK protein in a concentration-dependent manner. Furthermore, the aerobic glycolysis mediated by PFKFB3 and collagen synthesis in LPS-treated MRC-5 cells was gradually inhibited with increasing concentrations of metformin. However, this inhibitory role of metformin on PFKFB3-meditaed aerobic glycolysis and collagen synthesis was prevented by treatments with 3BDO and compound C, which are specific mTOR activator and AMPK inhibitor, respectively. Taken together, the findings from this study suggested that metformin may prevent PFKFB3-associated aerobic glycolysis from enhancing collagen synthesis in lung fibroblasts via regulating the AMPK/mTOR pathway.

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A FOX-like Mechanism Regulating Lung Fibroblasts: Are We Getting There?

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