RANKL Mediates Muscle Atrophy and Dysfunction in a Cigarette Smoke-induced Model of Chronic Obstructive Pulmonary Disease.

Skeletal muscle dysfunction is one of the important comorbidities of chronic obstructive pulmonary disease (COPD); however, the underlying mechanisms remain largely unknown. RANKL (receptor activator of nuclear factor κB ligand), a key mediator in osteoclast differentiation, was also found to play a role in skeletal muscle pathogenesis. Whether RANKL is involved in COPD-related skeletal muscle dysfunction is as-of-yet unknown. We examined the expression of RANKL/RANK in skeletal muscles from mice exposed to cigarette smoke (CS) for 24 weeks. Grip strength and exercise capacity as well as muscular morphology were evaluated in CS-exposed mice with or without anti-RANKL treatment. The expressions of protein synthesis- or muscle growth-related molecules (IGF-1, myogenin, and myostatin), muscle-specific ubiquitin E3 ligases (MuRF1 and atrogin-1), and the NF-κb inflammatory pathway were also evaluated in skeletal muscles. The effect of CS extract on RANKL/RANK expression and that of exogenous RANKL on the ubiquitin-proteasome pathway in C2C12 myotubes were investigated in vitro. Long-term CS exposure induced skeletal muscle dysfunction and atrophy together with upregulation of RANKL/RANK expression in a well-established mouse model of COPD. RANKL neutralization prevented skeletal muscle dysfunction and atrophy. RANKL inhibition decreased expressions of myostatin and MuRF1/Atrogin1 and suppressed the NF-κb pathway in skeletal muscles from CS-exposed mice. In in vitro experiments with C2C12 myotubes, CS extract induced expression of RANKL/RANK, and exogenous RANKL induced activation of the ubiquitin-proteasome pathway and NF-κb pathway via RANK. Our results revealed an important role of the RANKL/RANK pathway in muscle atrophy induced by CS exposure, suggesting that RANKL may be a potential therapeutic target in COPD-related skeletal muscle dysfunction.

Pentagalloyl glucose increases elastin deposition, decreases reactive oxygen species and matrix metalloproteinase activity in pulmonary fibroblasts under inflammatory conditions.

Emphysema is characterized by degradation of lung alveoli that leads to poor airflow in lungs. Irreversible elastic fiber degradation by matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) activity leads to loss of elasticity and drives the progression of this disease. We investigated if a polyphenol, pentagalloyl glucose (PGG) can increase elastin production in pulmonary fibroblasts. We also studied the effect of PGG treatment in reducing MMP activity and ROS levels in cells. We exposed rat pulmonary fibroblasts to two different types of inflammatory environments i.e., tumor necrosis factor-α (TNF-α) and cigarette smoke extract (CSE) to mimic the disease. Parameters like lysyl oxidase (LOX) and elastin gene expression, MMP-9 activity in the medium, lysyl oxidase (LOX) activity and ROS levels were studied to assess the effect of PGG on pulmonary fibroblasts. CSE inhibited lysyl oxidase (LOX) enzyme activity that resulted in a decreased elastin formation. Similarly, TNF-α treated cells showed less elastin in the cell layers. Both these agents caused increase in MMP activity and ROS levels in cells. However, when supplemented with PGG treatment along with these two inflammatory agents, we saw a significant increase in elastin deposition, reduction in both MMP activity and ROS levels. Thus PGG, which has anti-inflammatory, anti-oxidant properties coupled with its ability to aid in elastic fiber formation, can be a multifunctional drug to potentially arrest the progression of emphysema.

Cigarette smoke extract-induced BEAS-2B cell apoptosis and anti-oxidative Nrf-2 up-regulation are mediated by ROS-stimulated p38 activation.

Cigarette smoke contains reactive oxygen (ROS) that can cause oxidative stress. It increases the number of apoptotic and necrotic lung cells and further induces the development of chronic airway disease. In this study, we investigated the effects of cigarette smoke extract (CSE) on apoptosis in human bronchial epithelial cells (BEAS-2B). CSE exposure induced ROS generation and p38 mitogen-activated protein kinase (MAPK) activation that are associated with the activation of apoptosis-regulating signal kinase 1 (ASK-1). N-acetylcysteine (a general antioxidant) attenuated the CSE-induced ASK-1 and p38 MAPK activation and cell apoptosis, suggesting a triggering role of ROS in ASK-1/p38 MAPK activation during apoptotic progression. In contrast, the inhibition and knockdown of p38 attenuated the expression of anti-oxidant master NF-E2-related factor 2 (Nrf-2) and CSE-induced apoptosis, suggesting that p38 MAPK modulates Nrf-2 expression and presumably prevents cell apoptosis. Taken together, the data presented in this manuscript demonstrate that the ROS-dependent ASK-1/p38 signaling cascade regulates CSE-induced BEAS-2B cell apoptosis. In addition, anti-oxidative Nrf-2 is also up-regulated by the ROS/p38 signaling cascade in this progression.

Berberine Attenuates Cigarette Smoke Extract-induced Airway Inflammation in Mice: Involvement of TGF-ß1/Smads Signaling Pathway.

Although several studies confirmed that berberine may attenuate airway inflammation in mice with chronic obstructive pulmonary disease (COPD), its underlying mechanisms were not clear until now. We aimed to establish an experiment mouse model for COPD and to investigate the effects of berberine on airway inflammation and its possible mechanism in COPD model mice induced by cigarette smoke extract (CSE). Twenty SPF C57BL/6 mice were randomly divided into PBS control group, COPD model group, low-dose berberine group and high-dose berberine group, 5 mice in each group. The neutrophils and macrophages were examined by Wright's staining. The levels of inflammatory cytokines TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay. The expression levels of TGF-ß1, Smad2 and Smad3 mRNA and proteins in lung tissues were respectively detected by quantitative real-time polymerase chain reaction and Western blotting. It was found that CSE increased the number of inflammation cells in BALF, elevated lung inflammation scores, and enhanced the TGF-ß1/Smads signaling activity in mice. High-dose berberine restrained the alterations in the COPD mice induced by CSE. It was concluded that high-dose berberine ameliorated CSE-induced airway inflammation in COPD mice. TGF-ß1/Smads signaling pathway might be involved in the mechanism. These findings suggested a therapeutic potential of high-dose berberine on the CSE-induced airway inflammation.

The novel compound Sul-121 inhibits airway inflammation and hyperresponsiveness in experimental models of chronic obstructive pulmonary disease.

COPD is characterized by persistent airflow limitation, neutrophilia and oxidative stress from endogenous and exogenous insults. Current COPD therapy involving anticholinergics, ß2-adrenoceptor agonists and/or corticosteroids, do not specifically target oxidative stress, nor do they reduce chronic pulmonary inflammation and disease progression in all patients. Here, we explore the effects of Sul-121, a novel compound with anti-oxidative capacity, on hyperresponsiveness (AHR) and inflammation in experimental models of COPD. Using a guinea pig model of lipopolysaccharide (LPS)-induced neutrophilia, we demonstrated that Sul-121 inhalation dose-dependently prevented LPS-induced airway neutrophilia (up to ~60%) and AHR (up to ~90%). Non-cartilaginous airways neutrophilia was inversely correlated with blood H2S, and LPS-induced attenuation of blood H2S (~60%) was prevented by Sul-121. Concomitantly, Sul-121 prevented LPS-induced production of the oxidative stress marker, malondialdehyde by ~80%. In immortalized human airway smooth muscle (ASM) cells, Sul-121 dose-dependently prevented cigarette smoke extract-induced IL-8 release parallel with inhibition of nuclear translocation of the NF-κB subunit, p65 (each ~90%). Sul-121 also diminished cellular reactive oxygen species production in ASM cells, and inhibited nuclear translocation of the anti-oxidative response regulator, Nrf2. Our data show that Sul-121 effectively inhibits airway inflammation and AHR in experimental COPD models, prospectively through inhibition of oxidative stress.