Liujunzi Tang, a famous traditional Chinese medicine, ameliorates cigarette smoke-induced mouse model of COPD

Author’s Accepted Manuscript Liujunzi Tang, a famous traditional Chinese medicine, ameliorates cigarette smoke-induced mouse model of COPD Rui Zhou, Fen Luo, Hui Lei, Kai Zhang, Jingyan Liu, He He, Jin Gao, Xiayun Chang, Ling He, Hui Ji, Tianhua Yan, Tong Chen www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(16)30896-0 http://dx.doi.org/10.1016/j.jep.2016.09.036 JEP10436

To appear in: Journal of Ethnopharmacology Received date: 26 March 2016 Revised date: 20 July 2016 Accepted date: 18 September 2016 Cite this article as: Rui Zhou, Fen Luo, Hui Lei, Kai Zhang, Jingyan Liu, He He, Jin Gao, Xiayun Chang, Ling He, Hui Ji, Tianhua Yan and Tong Chen, Liujunzi Tang, a famous traditional Chinese medicine, ameliorates cigarette smokeinduced mouse model of COPD, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.09.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Liujunzi Tang, a famous traditional Chinese medicine, ameliorates cigarette smoke-induced mouse model of COPD Rui Zhoua, 1, Fen Luoa, 1, Hui Leib, Kai Zhangc, Jingyan Liua, He Hea, Jin Gaoa, Xiayun Changa, Ling Hed, Hui Jid, Tianhua Yana,*, Tong Chena,d** a

Department of Physiology and Pharmacology, China Pharmaceutical University, Nanjing

210009, China b

Pharmaceutical Animal Experiment Center of China Pharmaceutical University，Nanjing

211198, China c

Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University,

Nanjing 210009, China d

Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, China

Tianhua Yan (E-mail:[email protected]) Tong Chen(E-mail:[email protected]) *Corresponding authors:

Abstract Ethnopharmacological relevance: Liujunzi Tang is a traditional herbal medicine widely used in East Asia and clinically applied to treat Phlegm-Heat Syndrome. The purpose of the present study was to investigate the protective effects of Liujunzi Tang on cigarette smoke-induced (CS) mouse model of chronic obstructive pulmonary disease (COPD) and explore its potential molecular mechanism. Materials and methods The mice received 1 h of cigarette smoke for 8 weeks. The serum levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA) kits. Superoxide dismutase (SOD) and malondialdehyde (MDA) were tested by biochemical methods. Histopathological alteration was observed by

1

Rui Zhou and Fen Luo Contributed equally to this work.

hematoxylin–eosin (H&E) staining. Additionally, the expressions of nuclear transcription factor-κB (NF-κBp65) and (inhibitor of NF-κB)IκB-α were determined by western blot and immunohistochemistry analysis. Results Liujunzi Tang enhanced the activities of antioxidant enzymes and attenuated the levels of lipid oxidative production, meanwhile significantly inhibited the generations of inflammatory cytokines by inhibiting the phosphorylation of IκB-α and NF-κB. Conclusion Our findings indicated that Liujunzi Tang exhibited the protective effect on cigarette smoke-induced COPD mice by anti-inflammatory and anti-oxidative properties through the inhibition of NF-κB activation.

Keywords: Liujunzi Tang ; Cigarette Smoke; Anti-oxidation; NF-κB

1. Introduction Chronic Obstructive Pulmonary Disease (COPD) remains a leading cause of morbidity and mortality around the world. Cigarette smoking is established as a major risk factor especially in developing countries where smoking prevalence is rising and air pollution is serious (Woodruff et al., 2015). COPD is characterized by the limitation of airflow and chronic exposure of genetically susceptible individuals to environmental factors (Han et al., 2010). The airflow limitation is usually progressive and associated with an abnormal

inflammatory response to noxious particles or gases (Pauwels et al., 2001). Despite increasing patients suffering the disease, its pathophysiological mechanism is still unclear. Current treatments for COPD are primarily focused on targeting the symptom of airflow limitation and specific inflammatory pathways (Rennard and Drummond, 2015). Corticosteroids and bronchodilators have beneficial effect on the treatment and/or prevention of COPD. However, except the risk of pneumonia and systemic side-effects (ie, bone fractures), COPD patient have resistance to the corticosteroid therapy. Thus even high doses of inhaled corticosteroids could not achieve an optimum treatment (Price et al., 2013). Several mechanistic concepts have been implicated in the pathogenesis of COPD. The hallmark of COPD is development of exaggerated chronic pulmonary inflammation in response to inhalation of cigarette smoke (Tsuji et al., 2004). Tobacco smoke contains reactive oxygen species (ROS) and many different chemical components, which activated non-specific immune system then mediates the processes of inflammation, repairment, fibrosis and proteolysis (Oostwoud et al., 2016). NF-κB is a key modulator of inflammatory responses and governs the transcriptions of TNF-α, IL-1β, IL-6 (Chen et al., 2015b). Similarly, the inhibition of NF-κB contributes to the suppression of lung neutrophilia and cytokine generation in LPS-induced acute lung injury (Shilin et al., 2016). Cigarette smoke-induced release of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) cause the alterations including epithelial desquamation, goblet cell metaplasia, angiogenesis and fibrosis (Song et al., 2015). These changes occur in airways and are associated with chronic obstructive pulmonary disease (Oostwoud et al., 2016). In addition, oxidative stress plays a key role in the progression of the inflammatory process (Chen et al., 2016a). It is implicated in initiating inflammatory responses through the activation of transcriptional factors including NF-κB signaling pathway, activator protein-1 (AP-1) and gene expression of pro-inflammatory mediators (Zhu et al., 2015). It has also been proved in amplifying the inflammatory response and might inhibiting anti-inflammatory effects of corticosteroids (Chen et al., 2015c). Moreover, oxidative stress affects remodelling of extracellular matrix, mitochondrial respiration and infiltration of surfactants to alveoli (Fourcade et al., 2015).

Liujunzi Tang, which is also called rikkunshito (RKT) in Japan, was originally recorded in ‘Yi Xue Zheng Zhuan’, an important medical writings in ancient China since Ming Dynasty and ‘Shi Yi De Xiao Fang’, another valuable medical writings in the Yuan Dynasty. Liujunzi Tang is a traditional complex prescription comprising of following ingredients: Ginseng (9g), Atractylodes (9 g), Poria cocos (9 g), Glycyrrhizae (6 g), Pericarpium Citri Reticulatae (3 g), Pinellia ternata (4.5 g). Liujunzi Tang is used for eliminating dampness, sputum and preventing chronic gastritis. During the recent decades, it has been clinically used in the treatment of respiratory inflammation tonsillitis and acute pharyngitis. Experimental studies demonstrated that Liujunzi Tang upregulated glucocorticoid receptor (GCR) density and improved adrenocortical function in ovalbumin-induced asthma (Bao et al., 1999), as well as reduced lung fibrosis in bleomycin-induced acute lung injury (Tsubouchi et al., 2014). Moreover, former literature have shown that mixed herbal formula that contain Liujunzi Tang suppressed nasal mucosa inflammation in patients with H-IgE allergic rhinitis (Yang et al., 2002). Liujunzi Tang also attenuated the allergic reaction by modifying the physiologic function of the DC-CD4(+) T cell interaction (Yang et al., 2016). However, the effects and mechanisms of Liujunzi Tang on cigarette smoke-induced mouse model of chronic obstructive pulmonary disease are still not well-understood. Therefore, based on its anti-inflammatory and anti-oxidative properties, there was possible expectation that Liujunzi Tang could reduce pulmonary inflammation in an animal model of COPD by inhibiting the activation of nuclear factor NF-κB and excessive oxidative stress. Liujunzi Tang would be a promising alternative medicine for COPD management.

2. Materials and methods

2.1. Plant materials and plant extracts

Liujunzi Tang consists of the following ingredients: Ginseng (9 g), Atractylodes (9 g), Poria cocos (9 g), Glycyrrhizae (6 g), Pericarpium Citri Reticulatae (3 g), Pinellia ternata

(4.5 g). Each botanic medicine was purchased from pharmaceutical company and was identified as eligible and pure medicinal materials. These herbs were chopped into small pieces and immersed for 1 h with 8 times volume of water. The medicinal materials were decocted twice at boiling temperature for half an hour. The extract was immediately filtered through a two-layer mesh and the decocted liquids were collected. The yield of the extract is 18.3% (w/w). Finally, it was concentrated to 0.3 g/ml, placed in the sterile bottle and stored in the refrigerator. The Liujunzi Tang aqueous extract was intragastrically given to mice and also called Liujunzi Tang in the following text. 2.2. Chromatographic Conditions Chromatographic analysis was performed on a Waters Acquity UPLC system (Waters Corp., Milford, MA, USA), consisting of a binary pump solvent management system, an online degasser, and an auto-sampler. An ACQUITY UPLC BEH C 18 (100 mm × 2.1 mm, 1.7 μm) column was applied for all analyses. And the column temperature was maintained at 35 °C. The mobile phase was composed of (A) formic acid aqueous solution (0.1%) and (B) acetonitrile using a gradient elution of 10%–50% B at 0–5 min, 50%–95% B at 5–10 min, 95% B at 10–15 min, 95%–100% B at 15–18 min. 2.3. Mass Conditions Mass spectrometry detection was performed using a Xevo Triple Quadrupole MS (Waters Corp.) equipped with an electro spray ionization source (ESI). The ESI source was set in both positive and negative ionization mode. The parameters in the source were set as follows: capillary voltage 3.0 kV; source temperature 150 °C; desolvation temperature 550 °C; cone gas flow 50 L/h; desolvation gas flow 1000 L/h.

2.4. Reagents

The material Liujunzi Tang was purchased from Nanjing pharmaceutical company. Dexamethasone (Dex) was provided by Simcare Drug Store (Nanjing, China). Cigarettes were purchased from retail. TNF-α, IL-6 and IL-1β enzyme-linked immuno-sorbent assay (ELISA) kits were supplied by Nanjing KeyGEN Biotech. Co., Ltd. (Nanjing, China). MDA and SOD kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China).

IκBα (#4841), p-IκBα (#2859), NF-κBp65(#8242)and p-NF-κBp65(#3033), GAPDH(#5174) antibodies were produced by Cell Signaling Technology (Danvers, USA).

2.5. Animals and treatments

60 male ICR mice (6-8 weeks, China Comparative Medicine Centre of Yangzhou University) initially weighing 20-22 g were used in this study. All animals were housed in a specific pathogen-free (SPF) laboratory in the Animal Center of China Pharmaceutical University at 22 ± 1°C temperature and 40-50% humidity under a 12 h light/dark cycle with free access to water and standard laboratory chow. All of the animal experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

2.6. Experiment protocols

All groups except control group were exposed 5 days a week to the mainstream cigarette smoke of 5 cigarettes (Reference cigarette 3R4F without filter, University of Kentucky, Lexington, KY, USA), 4 times a day with a 10 minute smoke free interval between exposures. A standard smoking apparatus was used with the smoking chamber adapted for a group of mice. A smoke/air ratio of 1/6 was obtained. CS exposure period was 8 weeks and control mice were exposed to room air at the same time. From the fifth week of cigarette smoking stimulation, the mice in dexamethasone (Dex) group were treated with 2 mg/kg/d dexamethasone by intragastric administration for 4 weeks. The mice in low and high dose group of Liujunzi Tang were treated with 1.5g/kg/d and 3g/kg/d Liujunzi Tang by intragastric administration for 4 weeks. Simultaneously, the mice in control and model group were intragastrically treated with the same amount of distilled water.

2.7. Lung homogenate

4 h after the last drug administration, blood samples were collected from the orbit and

mice were sacrificed. The blood was centrifuged at 4500 rpm for 15 min to obtain serum sample. The supernatant was collected and set aside at -80 °C for further assay. The lung were harvested and rinse the tissues with ice-cold PBS to remove excess blood thoroughly. Then each 100 mg tissue was homogenized with 900 μM PBS. After being centrifuged at 12,000 rpm for 10 min at 4 °C, the supernatant of the homogenate was collected into tubes and stored at -80 °C.

2.8. Biochemical analysis

Mouse in each group was sacrificed after orbital blood and lung were immediately removed. Supernatant of the lungs homogenate was collected as above and the protein content was measured by BCA Protein Assay Kit (Beyotime, Nanjing, China). The levels of pro-inflammatory cytokines IL-6, IL-1β and TNF-α in the lungs and serum were measured by commercially ELISA kits according to the manufacturer’s instructions. The optical density was taken spectrophotometrically at 450 nm.

2.9. Antioxidant detection

The levels of malondialdehyde (MDA), superoxide dismutase (SOD) in serum were determined using MDA and SOD kits (Nanjing Jiancheng Bioengineering Institute) following the manufacturer’s instructions. The absorbance values were detected at 532 nm and 550 nm, respectively.

2.10. Histological examination

At the end of the experiment, the tissues were washed in phosphate buffer and fixed in 10% (v/v) neutral buffered formalin. Then, the tissues were embedded in paraffin, sliced into 4 μm thicknesses, deparaffinized in xylene and rehydrated in a decreasing concentration gradient of ethanol. Afterwards, the samples were stained with Hematoxylin-Eosin (HE) solution for the examination of histopathology. The observation was carried out under a common optical

microscope in a blind manner. Lung cell count based on a 5-point scoring system was performed described to estimate the severity of leukocyte infiltration. The scoring system was as follows: 0, no cells; 1, a few cells; 2, a ring of cells one cell layer deep; 3, a ring of cells two to four cell layers deep; and 4, a ring of cells more than four cell layers deep.

2.11. Western blot analysis

Lungs in each group were collected immediately after the therapy. Total protein was extracted, and the concentrations were measured by a BCA procedure. The samples were separated on SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in Tris buffer saline–Tween20 (TBST) and then incubated with the respective antibodies overnight at 4℃ with. After washing with TBST, the membranes were incubated with a horseradish peroxidase conjugated secondary antibody (1:1000) for 2 h at room temperature. The membranes were visualized using the enhanced chemiluminescence detection system (Pierce, USA). The level of GAPDH was used as an internal control. Relative intensities were quantified using Quantity One (Bio Rad).

2.12. Statistical analysis

The data are expressed as the means ± SDs and analyzed with one-way analysis of variance (ANOVA) with Tukey multiple comparison test. The P value less than 0.05 was considered significant.

3. Result 3.1. UPLC-MS profile of Liujunzi Tang The major components of Liujunzi Tang were analyzed by UPLC-MS (Fig. 1). As compared with standard reference compounds, six compounds were identified and determined: 1:

Succinic Acid; 2: Hesperidin; 3: Ginsenoside Rb1; 4: glycyrrhizic acid I; 5: 2-Atractylenolide; 6: Pachymic acid . The contents of these compounds in Liujunzi Tang were observed as: 0.031 mg/g, 5.21 mg/g, 0.612 mg/g, 4.241 mg/g, 0.013 mg/g, 0.011 mg/g.

3.2. Effects of Liujunzi Tang on inflammatory cytokines in lung tissues and serum

ELISA assay was used to investigate the effects of Liujunzi Tang on the levels of TNF-α, IL-6 and IL-1β in CS-induced COPD mice model. Pro-inflammatory cytokines in lung tissue showed that TNF-α, IL-6 and IL-1β were overproduced in model group compared with those in control group. However, the productions of TNF-α, IL-6 and IL-1β in Dexamethasone (Dex, 2 mg/kg) group was potently reduced, while those in Liujunzi Tang (1.5g/kg) and Liujunzi Tang (3g/kg) groups were also slightly decreased. (Fig.1) Additionally, the levels of TNF-α, IL-6 and IL-1β in CS-treated mice were dramatically higher than those in control mice. By contrast, the generations of inflammatory cytokines in mice treated with Dex(2 mg/kg) and Liujunzi Tang (3g/kg) were significantly inhibited, which were slightly more pronounced than those in rats administrated with Liujunzi Tang (1.5g/kg). (Fig.2)

3.3. Effect of Liujunzi Tang on antioxidant activity

The CS-induced COPD model group significantly reduced the levels of SOD and CAT in lung tissues as compared with control group. However, animals with the treatment of Liujunzi Tang (1.5, 3g/kg) significantly increased the activities of SOD and CAT in lung tissues compared with those in the model group. Furthermore, the treatment with Liujunzi Tang (1.5, 3 g/kg) significantly increased the levels of SOD and CAT in lung tissues compared with those in model group. The results demonstrated that Liujunzi Tang could enhance the antioxidant capacity in the process of COPD. (Fig.3)

3.4. Effect of the Liujunzi Tang on lipid peroxidation

The CS-induced COPD model group significantly increased the content of MDA in lung tissues compared with that in control group. By contrast, the Liujunzi Tang (1.5, 3 g/kg) group both significantly decreased the level of MDA compared with that in model group. The results showed Liujunzi Tang significantly reduced MDA content, a product of lipid peroxidation, and further attenuated the peroxidation damage induced by cigarette smoke. (Fig.4)

3.5．Expression of IκB-α and p65 in lung tissues

Next, we examined changes in the phosphorylations of IκB-α and NF-κBp65 by western blot. As shown in Fig.5, CS-injured mice had obvious up-regulated p-IκB-α and p-p65 in lung tissues. Notably, treatments with Liujunzi Tang (1.5, 3 g/kg) and Dex(2 mg/kg) effectively suppressed the expressions of p-IκB-α and p-p65 in CS-challenged mice. Our analytical results suggested that Liujunzi Tang inhibited the phosphorylation and degradation of IκB-α, which consequently inhibited the activation of NF-κBp65

3.6. Histopathological examination of lung tissues

As revealed in Fig.6, scarce obvious histological alteration was viewed in lung specimen of control group. The pulmonary pathology of CS group showed evident epithelial cells injury of bronchi, goblet epithelium cells metaplasia, inflammatory cells infiltration, mucus secretion and cell blocking in bronchial lumen. On the contrary, mice treated with Liujunzi Tang (1.5g/kg and 3g/kg) both exhibited a weakened degree of pathological changes compared with that in the CS group. We blindly scored an index in representative images to quantify these histopathological alterations. The CS group showed an increased index compared with that in control group. However, the Liujunzi Tang (1.5 g/kg and 3 g/kg) groups showed markedly decreased indices compared with that in CS group. The results of hematoxylineosin staining indicated that the COPD model was successfully established and Liujunzi Tang obviously improved the pathological injury of COPD.

4 Discussion Although many details remain to be delineated, oxidative stress and inflammatory response play critical roles in the pathogenesis of COPD patients (Di Stefano et al., 2004). In the present study, we demonstrated that Liujunzi Tang ameliorated the oxidative stress and inflammatory condition of COPD induced by cigarette smoke. To the best of our knowledge, this was the first report to demonstrate Liujunzi Tang could protect lungs from CS exposure damage in the mouse model of COPD. The cigarette exposure-induced COPD has led to much experimental work to define the inflammatory process involved in the disease (Wang et al., 2015). Metcalfe et al proposed that cigarette smoke provoked severe inflammation mediated by NF-κB-dependent production of cytokines (Metcalfe et al., 2014). Under normal condition, NF-κB presents as inactive cytoplasmic form binding with the repressor of NF-κB (IκBs). Nevertheless, CS stimulation conduces to the phosphorylation and degradation of IκBα (Chen et al., 2015a). IKK-mediated IκB-α degradation is a key step in NF-κB activation, which leads to the translocation of p65 subunit from the cytoplasm to the nucleus (Huang et al., 2015). Consequently, NF-κBp65 binds to promoter regions of pro-inflammatory genes, thus inducing the transcription of various inflammatory mediators including IL-1𝛽, IL-6 and TNF-𝛼 (Jiang et al., 2015). Those pro-inflammatory cytokines and chemokines are responsible for cell recruitment, survival and proliferation. They recruiting neutrophils to the lung tissue causing acute lung injury or asthma (Chen et al., 2015d). However, mice treated with Liujunzi Tang effectively inhibited the elevated phosphorylation of NF-κB with a suppression in phosphorylated IκB and reduction in pro-inflammatory mediators in lung tissues. Increased concentrations of circulating cytokines (L-1𝛽, IL-6 and TNF-𝛼), adipokines (leptin, ghrelin) and acute-phase proteins (C-reactive protein, fibrinogen) are observed in most of the disease (Broekhuizen et al., 2006). Our data showed Liujunzi Tang might protect against cigarette smoke-induced COPD by inhibiting NF-κB activation and cutting down the production of these pro-inflammatory cytokines both in the lungs and serum.

Although the lungs have a well-developed pulmonary and systemic antioxidant defense system, reactive oxygen species from cigarettes and inflammation reactions creates an oxidant/antioxidant imbalance resulting in oxidative stress, which is thought to play an important role in the pathogenesis of COPD (Tomaki et al., 2007). Several studies have demonstrated that oxidative stress can cause deleterious effects in the body including DNA damage, protein denaturation, and especially the lipid peroxidation (Rahman and Adcock, 2006). As the lipid peroxidation products, MDA is considered as a biomarker to evaluate the degree of lipid peroxidation and is highly related to pulmonary dysfunction (Jing et al., 2015). Moreover, it can affect remodeling of mitochondrial respiration, extracellular matrix, and increase the strength of alveoli (Chen et al., 2016b). SOD is an enzyme that exists in cells for removing oxyradicals, whose activity variation may represent the degree of tissue injury (Ma et al., 2014). Extracellular SOD can modulate neutrophil inflammation by reducing cytokine released from macrophages (Jiang et al., 2016a). Our investigation showed that cigarette smoke-induced oxidative stress came along with accelerating lipid peroxidation and reducing the activities of SOD and CAT. However, intragastrical administration of Liujunzi Tang could restore the activities of SOD, MDA and reduce MDA content. These results indicated that the anti-oxidation property of Liujunzi Tang was due to its role to suppress ROS formation, protect antioxidant enzyme system, and thus lead to the inhibition of lipid peroxidation. Reactive oxygen species from cigarettes potentially induce NF-κB through several distinct mechanisms. Firstly, oxidants can activate MAPK proteins which phosphorylate and activate I kappaB kinase (IKK) (Chang et al., 2015). Moreover, H2O2 can directly activate IKKs through phosphorylation of serine residues in their activation loops (Kamata et al., 2002). Both of these pathways will target IκB for proteasomal degradation. Several studies also have demonstrated that redox factors exert profound effects on NF-κB signaling (Jiang et al., 2016b). Treatment with lipid peroxidation products or depletion of reduced glutathione induce the ubiquination and subsequent proteasomal degradation of IκB (Ginn-Pease and Whisler, 1996). Similarly, H2O2 administration has been shown to produce the activation of NF-κB in several cell lines (Hua et al., 2015). Therefore, the increased MDA content and enhanced SOD, CAT activities in our study might explain the observed upregulation of NF-κB phosphorylation caused by the levels of pro-inflammatory mediators following CS,

which further augmented the inflammatory reaction. In conclusion, we found that Liujunzi Tang exhibited protective effect on cigarette smoking-induced COPD mice by inhibiting inflammatory process and oxidative stress through suppressing NF-κB pathway. Further studies are warranted before clinical application of Liujunzi Tang.

Conflict of interest

The authors have declared that there is no conflict of interest. Author contributions Tianhua Yan and Tong Chen designed research and wrote the paper; Rui Zhou, Luo Fen and Kai Zhang performed the mice experiment; Gao Jin and Chang Xiayun analyzed data; Liu Jingyan and He He performed biochemical analysis.

Acknowledgements

I will extend my thanks to Hui Wang for careful review. The study was supported by Jiangsu Province Program for Culture and Creation Project (CX09B-292Z).

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Figure legends Fig 2. UPLC-MS profile of Liujunzi Tang. Representative UPLC-MS of Liujunzi Tang: Representative chromatograms of Liujunzi Tang(A); Representative MS of Liujunzi Tang(B). 1: Succinic Acid; 2: Hesperidin; 3: Ginsenoside Rb1; 4: glycyrrhizic acid I; 5: 2-Atractylenolide; 6: Pachymic acid .

Fig 2. Effects of Liujunzi Tang on inflammatory cytokines (TNF-α, IL-1β, IL-6) in lung tissues and serum. Mice were exposed to cigarette smoking for 8 weeks and intragastrically treated with 1.5g/kg/d and 3g/kg/d Liujunzi Tang from the fifth week for continuous 4 weeks. All protein levels were measured by ELISA and expressed as means ± SDs. A:Control; B:CS; C:CS+Dex(2mg/kg); D:CS+Liujunzi Tang(1.5g/kg); E:CS+Liujunzi Tang(3g/kg). Compared with control: ##P<0.01; Compared with model: **P<0.01.

Fig 3. Effect of Liujunzi Tang on antioxidant activity. Mice were exposed to cigarette

smoking for 8 weeks and intragastrically treated with 1.5g/kg/d and 3g/kg/d Liujunzi Tang from the fifth week for continuous 4 weeks. SOD and CAT in serum representing degree of tissue injury were tested by SOD and CAT kits. Values are expressed as means±SDs. Compared with control: ##P<0.01; Compared with model: **P<0.01.

Fig 4. Effect of the Liujunzi Tang on lipid peroxidation. Mice were exposed to cigarette smoking for 8 weeks and intragastrically treated with 1.5g/kg/d and 3g/kg/d Liujunzi Tang from the fifth week for continuous 4 weeks. MDA in serum representing the degree of oxidative stress were measured by MDA kit. Values are expressed as means±SDs. Compared with control: ##P<0.01; Compared with model: **P<0.01.

Fig 5. Effect of Liujunzi Tang on NF-κB and IκB-α activation in mouse model of COPD. Mice were exposed to cigarette smoking for 8 weeks and intragastrically treated with 1.5g/kg/d and 3g/kg/d Liujunzi Tang from the fifth week for continuous 4 weeks. Lung tissues were collected and NF-κB p65, IκB-α , p-NF-κB p65 and p-IκB-α expression levels in lung tissue were analyzed by western blot. (A) western blot image. (B) quantified expression level of NF-κB p65 and IκB-α in lung tissue normalized against p-NF-κB and p-IκB-α respectively.

A:Control;

B:CS;

C:CS+Dex(2mg/kg);

D:CS+Liujunzi

Tang(1.5g/kg);

E:CS+Liujunzi Tang(3g/kg). Values are expressed as means±SDs. Compared with control: ##P<0.01; Compared with model: **P<0.01.

Fig 6. Effect of Liujunzi Tang on cigarette smoke-induced pulmonary pathological changes (n=3). Mice were exposed to cigarette smoking for 8 weeks and intragastrically treated with 1.5g/kg/d and 3g/kg/d Liujunzi Tang from the fifth week for continuous 4 weeks. Histopathological examination of lung tissues by HE (200×) .

Figure 1 2

4

3

1

人参 9克；白术 9克；茯苓 9克；炙甘草 6 5 克；陈皮 3克；半夏 4.5克。

1: Succinic Acid; 2: Hesperidin ; 3: Ginsenoside Rb1 ; 4: glycyrrhizic acid I; 5: 2-Atractylenolide; 6: Pachymic acid

6

117

100

609

100

Hesperidin

%

%

Succinic Acid

235

301 610

236 118

0 100

317

153

200

300

417 463

350

400

! 625 637 721 743

549

500

600

700

! ! ! 813 863 917 975 997 113 2 115 9

800

900

1000

1100

! ! 121 3 1273

1200

1300

117

m/z

1400

0 100

286

206

200

1219 1220 ! ! 1218 1259 1272 1109

611

302 ! ! 1398 146 8

! 304 385 431

300

400

507

607 649 672 813 707 851

500

600

700

800

900

1000

1100

1200

1300

! 1421

1400

m/z

1108

100

821

100

Ginsenoside Rb1 glycyrrhizic acid 1109

%

%

351

553

822 1110 783 784 829

554 104 161 220

0 100

200

301

323366

300

472

400

1111 946

553 555 711 765 603

500

600

700

800

1060 1078

900

1000

1100

11541226

1200

1312

1300

1410

1400

1465

m/z 337 117 221265 0 100 200 300

485 513

400

500

643 661

600

757

700

831 901 955

800

900

1166 1232 983 11241138 1273

1000

1100

1200

1300

m/z

1400

231

100

2-Atractylenolide

527

100

%

%

Pachymic acid

528

232 1056

! ! 132 181

0 100

200

233

325 300

! 459 521 545

400

500

! ! 630 676 709 771 847 600

700

800

! 917 900

! 1138

1019 1000

1100

! ! 1229 1301

1200

1300

1057

! 1337

! 529

m/z 1400

117

0 100

198

265

200

293 311353 477 494

300

400

500

612

627 652

600

1058 ! 1055 1078 1146 1218

688

700

m/z 800

900

1000

1100

1200

1300

1400

Figure 2

Figure 3

Figure 4

Figure 5

A P-p65 p65 IκB-α P-IκB-α GAPDH

B

C

D

E

Figure 6

C

on tr ol

S+ C S S+ D e Li x( uj 2m un g/ zi kg Ta C ) S+ ng Li (1 uj .5 g/ un kg zi ) Ta ng (3 g/ kg )

C

C

Inflammation scores 3

##

2

**

1

**

**

0

*Graphical Abstract

COPD

Liujunzi Tang