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The standardized herbal formula, PM014, ameliorated cigarette smoke-induced lung inflammation in a murine model of chronic obstructive pulmonary disease
© Jung et al.; licensee BioMed Central Ltd. 2013
Received: 15 April 2013
Accepted: 29 August 2013
Published: 5 September 2013
In this study, we evaluated the anti-inflammatory effect of PM014 on cigarette smoke induced lung disease in the murine animal model of chronic obstructive pulmonary disease (COPD).
Mice were exposed to cigarette smoke (CS) for 2 weeks to induce COPD-like lung inflammation. Two hours prior to cigarette smoke exposure, the treatment group was administered PM014 via an oral injection. To investigate the effects of PM014, we assessed PM014 functions in vivo, including immune cell infiltration, cytokine profiles in bronchoalveolar lavage (BAL) fluid and histopathological changes in the lung. The efficacy of PM014 was compared with that of the recently developed anti-COPD drug, roflumilast.
PM014 substantially inhibited immune cell infiltration (neutrophils, macrophages, and lymphocytes) into the airway. In addition, IL-6, TNF-α and MCP-1 were decreased in the BAL fluid of PM014-treated mice compared to cigarette smoke stimulated mice. These changes were more prominent than roflumilast treated mice. The expression of PAS-positive cells in the bronchial layer was also significantly reduced in both PM014 and roflumilast treated mice.
These data suggest that PM014 exerts strong therapeutic effects against CS induced, COPD-like lung inflammation. Therefore, this herbal medicine may represent a novel therapeutic agent for lung inflammation in general, as well as a specific agent for COPD treatment.
Chronic obstructive pulmonary disease (COPD) is a common public health concern worldwide, and the incidence of COPD is increasing globally [1, 2]. COPD is characterized by progressive and irreversible airway obstruction . Chronic inflammation contributes to a decline in pulmonary function associated with chronic bronchitis, mucus hypersecretion, and emphysema via the release of pro-inflammatory mediators, reactive oxygen species, and tissue degradation enzymes . Subsequently, these pulmonary changes result in abnormalities in gas exchange at the pulmonary level and respiratory failure . The pathology of COPD differs markedly from that of asthma [6–8]. In larger airways, there is evidence of neutrophilic rather than eosinophilic inflammation, as indicated by an increased number of neutrophils in BAL fluid. Current conventional treatment is aimed at relieving symptoms, preventing recurrent exacerbation, preserving optimal lung function and enhancing overall quality of life . Although many drugs are used to treat COPD, the adverse effects associated with several classes of drugs, such as steroids, have increased the need for alternative treatments, such as herbal medicines [10, 11].
Because COPD is a chronic inflammatory disorder, it is essential to determine whether novel anti-inflammatory agents can halt or slow the decline in lung function that occurs in response to this disease when selecting candidate drugs. Several studies have demonstrated that compounds derived from plants have anti-inflammatory or immune-modulating properties , and several herbal medicines, including Panax ginseng and Salvia miltiorrhiza, have been used for COPD treatment .
PM014 is modified from Chung-Sang-Bo-Ha-Tang (CSBHT). The Chung-Sang-Bo-Ha-Tang (CSBHT) has been especially used to treat chronic pulmonary diseases in Korea for centuries . Previously, we developed the formulation of PM014 based on the series of in vitro and in vivo screening efforts. The results showed that PM014 possessed potent anti-inflammatory effects in both lipopolysaccharide (LPS)-induced and elastase/LPS induced acute lung inflammation murine models . However, the previous study only suggested the prophylactic effects of PM014 in lung injury. In addition, the lung inflammation inducer did not reflect the actual environment. To overcome these limitations, we evaluated PM014 to determine if it had therapeutic effects on lung inflammation in a mouse model of CS-induced lung neutrophilia that mimicked a COPD-like lung injury . CS-associated chronic obstructive pulmonary disease (COPD) is characterized by inflammation, changes affecting small airways and the development of emphysema . The main pathological characteristics of CS-associated COPD are inflammation along the bronchus and bronchioles, fibrosis, smooth muscle hypertrophy, goblet cell hyperplasia, small airway and vascular remodeling, and development of centrilobular emphysema . In addition, CS is a very strong environmental risk factor that is linked to rheumatoid arthritis (RA) [19, 20] and other autoimmune diseases [21–23]. CS is a toxic and carcinogenic mixture of more than 5,000 chemicals . Of these chemicals, approximately 400 have been quantified; at least 200 are toxic to humans and/or experimental animals; and over 50 have been identified as known, probable, or possible human carcinogens . Mainstream smoke typically contains large amounts of bacterial and fungal compounds, such as endotoxins (lipopolysaccharide, LPS, in the outer membrane of Gram-negative bacteria) and ergosterol (a specific fungal membrane lipid) .
Roflumilast is indicated as a treatment to reduce the risk of COPD exacerbations in patients with severe COPD associated with chronic bronchitis and a history of exacerbation . Roflumilast is a lipophilic, highly permeable molecule that exhibits rapid and nearly complete absorption after oral administration .
In the present study, we evaluated the anti-inflammatory effects of PM014 on CS-induced lung inflammation in mice and elucidated the possible mechanism by which PM014 suppresses CS-induced lung inflammation.
Composition and amount of PM014
Rehmannia Radix Preparata
Animal and maintenance conditions
Analysis of BAL cells
The mice were sacrificed via cervical vertebral dislocation. PBS (phosphate buffered saline) was slowly infused into the lungs and withdrawn via a cannula inserted into the trachea. The cell numbers were counted using a hemocytometer, and differential cell counts were performed on slides prepared by cytocentrifugation at 250 rpm for 3 min and Diff-Quick staining. Approximately 500 cells were counted. BAL fluid was then centrifuged, and the supernatants were kept at −80°C.
ELISA measurements of IL-6 and TNF-α and MCP-1 in bronchoalveolar lavage fluids
Protein concentrations were determined using a BCA kit (Pierce Biotechnology Inc., Rockford, IL, U.S.A.). IL-6, TNF-α, and MCP-1 concentrations were measured with a quantitative sandwich enzyme-linked immunoassay kit (BD, San Diego, CA, U.S.A.). A 96-well microtiter plate was incubated overnight at 4°C with anti-rat IL-6,TNF-α and MCP-1 monoclonal antibody in coating buffer, washed with PBS containing 0.05% tween 20 (Sigma, St. Louis, MO, U.S.A.) and blocked with 5% FBS in PBS for 1 hr at room temperature. Subsequently, the BAL fluid (100 μl) was incubated for 2 hr at room temperature. Then, secondary peroxidase-labeled biotinylated anti-rat IL-6, TNF-α, and MCP-1 monoclonal antibody was incubated in 5% FBS in PBS for 1 hr. Finally, the plates were treated with TMB substrate solution for 30 min, and the reaction was stopped by adding TMB stop solution (BD, San Diego, CA, U.S.A.). Optical density was measured at 450 nm in a microplate reader (SOFT max PRO software, Sunnyvale, CA, U.S.A.).
Preparation of lung tissues and histology
The lung tissues were removed from the mice, and the right lower lobes were removed for histological analysis. Four percent paraformaldehyde fixing solution was infused into the lungs. The specimens were dehydrated and embedded in paraffin. For histological examination, 4 μm sections of embedded tissue were cut on a rotary microtome, placed on glass slides, deparaffinized, and stained sequentially with hematoxylin and eosin (H&E). The severity of peribronchial inflammation was graded semi-quantitatively as previously described . Hyperplasia of the goblet cells within the bronchial epithelium was assessed by counting cells in periodic acid Schiff (PAS)-stained sections. Slides were mounted with Canada balsam (Showa Chemical Co. Ltd., Tokyo, Japan). PAS-positive cells in the epithelium and total epithelial cells were counted, and the percentage of PAS-positive cells was calculated. For quantitating air space in lung, the sections with the maximum parenchyma cross-sections were selected for morphometric analysis using a digitized image tool . Micrographs were obtained using Image Pro-Plus 5.1 software (Media Cybernetics, Inc. Silver Spring, MD, U.S.A.).
Data are presented as the means ± S.E.M. Data analysis was conducted using Graphpad Prism software (version 4, San Diego, CA, U.S.A.). The differences between study groups were determined by one-way ANOVA and the Newman-Keuls multiple comparison test. P < 0.05 was considered to be statistically significant.
The effects of PM014 on total and inflammatory cells levels in BAL fluid
2.80 ± 1.10
12.00 ± 1.79***
6.20 ± 1.10#
11.60 ± 2.97
11.20 ± 3.90
10.40 ± 4.78
5.67 ± 2.66##
0.00 ± 0.00
0.31 ± 0.02***
0.08 ± 0.03##
0.28 ± 0.11
0.26 ± 0.12
0.19 ± 0.05
0.06 ± 0.07###
2.78 ± 1.08
12.30 ± 1.21***
6.37 ± 1.20##
11.20 ± 2.85
11.17 ± 4.41
8.24 ± 2.40
4.70 ± 2.29###
0.01 ± 0.02
0.17 ± 0.03***
0.09 ± 0.04#
0.12 ± 0.05
0.13 ± 0.04
0.11 ± 0.06
0.03 ± 0.02###
The effect of PM014 on IL-6, TNF-α and MCP-1 in BAL fluids by CS-induced mice
The Effect of PM014 on histologic lung damage
The effect of PM014 on goblet cell hyperplasia in bronchial airways
Inflammation in COPD is complicated, with inflammatory and structural cells that release various mediators, including mediators such as LTB4, IL-8 and GCP-2, which were chemoattractant for neutrophil and chemokines such as MCP-1 and MIP-1α, which attract macrophage . An accumulation of inflammatory cells such as neutrophils, macrophages, dendritic cells CD+8T lymphocytes is seen . In our study, the infiltration of inflammatory cells in BAL fluid and in the lung parenchyma was observed following exposure to cigarettes. Neutrophils have been implicated in causing tissue damage in COPD through the release of a number of mediators, including proteases, such as elastases and matrix metalloproteinase, and oxidants and toxic peptides, such as defensins .
Concomitant with the influx of neutrophils, increased levels of the pro-inflammatory cytokines, TNF-α and IL-6 were observed in the BAL fluid by Cigarette smoke (CS) exposure induce mice model. In addition, the expression of inflammation-related cytokines, such as IL-6 and TNF-α was found to increase following CS exposure in the airway. Also, CS-induced goblet cell hyperplasia is associated with the development of bronchitis which is related to COPD, which depends on the degree of epithelial inflammation . Goblet cell hyperplasia is one of the morphogic changes in lung epithelium. In addition to that, there are various epithelial changes including submucosal gland hypertrophy associated with loss of ciliated epithelial cell number, leading to reduced mucociliary clearance, and mucus plug formation .
Cigarette smoke can contribute to the development of many human diseases, such as cardiovascular disease, lung cancer, asthma, and chronic obstructive pulmonary disease. Thousands of compounds are present in CS, including a large number of reactive oxygen species that can cause DNA damage and lead to the activation of poly (ADP-ribose) polymerase (PARP) enzyme . Other components of CS, such as nicotine and acrolein, have also been shown to exert direct genotoxic effects [37, 38].
In our study, the infiltration of inflammatory cells in BAL fluid was observed after CS exposure. Exposure to CS induced several pathological changes, such as inflammatory cell accumulation in the lung parenchyma, hyperplasia of goblet cells, hypersecretion of mucus, alveoli enlargement, and increases in collagenic and elastic structures in the alveolus. The pathological changes observed in this study were very similar to the clinical features of COPD patients . In this CS exposure model, the herbal mixture PM014 showed consistent efficacy comparable with that of the commercially available anti-COPD drug, roflumilast .
Herbal mixtures are widely used as traditional medicines to treat many different types of disease [40, 41]. In Korean traditional medicine, herbs are used as mixtures rather than as one herb by itself. PM014 is modified from Chung-Sang-Bo-Ha-Tang (CSBHT). The Chung-Sang-Bo-Ha-Tang (CSBHT) has been especially used to treat chronic pulmonary diseases in Korea for centuries. However, CSBHT contains 18 species of medicinal plants, and it is difficult to standardize the herbal formula . Therefore, CSBHT was modified to PM014, which contains 7 species of medicinal plants. Previously, we initially compared the effects of each herb and the PM014 herbal mixture in an acute LPS-induced lung injury model . Stemona sessilifolia, S. japonica and S. tuberose are the three original sources of Stemonae Radix specified in Chinese Pharmacopoeia (CP) and have been traditionally used as antitussive and insecticidal remedies . Asparagus cochinchinensis is used for treating lung- and spleen-related diseases . A bioactive flavonoid extracted from the root of Scutellaria baicalensis has anti-inflammatory and anti-angiogenic activities . Schisandra chinensis fruit especially alleviates cough and satisfies thirst. In modern pharmaceutical studies, Schisandra chinensis fruit has been reported to reduce hepatotoxicity [45–50]. The root of Rehmannia glutinosa (RR) is commonly used to reduce inflammation . The root cortex of Paeonia suffruticosa Andrews (PSA), also known as Moutan Cortex, is known to have anti-allergic and anti-inflammatory properties . Individual herb extracts in PM014 also attenuated the immune cell influx; however, treatment with the herbal mixture PM014 resulted in less recruitment of all immune cells toward the lungs than the individual herbal treatments . Therefore, it is assumed that using a mixture of 7 medicinal herbs is more effective with regard to synergism than using each of the 7 herbs separately. These results may suggest that PM014 is a powerful therapeutic agent, which reduces chronic accumulation of inflammatory cells induced by CS. In this study, PM014 (50, 100, 200 mg/kg wt) markedly reduced inflammatory cells in BAL fluid. Furthermore, when we compared the histological findings, PM014 (100 mg/kg wt) significantly reduced the numbers of lymphocytes, neutrophils, infiltrating macrophage and the level of goblet cell metaplasia. Epithelial basement membrane thickening and inflammation of the bronchiole also were remarkably inhibited. PM014 (100 mg/kg wt) also, markedly reduced levels of TNF-α, IL-6 and MCP-1 than CS group. However, PM014 (50 mg/kg wt) did not show significant effect than PM014 (100, 200 mg/kg wt) in TNF-α, IL-6, and MCP-1 production.
TNF-α, an early pro-inflammatory cytokine, is believed to trigger the activation of other pro-inflammatory cytokines, such as IL-6 and IL-8 . TNF-α also activates nuclear factor-κB, which increases IL-8 gene transcription, thereby inducing the release of IL-8 from the airway epithelium and neutrophils. IL-8, a CXC chemokine, is a neutrophil chemoattractant and activator . MCP-1 a monocyte selective chemokine which attracts monocytes to lung is increased in lung COPD patients . Macrophages mediate inflammation in COPD through the release of chemokines that attract neutrophils, monocytes and T-cells and releases serine proteases like matrix metalloproteinase (MMP-9) .
In our study, PM014 downregulated pro-inflammatory cytokine production in both acute and chronic lung inflammation. Therefore, treatment with PM014 may act steadily on downstream events, including the influx of inflammatory cells and the levels of mediators aggravating the inflammatory response. In addition, histopathological data implied that PM014 administration inhibited the progression of airspace enlargement and goblet cell hyperplasia. Theses result also demonstrated that PM014 could be sufficient to structural changes of lung, typical of COPD.
The results of this study provide evidence that treatment with PM014 exerts therapeutic effects against smoking-induced lung inflammation in mice. The remarkable anti-inflammatory effects exerted by PM014 suggest that it has the potential to be used in the treatment of COPD patients. However, further study to elucidate the mechanisms underlying the action of PM014 should be conducted to aid in the discovery of new therapeutic agents for COPD treatment.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (No. 2011–0006220) and Traditional Korean Medicine R&D Project, Ministry for Health &Welfare (B100053), Republic of Korea.
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