Journal of Ethnopharmacology 160 (2015) 41–51

Contents lists available at ScienceDirect

Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Stemona tuberosa prevented inflammation by suppressing the recruitment and the activation of macrophages in vivo and in vitro Dahae Lim a, Euijeong Lee a, Eunyoung Jeong b, Young-Pyo Jang b, Jinju Kim a,n a b

Department of Korean Physiology, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 10 July 2014 Received in revised form 17 November 2014 Accepted 17 November 2014 Available online 2 December 2014

Ethnopharmacological relevance: Stemona tuberosa (ST) is a traditional herbal medicine used for the treatment of various respiratory diseases in eastern Asia. Aim of the study: We investigated the anti-inflammatory effects of a ST water extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and in cigarette smoke (CS)-induced lung inflammation mouse models. Materials and methods: RAW 264.7 macrophages were treated with the ST extract and stimulated by LPS. The expressions of pro-inflammatory mediators were evaluated by using nitric oxide (NO) assay, enzyme-linked immunosorbent assay and Western blot analysis. After the C57BL/6 mice were exposed to CS, they were administrated with the ST extract. The accumulated inflammatory cells in the bronchoalveolar lavage fluid (BALF) were counted. Also, real-time polymerase chain reaction and hematoxylin and eosin staining were performed in lung tissues. Results: The ST extract treatment reduced the production of NO via blocking the expressions of cyclooxygenase-2 and inducible nitric oxide synthase protein in RAW 264.7 macrophages. In addition, ST extract treatment decreased the secretions of inflammatory cytokines and regulated NF-κB activation by inhibiting the phosphorylation of IκB and the mitogen-activated protein kinase pathway. Also, ST extract administration to mice reduced the infiltrations of macrophages into BALF and the histological inflammatory changes in lung tissues. Furthermore, administration of the ST extract regulated the levels of tumor necrosis factor-α, interleukin (IL)-6, IL-1β, monocyte chemoattractant protein-1 and matrix metalloproteinases-12 in the lungs. Conclusion: These findings suggested that ST extract attenuated pulmonary inflammatory responses by inhibiting the expression of diverse inflammatory mediators in vivo and in vitro. & 2014 Elsevier Ireland Ltd. All rights reserved.

Chemical compounds studied in this article: Croomine (PubChem CID: 3085457) Stemoninine (PubChem CID: 15983991) Neotuberostemonine (PubChem CID: 11667940) Tuberostemonine (PubChem CID: 100781) Tuberostemonone (PubChem CID: 6426912) 6-Hydroxycroomine (Tuberospironine) N-Oxytuberostemonine Tuberostemonine K Tuberostemonine H Keywords: Cigarette smoke Lipopolysaccharide Macrophages Pulmonary inflammation Stemona tuberosa

1. Introduction Lung inflammatory responses, characterized by the accumulation of immune cells, are associated with many respiratory diseases, including chronic obstructive pulmonary disease and emphysema (Barnes et al., 2003). Cigarette smoke (CS) is a major cause of pulmonary inflammatory disease (Yoshida and Tuder, 2007). CS exposure induces the recruitment of inflammatory cells, including macrophages and neutrophils, into the lungs (Churg et al., 2008). Inflammatory cells that accumulate in the alveoli and

Abbreviations: BALF, bronchoalveolar lavage fluid; COPD, chronic obstructive pulmonary disease; COX-2, cyclooxygenase-2; CS, cigarette smoke; iNOS, inducible nitric oxide synthase; MAPKs, mitogen-activated protein kinases; MCP-1, monocyte chemoattractant protein-1; MMP-12, matrix metalloproteinases-12; ST, Stemona tuberosa; TNF-α, tumor necrosis factor-α n Corresponding author. Tel.: þ 82 2 961 9437; fax: þ82 2 968 0560. E-mail address: [email protected] (J. Kim). http://dx.doi.org/10.1016/j.jep.2014.11.032 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

bronchiole secrete pro-inflammatory mediators such as cytokines, chemokines and matrix metalloproteinases (MMP), which participate in the inflammatory response (Tetley, 2002; Bhalla et al., 2009). Activation of these inflammatory mediators by repeated CS-exposure aggravates the inflammatory response, and induces tissue destruction, causing pulmonary inflammatory disease. Macrophages are immune cells that participate in the inflammatory process through the secretion of various pro-inflammatory mediators (Fujiwara and Kobayashi, 2005). Lipopolysaccharide (LPS) is an endotoxin that elicits immune responses by promoting the secretion of nitric oxide (NO), pro-inflammatory cytokines, and chemokines in macrophages. As a component of CS, LPS is involved in pulmonary inflammation (Hasday et al., 1999). Macrophages activated by LPS express cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS); these enzymes generate various pro-inflammatory mediators (Zamora et al., 2000; Kuwano et al., 2004). The expression of iNOS and COX-2 is regulated by activation of the nuclear transcription factor-κB (NF-κB) via phosphorylation

42

D. Lim et al. / Journal of Ethnopharmacology 160 (2015) 41–51

of IκB and mitogen-activated protein kinases (MAPKs) (Tak and Firestein, 2001; Bar-Shai et al., 2006). Thus, NF-κB activation and MAPKs phosphorylation can represent important targets in the treatment of inflammatory disease. Dongui-Bogam is a Korean traditional medical book written in 1610. In Dongui-Bogam, various medicinal herbs for treating respiratory diseases were introduced (Bae et al., 2012; Lee et al., 2013). One of them was Stemona tuberosa (ST) which was explained to be used for curing the ‘lung atrophy’ (an oriental medical term about chronic wasting lung disease resulting from chronic cough) and the ‘lung heat’ (a term for inflammatory conditions of the lung). Accordingly, ST has been continuously used as the herbal medicine to treat pertussis, chronic cough and asthma in China as well as in Korea. Recently, the function of ST as respiratory depressant and antitussive has been reported (Xu et al., 2010). Also, some studies have demonstrated that Stemona alkaloids isolated from ST have an antitussive activity (Chung et al., 2003; Zhou et al., 2009). However, the effects of ST on pulmonary inflammatory disease have not been reported to date. We investigated the anti-inflammatory activities of a ST extract in LPSstimulated RAW 264.7 macrophages and a CS-induced pulmonary inflammation animal model.

and was equipped with an ESI source (Electrospray ionization, JEOL, USA). In the positive ion mode, the atmospheric pressure interface potentials were typically set to the following values: orifice 1 ¼ 80 V and ring lens and orifice 2 ¼10, 5 V, respectively. The ion guide potential and detector voltage were set to 2000 V and 2300 V, respectively. ESI parameters were set as follows: needle electrode¼ 2000 V, nitrogen gas was used as a nebulizer, desolvating and their flow rate were 1 and 3 L/min, desolvating chamber temperature ¼250 1C orifice 1 temperature¼80 1C. Mass scale calibration was accomplished with Yokudelna (Koyo Science Co., Ltd., Japan) for accurate mass measurements and calculations of the elemental composition. MS acquisition was set with a scan range of m/z 50–1000. 2.3. Cell culture The mouse macrophage RAW 264.7 cell line was purchased from ATCC (Manassas, VA, USA). The cells were maintained in 100% humidity and 5% CO2 at 37 1C in Dulbecco's modified Eagle's medium (DMEM) (Welgene, Daegu, Korea) supplemented with 10% v/v fetal bovine serum (FBS) (Welgene, Korea) and 1% penicillin–streptomycin. 2.4. Cell viability assay

2. Materials and methods 2.1. Sample preparation for HPLC analysis Acetonitrile was HPLC grade and purchased from Duksan pure chemicals Co. (Ansan, South Korea) and triethylamine was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). ST water extract was purchased from the Sun Ten Pharmaceutical Co. (Taipei, Taiwan) ST was extracted with 100 1C water, and the lyophilized extract was granulated. High purity nitrogen gas was provided by Shinyang Oxygen Co. (Seoul, South Korea). Five grams of ST extract was refluxed with 100 ml of 95% ethanol for 1 h. The extracted solution was filtered with filter paper (Hyundai Micro Co., Ltd., Korea) and condensed under reduced pressure to afford a residue which was dissolved in 50 ml of 4% HCl. The filtrate was basified with ammonia water (Duksan Pure Chemical Co., Ltd., Korea) to pH 9 and extracted with diethyl ether. The diethyl ether extract was concentrated using a rotatory vacuum evaporator and yield 18.15 mg of brown powder (EYELA, Japan). The residual was dissolved in 2 ml methanol and passed through a C18 Sep-Pak cartridge (Waters Co., Massachusetts, USA) and filtered through a 0.2 μm Whatman syringe filter before being injected to HPLC. 2.2. HPLC–ESI–MS analysis The high performance liquid chromatography (HPLC) system was operated by Empower software (Milford, USA) and consisted of Waters model 515 pump, a 717 autosampler and a 2487 dual λ absorbance detector. The Atlantis C18 column (4.6  150 mm2, 3 m) was selected for the analysis. The monitoring wavelength was set to 254 nm. The mobile phase was comprised of acetonitrile with triethylamine (0.1%, solvent A) and water (solvent B). All solvents were filtered through a 0.45 μm filter. The gradient program was 0–10 min, 40% of solvent A; 10–12 min, linear from 40% to 50% of solvent A; 12–28 min, 50% of solvent A; 28–30 min, linear from 50% to 400% of solvent A, at a flow rate of 0.6 ml per min. The injection volume was 10 ml. The analysis condition for Stemona alkaloids were modified from the method previously reported (Li et al., 2007). AccuTOFs single-reflectron time-of-flight mass spectrometer was operated with Mass Center system version 1.3.7b (JEOL, USA)

Cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RAW 264.7 cells were seeded onto 96-well plates (1  105 cells/ml) and incubated at 37 1C for 24 h. The cells were then treated with the indicated concentrations of ST extract (1, 10 and 100 mg/ml). After 24-h incubation, 50 ml of MTT solution (2 mg/ml) were added to each well, followed by incubation for 4 h. Formazan crystals were dissolved in 100 μl of DMSO, and the cellular viability was measured at 570 nm using an enzyme linked immunosorbent assay (ELISA) reader (Molecular Devices, Downingtown, PA, USA). 2.5. NO production assay RAW 264.7 cells were seeded onto a 12-well plate (5  105 cells/ml) and incubated at 37 1C for 24 h. The cells were pretreated with ST extract (1, 10 and 100 μg/ml) for 1 h and then stimulated with 1 μg/ml of LPS (Sigma-Aldrich) for 24 h. NO concentrations in the supernatant were measured using the Griess reagent system (Promega, Madison, WI, USA). 2.6. Enzyme-linked immunosorbent assay (ELISA) RAW 264.7 cells were seeded onto a 12-well plate (5  105 cells/ml) and incubated at 37 1C for 24 h. The cells were pretreated with ST extract (1, 10 and 100 μg/ml) for 1 h and then stimulated with 1 μg/ml of LPS for 24 h. Levels of TNF-α, IL-6, IL-1β and MCP-1 in the supernatant were measured by ELISA using a commercial kit (OptELA™ Kits; BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions as previously described (Cheung et al., 2013). 2.7. Western blot analysis Cells were lysed by RIPA buffer containing proteinase inhibitors. Protein concentrations were measured using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). The proteins (20 μg/lane) were resolved by SDS–polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. The membranes were incubated overnight with the following TBS-T diluted primary antibodies: β-actin, NF-κB, PCNA, iNOS, COX-2, IκB and p-IκB (at a dilution of 1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA), and ERK, JNK, p38, p-ERK, p-JNK and p-p38 (at a dilution of 1:1000)

D. Lim et al. / Journal of Ethnopharmacology 160 (2015) 41–51

(Cell Signaling Technology, Danvers, MA, USA). Blots were washed three times in TBS-T buffer, and then incubated with secondary antibodies for 2 h. After washing, the blots were developed using an enhanced chemiluminescence Western blot analysis system (AbClon Inc., Seoul, South Korea) as previously described (Cheung et al., 2013). 2.8. Animal treatment C57BL/6 mice (aged 6–7 weeks; Orient Bio Inc., Seongnam-si, Korea) were maintained under pathogen-free conditions. All animal experimental protocols complied with the Committee for the Care and Use of Laboratory Animals (College of Pharmacy, Kyung Hee University; KHU-2011-05-1). Mice were divided into four groups (n ¼5): (1) fresh air þDW, (2) CS (reference cigarettes 3R4F, University of Kentucky, Lexington, Kentucky, USA)þ DW, (3) CS þdexamethasone (DEX, 1 mg/kg, p.o.) as an active control (Ra et al., 2010), and (4) CS þST extract (ST, 100 mg/kg, p.o.). Mice were exposed to fresh air or to CS from the reference cigarettes, 3R4F (University of Kentucky, USA) using a smoking apparatus. The mice were exposed to CS five times per week for 3 weeks. The mice were placed in the smoking chamber, which was filled with smoke from a lighted 3R4F cigarette, via the smoking apparatus. Mice were exposed to CS for 30-min periods, after which they were rested in a fresh air environment for 1 h. Three cigarettes were used, and thus the mice were exposed to CS for 1.5 h per day. Administration of DEX and ST extract were performed by oral gavages after the CS exposure as previously described (Lee et al., 2014). Mice were sacrificed after 3 weeks for further experiments.

43

CCG GACTCCGC-30 ; for IL-6, forward, 50 -TGCTGG TGACAACCACGGCCT-30 and reverse, 50 -ACAGGTCTG TTG GGAGTGGTATCCT30 ; for IL-1β, forward, 50 -ACCTGCTGGTGTGTGACGTT-30 and ; reverse, 50 -TCGTTGCTTGGTTCTCCTTG-30 ; for MCP-1, forward, 50 -TCACAGTTGCCGGCT GGAGC-30 and reverse, 50 -CAGCAGGTGAGTGGGGCG TT-30 ; for MMP-12, forward, 50 -GGC CATTCC TTG GGGCTGCA-30 and reverse, 50 -GGG GGTTTCACT GGG GCTCC-30 ; and for GAPDH, forward, 50 -TCTCAGGTGCCGCCTGGAGA-30 and reverse, 50 -TGGGCCCTCAGATGCCTGCT-30 (Cosmogentech Ltd., Seoul, Korea). A dissociation curve analysis of TNF-α, IL-6, IL-1β, MCP-1, MMP-12 and GAPDH showed a single peak. PCRs were carried out for 40 cycles using the following conditions: denaturation at 95 1C for 10 s, annealing at 60 1C for 10 s, and elongation at 72 1C for 12 s. The mean Ct value of the gene of interest was calculated from triplicate measurements and normalized to the mean Ct value of GAPDH, which was used as the control. 2.12. Statistical analysis Statistical analysis of the data was carried out using GraphPad Prism version 4.00 (GraphPad Software Inc., San Diego, CA, USA). Data were presented as means 7standard error of the mean (SEM), and multiple comparisons were performed by one-way ANOVA. Differences with p o0.05 were considered statistically significant (Lee et al., 2014).

3. Results 3.1. Establishment of HPLC profile

2.9. Histology analysis Lung specimens were obtained from the mice and fixed in 10% formaldehyde for 24 h. The lung tissue was embedded in paraffin and sectioned at 4-mm thickness using a rotary microtome. Tissue paraffin sections were stained with hematoxylin and eosin (H&E) solution for morphometric analysis. H&E stained lung tissue sections were examined using light microscopy at a magnification of 100  . A lung injury score for the H&E-stained lung tissue sections was given for perivascular and peribronchial cell infiltration according to Srivastava et al. (2010): 0, not present; 1, very slight; 2, slight; 3, moderate; 4, moderate to marked; and 5, marked. This method adhered to the blinded principle (Srivastava et al., 2010). 2.10. Bronchoalveolar lavage fluid (BALF) analysis After the mice were sacrificed, PBS was infused into the lungs and withdrawn via a cannula inserted into the trachea. The cell concentrations in BALF were measured using a hemocytometer, and differential cell counts were performed on slides prepared by cytocentrifugation and Diff-Quick staining using light microscopy as previously described (Lee et al., 2014). 2.11. Real-time polymerase chain reaction (PCR)

Although the identification and quality of medicinal herb used in this study was guaranteed by Sun Ten Pharmaceutical Co., HPLC–ESI–MS measurement were conducted on ST water extract to reconfirm the biological origin. In order to identify the origin for the ST extract, we developed HPLC–ESI–MS method slightly modified from a previous report (Li et al., 2007). Most Stemona alkaloids are insensitive to direct UV detection because of the absence of appropriate chromophore but they are easily detectable by ESI–MS. Therefore ST extract were subjected to HPLC/ESI–MS (Fig. 1). 3.2. Identification of phytochemicals by HPLC–ESI–MS The retention time, observed mass, mass difference and proposed compounds of 9 peaks are listed in Table 1. Fig. 2 shows the HPLC–ESI–MS total ion current (TIC) chromatograms for ST extract. According to the protonated molecular ion mass number and retention time comparison with the previous report, these peaks were identified as croomine (m/z 322.206) (peak 3), tuberostemonine K (m/z 376.246) (peak 5), stemoninine (m/z 390.224) (peak 6), neotuberostemonine (m/z 376.246) (peak 7), tuberostemonine (m/z 376.246) (peak 8) and tuberostemonine H (m/z 376.246)

Real-time PCR was performed to measure mRNA levels of TNF-

α, IL-6, IL-1β, MCP-1 and MMP-12 using a Thermal Cycler Dice™

Real Time (RT) PCR system (Takara, Katsushika, Japan) as previously described (Lee et al., 2014). Lung tissue was lysed using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), after which total RNA was isolated according to the manufacturer's instructions. RNA (2 mg) was transcribed using M-MuLV Reverse Transcriptase (Invitrogen, USA) and random hexamers. The synthesized cDNA was used as a template for PCR amplification. The primers used for the SYBR Green real-time PCR were as follows: for TNF-α, forward, 50 -CAA GGG ACAAGGCTGCCCCG-30 and reverse, 50 -TAGACCTGC

Fig. 1. TIC chromatograms from LC/ESI–MS analysis of the Stemona tuberosa extract.

44

D. Lim et al. / Journal of Ethnopharmacology 160 (2015) 41–51

Table 1 The observed and calculated mass numbers of LC/ESI–MS peaks of Stemona tuberosa extract. Peak no.

Rt (min)

Theoretical mass [Mþ H] þ

Observed mass [M þH] þ

Mass difference (mmu)

Identification

Reference

Tuberostemonone Tuberostemoninol 6-Hydroxycroomine Tuberospironine Croomine N-Oxytuberostemonine Tuberostemonine K Stemoninine Neotuberostemonine Tuberostemonine Tuberostemonine H

Lin et al. (1992) Han Lin et al. (1994) Schinnerl et al. (2005) Jiang et al. (2006) Jiang et al. (2006) Dong et al. (2012) Jiang et al. (2006) Jiang et al. (2006) Jiang et al. (2006) Jiang et al. (2006) Jiang et al. (2006)

1

3.836

406.22239

406.22244

 0.52

2

6.459

338.19618

338.19923

2.48

3 4 5 6 7 8 9

13.875 15.299 19.273 22.941 26.975 28.100 29.578

322.20127 392.24313 376.24821 390.22748 376.24821 376.24821 376.24821

322.20577 392.24128 376.24688 390.22428 376.24544 376.24562 376.24625

3.94  2.42  1.90  3.99  3.52  3.31  2.65

(peak 8) (Zhou et al., 2006). Although the same analysis condition from the previous analysis was applied to this study, the retention times of peaks were delayed (Zhou et al., 2006). This seems to be due to the different separation efficiency from different column manufacturer. TIC chromatogram was slightly different by comparison of its fingerprint with literature (Zhou et al., 2006). From the TIC mass chromatogram in this study, stemonine (peak 6) was the major Stemona alkaloid. The peak 1 was supposed to be tuberostemoninol or tuberostemonone (Lin et al., 1992; Han Lin et al., 1994) and peak 2 was supposed to be 6-hydroxycroomine or tuberospironine. Since these compounds have same molecular weight, it was not able to identify exact structure of the peak only by HPLC–MS study. The mass spectrum of peak 4 showed protonated molecular ion of m/z 392 and this molecular weight is corresponding to N-oxytuberostemonine (Dong et al., 2012). In order to elucidate the exact identity of the peaks, further sets of experiment including semi-quantitative scale isolation and spectroscopical analysis are needed. At 28 min, two peaks which have the same molecular weight were observed. They were regarded as isomers and tuberostemonine K, neotuberostemonine, and tuberostemonine H were only reported as having same molecular weight in Stemona tuberosa (Zhou et al., 2006; Li et al., 2007; Schinnerl et al., 2007). The overall content of these alkaloids in commercial granule was ca. 3.6 ppm which was calculated by alkaloids specific fractionation method. The chemical profiles of the extract indicate that the biological source of this commercial herbal extract is Stemona tuberosa. 3.3. ST extract decreased NO production in LPS-stimulated RAW 264.7 macrophages The cytotoxicity of the ST extract on RAW cells was investigated by MTT assay. The ST extract exhibited no cytotoxicity up to 100 mg/ml (Fig. 3A). Thus, we performed subsequent experiments using 1, 10, and 100 mg/ml. The amount of NO, as a marker of the inflammatory response, was measured using Griess reagent. As opposed to the negative control (inactivated cells), the positive control, stimulated by LPS, had significantly increased levels of NO production. Compared with the positive control, cells treated with 10 and 100 mg/ml ST extract showed 22% and 87%, respectively, reductions in NO production (Fig. 3B). 3.4. ST extract reduced production of pro-inflammatory cytokines and chemokines in LPS-stimulated RAW 264.7 macrophages TNF-α, IL-6, IL-1β and MCP-1 levels were measured by ELISA using a commercial kit (OptELATM Kits; BD Biosciences, San Diego, CA, USA). Compared with the negative control, the positive control had significantly increased levels of TNF-α, IL-6, IL-1β and MCP-1. Compared with the positive control, cells treated with 10 and 100 mg/ml ST extract showed 63% and 94% reductions in TNF-α

production, respectively (Fig. 4A). For IL-6, compared with the positive control, cells treated with 10 and 100 mg/ml of ST extract showed 64% and 97% reductions, respectively (Fig. 4B). As seen in Fig. 4C, compared with the positive control, cells treated with 1, 10 and 100 mg/ml of ST extract exhibited a significant and dosedependent decrease in IL-1β production, (18%, 70%, and 92% for 1, 10 and 100 mg/ml treatments, respectively). For MCP-1, compared with the positive control, cells treated with 10 and 100 mg/ml ST extract showed reductions of 68% and 90%, respectively (Fig. 4D). 3.5. ST extract reduced iNOS and COX-2 protein expression in LPSstimulated RAW 264.7 macrophages We determined the iNOS and COX-2 protein levels by Western blot analysis. Cells were pretreated with ST extract (1, 10 and 100 μg/ml) for 1 h and were then stimulated with 1 μg/ml LPS for 6 h. As shown in Fig. 5, compared with the negative control, the positive control had significantly increased levels of iNOS and COX-2. Compared with the positive control, cells treated with 10 and 100 mg/ml ST extract showed reductions in iNOS expression of 24% and 57%, respectively (Fig. 5B). Compared with the positive control, cells treated with 100 mg/ml ST extract showed a 36% decrease in COX-2 expression (Fig. 5C). 3.6. ST extract inhibited NF-κB activation and IκB phosphorylation in LPS-stimulated RAW 264.7 macrophages We assessed NF-κB and IκB phosphorylation levels by Western blot analysis. Cells were pretreated with ST extract (1, 10 and 100 μg/ml) for 1 h and then stimulated with 1 μg/ml LPS for 30 min. The cytosol and nucleus were isolated, and the level of NF-κB in the nuclear extract was determined. Compared with the negative control, LPS significantly increased NF-κB expression. Compared with the positive control, cells treated with 1, 10 and 100 mg/ml ST extract showed significant dose-dependent inhibitions of NF-κB expression (18%, 43% and 76%, respectively) (Fig. 6A and C). Furthermore, we investigated the IκB phosphorylation levels in the cytosol. Compared with the negative control, LPS significantly increased IκB phosphorylation. However, 1, 10 and 100 μg/ml ST extract inhibited phosphorylation of IκB in a dosedependent manner by 23%, 53% and 63%, respectively (Fig. 6B and D), which led to reductions in IκB degradation by 61%, 51% and 33%, respectively (Fig. 6B and E). 3.7. ST extract inhibited the phosphorylation of MAPKs in LPSstimulated RAW 264.7 macrophages We examined the phosphorylation levels of ERK, JNK and p38 by Western blot analysis. Cells were pretreated with ST extract

D. Lim et al. / Journal of Ethnopharmacology 160 (2015) 41–51

45

Fig. 3. Effects of Stemona tuberosa extract on cell viability and nitric oxide (NO) production in RAW 264.7 macrophages. (A) Cells were incubated with various concentrations of ST extract for 24 h. Cell viability was measured by MTT assay. (B) Cells were pretreated with ST extract (1, 10 and 100 mg/ml) for 1 h, and then stimulated with 1 μg/ml LPS for 24 h. The NO concentration in the medium was measured using Griess reagent. Values represent the means 7 SEM. Statistical analysis was by one-way ANOVA; **p o 0.01; ***p o 0.001.

3.8. ST extract reduced the extent of CS-induced histological changes in the lung of CS-exposed mice After confirmation that ST extract had an anti-inflammatory effect, we investigated the effects of ST extract on pulmonary inflammatory responses in a CS-exposed mouse model. H&Estained lung tissue sections were examined to assess the extent of inflammation with immune cell infiltration in the lungs of mice exposed to CS. Compared with the control group, the CS group showed histological changes with infiltration of cells surrounding the perivascular and peribronchial regions. Administration of DEX and ST extract reduced the extent of CS-induced histological inflammatory changes. The administration of DEX (1 mg/kg, p.o.) was used as an active control (Fig. 8).

Fig. 2. EMI–MS spectra of nine peaks from Stemona tuberosa extract.

(1, 10 and 100 μg/ml) for 1 h and then stimulated with 1 μg/ml LPS for 15 min. As shown in Fig. 7, compared with the negative control, LPS significantly increased the phosphorylation of ERK, JNK and p38. The cells treated with 1, 10 and 100 mg/ml ST extract showed a significant and dose-dependent decrease in the phosphorylation of ERK (19%, 31% and 65%: Fig. 7A and D), JNK (3%, 21% and 75%: Fig. 7B and E), and p38 (22%, 27% and 68%: Fig. 7C and F).

3.9. ST extract decreased levels of inflammatory cell accumulation in the BALF The levels of inflammatory cells accumulated in the BALF of CS-exposed mice were examined. Compared with the control group, CS exposure significantly increased the levels of total cells, macrophages and neutrophils. As compared with the CS group, DEX significantly reduced the levels of total cells (81.5%), macrophages (81.9%) and neutrophils (81.3%). Similarly, ST extract significantly decreased the levels of total cells (65.3%), macrophages (69.7%) and neutrophils (68.4%). Administration of DEX (1 mg/kg, p.o.) was used as an active control (Fig. 9).

46

D. Lim et al. / Journal of Ethnopharmacology 160 (2015) 41–51

Fig. 4. Effects of Stemona tuberosa extract on the production of cytokines and chemokines in LPS-stimulated RAW 264.7 macrophages. Cells were pretreated with ST extract (1, 10 and 100 mg/ml) for 1 h, and then exposed to 1 mg/ml LPS for 24 h. The amount of TNF-α (A), IL-6 (B), IL-1β (C), and MCP-1 (D) in the medium was measured by an ELISA. Values represent the means7 SEM. Statistical analysis was by one-way ANOVA; *po 0.05, ***p o 0.001.

Fig. 5. Effects of Stemona tuberosa extract on iNOS and COX-2 expression in LPS-stimulated RAW 264.7 macrophages. Cells were pretreated with ST extract (1, 10 and 100 mg/ ml) for 1 h, and then stimulated with 1 μg/ml LPS for 6 h. (A) iNOS and COX-2 expressions were analyzed by Western blot. β-Actin was used as internal control. Relative protein levels of iNOS (B) and COX-2 (C) were quantified using the ImageJ software (http://imagej.nih.gov/ij/), and normalized to the control protein levels. Values represent the means 7 SEM. Statistical analysis was by one-way ANOVA; ***p o 0.001.

3.10. ST extract inhibited TNF-α, IL-6, IL-1β, MCP-1 and MMP-12 mRNA levels in lung tissue Messenger RNA levels of inflammatory mediators in the lung tissue were measured by real-time PCR. As shown in Fig. 10,

compared with the controls, CS significantly increased the mRNA levels of TNF-α, IL-6, IL-1β, MCP-1 and MMP-12. Compared with the CS group, DEX significantly reduced CS-induced upregulation of these mRNAs (39.9%, 54.9%, 71.2%, 58.6%, and 53.1%, respectively). The ST group showed a similar mRNA expression pattern

D. Lim et al. / Journal of Ethnopharmacology 160 (2015) 41–51

47

Fig. 6. Effects of Stemona tuberosa extract on NF-κB activation in LPS-stimulated RAW 264.7 macrophages. Cells were pretreated with ST extract (1, 10 and 100 mg/ml) for 1 h, and then stimulated with 1 μg/ml LPS for 30 min. NF-κB, IκB and p-IκB levels were determined by Western blot. (A) NF-κB levels in nuclear extract. (C) Relative NF-κB protein levels were quantified using the ImageJ software, and normalized to proliferating cell nuclear antigens (PCNA), internal controls, and protein levels. (B) p-IκB and IκB levels in the cytosol. Relative protein levels of p-IκB (D) and IκB (E) were normalized to β-actin, the internal control and protein levels. Values represent the means 7 SEM. Statistical analysis was by one-way ANOVA; ***p o 0.001.

(57.2%, 54.2%, 90.4%, 51.9%, and 65.5%, respectively). The administration of DEX (1 mg/kg, p.o.) was used as an active control.

4. Discussion CS contains a harmful mix of more than 4000 different compound and LPS is one component of the CS (Hasday et al., 1999). CS is a leading cause of disease affecting the heart, lungs and liver. Smoking in particular, is implicated in many inflammatory pulmonary disorders, including lung cancer, COPD and emphysema (Yoshida and Tuder, 2007; Bhalla et al., 2009). The acute or chronic inflammation associated with these diseases is a biological response to harmful stimuli of CS. After CS inhaled, activated immune cells, especially macrophage in pulmonary, release a variety of pro-inflammatory mediators that participate in the inflammatory process (Fujiwara and Kobayashi, 2005). Thus, regulation of pro-inflammatory mediators is regarded as a therapeutic mechanism for many inflammatory diseases. ST is a traditional herbal medicine used for the treatment of various respiratory diseases in eastern Asia. Recently, a few scientists have investigated the activity of Stemona alkaloids. Xu et al. (2010) have reported that four major alkaloids of ST (neotuberostemonine, tuberostemonine, croomine and stemonine) significantly inhibited cough responses. Furthermore, they have demonstrated that neotuberostemonine, tuberostemonine and stemoninine act on peripheral cough reflex pathway, and croomine acts on central reflex pathway. Zhou et al. (2009) isolated the tuberostemonine-type alkaloids such

as neotuberostemonine, tuberostemonine and tuberostemonine H from ST and reported that those tuberostemonine-type alkaloids have antitussive activity. However, the activity of total water extract ST on pulmonary inflammation has not been investigated yet. Therefore, in this study, the inhibitory effects of ST water extract on the production of LPS-induced inflammatory mediators in RAW 264.7 macrophages were confirmed. In addition, using an animal experimental model, we demonstrated that ST attenuated CSinduced lung inflammation. The inflammatory process is regulated by NF-κB, a major transcription factor that regulates the expression of genes associated with the inflammatory response. NF-κB exists in the cytoplasm in an inactive form where it is bound to IκB, a regulatory protein, preventing its translocation. In the presence of inflammatory signals, IκB becomes phosphorylated, and this results in degradation of IκB, as well as translocation of NF-κB into the nucleus (Guha and Mackman, 2001; Tak and Firestein, 2001; Lawrence, 2009). Translocated NF-κB triggers the expression of iNOS and COX-2, the enzymes responsible for generating pro-inflammatory mediators. COX-2 regulates macrophage cytokine production (Williams and Shacter, 1997), and iNOS participates in the lung inflammatory response by destroying the alveoli and causing emphysema (Seimetz et al., 2011). NO generated by iNOS enhances the production of pro-inflammatory cytokines, and aggravates inflammatory and remodeling processes in the inflammation of the airways (Deakin et al., 1995; Ichinose et al., 2000). Our results have shown that ST extract suppressed expression of iNOS and COX-2, and the subsequent production of NO, inflammatory cytokines, and chemokines, by inhibiting

48

D. Lim et al. / Journal of Ethnopharmacology 160 (2015) 41–51

Fig. 7. Effects of Stemona tuberosa extract on phosphorylation of MAPKs in LPS-stimulated RAW 264.7 macrophages. Cells were pretreated with ST extract (1, 10 and 100 mg/ ml) for 1 h, and then stimulated with 1 μg/ml LPS for 15 min. ERK (A), JNK (B), and p38 (C) phosphorylation were analyzed by Western blot. Relative p-ERK (D), p-JNK (E), and p-p38 protein levels were quantified using the ImageJ software, and normalized to the levels of ERK, JNK, and p38, respectively. Values represent the means 7 SEM. Statistical analysis was by one-way ANOVA; **p o 0.01, ***po 0.001.

Fig. 8. Effects of Stemona tuberosa extract on the histological changes in the lungs of cigarette smoke (CS)-exposed mice. Lung tissue was fixed, sectioned at 4 mm, and stained with H&E solution (100  magnification). (A) CON, (B) CS, (C) DEX and (D) ST. (E) The lung inflammation index was scored by a blinded observer. CON: normal control mice; CS: CS-exposed mice without drug treatment; DEX: CS-exposed mice with dexamethasone treatment; ST: CS-exposed mice with ST extract treatment. Values represent the means 7 SEM. Statistical analysis was by one-way ANOVA; ***po 0.001 (n¼ 5 in each group).

D. Lim et al. / Journal of Ethnopharmacology 160 (2015) 41–51

49

Fig. 9. Effects of Stemona tuberosa extract on inflammatory cell profiles in the bronchoalveolar lavage fluid of cigarette smoke (CS)-exposed mice. Cells were isolated by centrifugation, and enumerated by light microscopy after Diff-Quick staining. (A) Total cells, (B) macrophages and (C) neutrophils. CON: normal control mice; CS: CS-exposed mice without drug treatment; DEX: CS-exposed mice with dexamethasone treatment; ST: CS-exposed mice with ST extract treatment. Values represent the means 7 SEM. Statistical analysis was by one-way ANOVA; **p o0.01, ***p o 0.001 (n¼5 per group).

Fig. 10. Effects of Stemona tuberosa extract on mRNA levels in the lung tissue of cigarette smoke (CS)-exposed mice. mRNA levels were measured by real-time PCR. GAPDH served as control for the normalization. (A) TNF-α, (B) IL-6, (C) IL-1β, (D) MCP-1 and (E) MMP-12. CON: normal control mice; CS: CS-exposed mice without drug treatment; DEX: CS-exposed mice with dexamethasone treatment; ST: CS-exposed mice with ST extract treatment. Values represent the means 7 SEM. Statistical analysis was by oneway ANOVA; *p o0.05, **p o0.01, ***p o 0.001 (n¼5 in each group).

activation of NF-κB and phosphorylation of IκB in LPS-stimulated macrophages. The activation of NF-κB by phosphorylation of IκB is regulated by cellular kinases such as MAPKs. MAPKs consist of extracellular

signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (p38 MAPK) (Kaminska, 2005). The phosphorylation of MAPKs in the LPSstimulated macrophages induces cellular responses such as the

50

D. Lim et al. / Journal of Ethnopharmacology 160 (2015) 41–51

production of the pro-inflammatory cytokines, iNOS and COX-2, by activating NF-κB (Guha and Mackman, 2001; Sun et al., 2008). Additionally, MAPK activation contributes to cytokine and MMP expression, inflammation, and fibrosis in the COPD (Mercer and D’Armiento, 2006). Therefore, NF-κB activation and MAPK phosphorylation can be important targets in the treatment of inflammatory disease. We investigated the effects of ST extract on the phosphorylation of MAPKs in LPS-stimulated macrophages, and the results showed that ST extract inhibited phosphorylation. This indicates that ST extract inhibits the production of proinflammatory mediators in activated macrophages, and that this inhibitory effect is caused by blocking activation of the NF-κB and MAPK pathways. We then investigated the effects of ST extract on pulmonary inflammation using CS exposure in mouse models. Exposure to CS induced an accumulation of inflammatory cells surrounding the perivascular and peribronchial regions. Cells recruited in the lungs released various inflammatory cytokines, chemokines and proteinases, which induced small airway inflammation and abnormal changes of the lung parenchymal tissue (Tetley, 2002). TNF-α is a pro-inflammatory cytokine involved in immune cell activation, mucus secretion, and destruction of lung parenchyma by proteinase release (Chung, 2001). IL-6 plays an important role in the regulation of the inflammatory response, and is present in high concentrations in the sputum, bronchoalveolar lavage and plasma of COPD patients (Pauwels et al., 2010). IL-1β is also a potent activator of alveolar macrophages, and stimulates the expression of MMPs, causing emphysema and small airway remodeling (Churg et al., 2009). MCP-1 is a chemoattractant for monocytes, and is involved in the recruitment of macrophages to the lungs in chronic inflammation (deBoer et al., 2000). MMP-12, an elastase produced by alveolar macrophages in response to CS, induces lung tissue destruction and remodeling via degradation of collagen and elastin (Nenan et al., 2005; Dean et al., 2008). Ultimately, the interaction of these inflammatory mediators leads to the inflammatory responses, causing alveolar destruction and pulmonary emphysema. Therefore, inhibition of the production of inflammatory mediators is considered a preventative treatment for pulmonary inflammatory disease. Our results demonstrated that administration of ST extract inhibited the CS-induced expression of TNF-α, IL-6, IL-1β, MCP-1 and MMP-12 in the lungs. Furthermore, ST extract reduced the histological inflammatory changes in the lungs, and the levels of total cells, macrophages and neutrophils in the BALF. These results suggest that ST extract attenuated the pulmonary inflammatory responses by inhibiting cell infiltration into the lungs and reducing production of inflammatory mediators. From the chemical profile study on ST extract, the biological origin of this commercial ST extract was confirmed as Stemona tuberosa but active principles of this bioactivity were not elucidate because bioactivity-guided isolation was beyond the scope of our study. However, from the previous reports and current analysis data, the major components of this herbal medicinal product were Stemona alkaloids and these alkaloids including stemonine were assumed to be major contributor on current anti-inflammatory activities. Since the anti-inflammatory pharmacological activity of the ST extract should be holistic summation of each synergic and countervail effect between all the components in the extract, it would be very difficult to define the pharmacological efficacy of ST extract in the molecular level. Therefore, further study would be needed. In summary, this study showed that ST extract reduced the production of pro-inflammatory mediators by inhibiting activation of the NF-κB and MAPK pathways in LPS-stimulated RAW 264.7 macrophages. It also suppressed CS-induced lung inflammation in mouse models. Further studies should be conducted in order to distinguish the most important compound in ST water extract, and

would lead us to find out the new therapeutic agents for CSinduced lung inflammation such as lung cancer, COPD and emphysema.

Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (No. NRF2014R1A1A305081) References Bae, H., Kim, R., Kim, Y., Lee, E., Jin Kim, H., Pyo Jang, Y., Jung, S.K., Kim, J., 2012. Effects of Schisandra chinensis Baillon (Schizandraceae) on lipopolysaccharide induced lung inflammation in mice. Journal of Ethnopharmacology 142, 41–47. Bar-Shai, M., Hasnis, E., Wiener-Megnazi, Z., Reznick, A.Z., 2006. The role of reactive nitrogen species and cigarette smoke in activation of transcription factor NF-kappaB and implication to inflammatory processes. Journal of Physiology and Pharmacology: An Official Journal of the Polish Physiological Society 57 (Suppl. 4), S39–S44. Barnes, P.J., Shapiro, S.D., Pauwels, R.A., 2003. Chronic obstructive pulmonary disease: molecular and cellular mechanisms. The European Respiratory Journal 22, 672–688. Bhalla, D.K., Hirata, F., Rishi, A.K., Gairola, C.G., 2009. Cigarette smoke, inflammation, and lung injury: a mechanistic perspective. Journal of Toxicology and Environmental Health. Part B: Critical Reviews 12, 45–64. Cheung, D.W., Koon, C.M., Wat, E., Ko, C.H., Chan, J.Y., Yew, D.T., Leung, P.C., Chan, W.Y., Lau, C.B., Fung, K.P., 2013. A herbal formula containing roots of Salvia miltiorrhiza (Danshen) and Pueraria lobata (Gegen) inhibits inflammatory mediators in LPSstimulated RAW 264.7 macrophages through inhibition of nuclear factor kappaB (NFkappaB) pathway. Journal of Ethnopharmacology 145, 776–783. Chung, H.S., Hon, P.M., Lin, G., But, P.P., Dong, H., 2003. Antitussive activity of Stemona alkaloids from Stemona tuberosa. Planta Medica 69, 914–920. Chung, K.F., 2001. Cytokines in chronic obstructive pulmonary disease. The European Respiratory Journal Supplement 34, 50s–59s. Churg, A., Cosio, M., Wright, J.L., 2008. Mechanisms of cigarette smoke-induced COPD: insights from animal models. American Journal of Physiology. Lung Cellular and Molecular Physiology 294, L612–L631. Churg, A., Zhou, S., Wang, X., Wang, R., Wright, J.L., 2009. The role of interleukin1beta in murine cigarette smoke-induced emphysema and small airway remodeling. American Journal of Respiratory Cell and Molecular Biology 40, 482–490. deBoer, W.I., Sont, J.K., van Schadewijk, A., Stolk, J., van Krieken, J.H., Hiemstra, P.S., 2000. Monocyte chemoattractant protein 1, interleukin 8, and chronic airways inflammation in COPD. The Journal of Pathology 190, 619–626. Deakin, A.M., Payne, A.N., Whittle, B.J., Moncada, S., 1995. The modulation of IL-6 and TNF-alpha release by nitric oxide following stimulation of J774 cells with LPS and IFN-gamma. Cytokine 7, 408–416. Dean, R.A., Cox, J.H., Bellac, C.L., Doucet, A., Starr, A.E., Overall, C.M., 2008. Macrophage-specific metalloelastase (MMP-12) truncates and inactivates ELR þCXC chemokines and generates CCL2, -7, -8, and -13 antagonists: potential role of the macrophage in terminating polymorphonuclear leukocyte influx. Blood 112, 3455–3464. Dong, W., Wang, P., Meng, X., Sun, H., Zhang, A., Wang, W., Dong, H., Wang, X., 2012. Ultra-performance liquid chromatography-high-definition mass spectrometry analysis of constituents in the root of Radix stemonae and those absorbed in blood after oral administration of the extract of the crude drug. Phytochemical Analysis 23, 657–667. Fujiwara, N., Kobayashi, K., 2005. Macrophages in inflammation. Current Drug Targets. Inflammation and Allergy 4, 281–286. Guha, M., Mackman, N., 2001. LPS induction of gene expression in human monocytes. Cellular Signalling 13, 85–94. Han Lin, W., Ma, L., Shen Cai, M., Barnes, R.A., 1994. Two minor alkaloids from roots of Stemona tuberosa. Phytochemistry 36, 1333–1335. Hasday, J.D., Bascom, R., Costa, J.J., Fitzgerald, T., Dubin, W., 1999. Bacterial endotoxin is an active component of cigarette smoke. Chest 115, 829–835. Ichinose, M., Sugiura, H., Yamagata, S., Koarai, A., Shirato, K., 2000. Increase in reactive nitrogen species production in chronic obstructive pulmonary disease airways. American Journal of Respiratory and Critical Care Medicine 162, 701–706. Jiang, R.-W., Hon, P.-M., Zhou, Y., Chan, Y.-M., Xu, Y.-T., Xu, H.-X., Greger, H., Shaw, P.-C., But, P.P.-H., 2006. Alkaloids and chemical diversity of Stemona tuberosa. Journal of Natural Products 69, 749–754. Kaminska, B., 2005. MAPK signalling pathways as molecular targets for antiinflammatory therapy – from molecular mechanisms to therapeutic benefits. Biochimica et Biophysica Acta 1754, 253–262. Kuwano, T., Nakao, S., Yamamoto, H., Tsuneyoshi, M., Yamamoto, T., Kuwano, M., Ono, M., 2004. Cyclooxygenase 2 is a key enzyme for inflammatory cytokineinduced angiogenesis. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology 18, 300–310.

D. Lim et al. / Journal of Ethnopharmacology 160 (2015) 41–51

Lawrence, T., 2009. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harbor Perspectives in Biology 1, a001651. Lee, E., Yun, N., Jang, Y.P., Kim, J., 2013. Lilium lancifolium Thunb. extract attenuates pulmonary inflammation and air space enlargement in a cigarette smokeexposed mouse model. Journal of Ethnopharmacology 149, 148–156. Lee, H., Yu, S.R., Lim, D., Lee, H., Jin, E.Y., Jang, Y.P., Kim, J., 2014. Galla chinensis attenuates cigarette smoke-associated lung injury by inhibiting recruitment of inflammatory cells into the lung. Basic & Clinical Pharmacology & Toxicology , http://dx.doi.org/10.1111/bcpt.12308. Li, S.-L., Jiang, R.-W., Hon, P.-M., Cheng, L., Xu, H.-X., Greger, H., But, P.P.-H., Shaw, P.-C., 2007. Quality evaluation of Radix stemonae through simultaneous quantification of bioactive alkaloids by high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors. Biomedical Chromatography 21, 1088–1094. Lin, W.-H., Ye, Y., Xu, R.-S., 1992. Chemical studies on new Stemona alkaloids, IV. Studies on new alkaloids from Stemona tuberosa. Journal of Natural Products 55, 571–576. Mercer, B.A., D’Armiento, J.M., 2006. Emerging role of MAP kinase pathways as therapeutic targets in COPD. International Journal of Chronic Obstructive Pulmonary Disease 1, 137–150. Nenan, S., Boichot, E., Lagente, V., Bertrand, C.P., 2005. Macrophage elastase (MMP-12): a pro-inflammatory mediator? Memorias do Instituto Oswaldo Cruz 100 (Suppl. 1), S167–S172. Pauwels, N.S., Bracke, K.R., Maes, T., Pilette, C., Joos, G.F., Brusselle, G.G., 2010. The role of interleukin-6 in pulmonary and systemic manifestations in a murine model of chronic obstructive pulmonary disease. Experimental Lung Research 36, 469–483. Ra, J., Lee, S., Kim, H.J., Jang, Y.P., Ahn, H., Kim, J., 2010. Bambusae caulis in Taeniam extract reduces ovalbumin-induced airway inflammation and T helper 2 responses in mice. Journal of Ethnopharmacology 128, 241–247. Schinnerl, J., Brem, B., But, P.P.-H., Vajrodaya, S., Hofer, O., Greger, H., 2007. Pyrroloand pyridoazepine alkaloids as chemical markers in Stemona species. Phytochemistry 68, 1417–1427. Schinnerl, J., Kaltenegger, E., Pacher, T., Vajrodaya, S., Hofer, O., Greger, H., 2005. New pyrrolo [1, 2-a] azepine type alkaloids from Stemona and Stichoneuron (Stemonaceae). Monatshefte für Chemie/Chemical Monthly 136, 1671–1680.

51

Seimetz, M., Parajuli, N., Pichl, A., Veit, F., Kwapiszewska, G., Weisel, F.C., Milger, K., Egemnazarov, B., Turowska, A., Fuchs, B., Nikam, S., Roth, M., Sydykov, A., Medebach, T., Klepetko, W., Jaksch, P., Dumitrascu, R., Garn, H., Voswinckel, R., Kostin, S., Seeger, W., Schermuly, R.T., Grimminger, F., Ghofrani, H.A., Weissmann, N., 2011. Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice. Cell 147, 293–305. Srivastava, D., Mehta, A.K., Arora, N., Gaur, S.N., Singh, B.P., 2010. Proteolytically inactive per a 10 allergen of Periplaneta americana modulates Th2 response and enhances IL-10 in mouse model. Journal of Clinical Immunology 30, 426–434. Sun, J., Ramnath, R.D., Zhi, L., Tamizhselvi, R., Bhatia, M., 2008. Substance P enhances NF-kappaB transactivation and chemokine response in murine macrophages via ERK1/2 and p38 MAPK signaling pathways. American Journal of Physiology. Cell Physiology 294, C1586–C1596. Tak, P.P., Firestein, G.S., 2001. NF-kappaB: a key role in inflammatory diseases. The Journal of Clinical Investigation 107, 7–11. Tetley, T.D., 2002. Macrophages and the pathogenesis of COPD. Chest 121, 156S–159S. Williams, J.A., Shacter, E., 1997. Regulation of macrophage cytokine production by prostaglandin E2. Distinct roles of cyclooxygenase-1 and -2. The Journal of Biological Chemistry 272, 25693–25699. Xu, Y.T., Shaw, P.C., Jiang, R.W., Hon, P.M., Chan, Y.M., But, P.P., 2010. Antitussive and central respiratory depressant effects of Stemona tuberosa. Journal of Ethnopharmacology 128, 679–684. Yoshida, T., Tuder, R.M., 2007. Pathobiology of cigarette smoke-induced chronic obstructive pulmonary disease. Physiological Reviews 87, 1047–1082. Zamora, R., Vodovotz, Y., Billiar, T.R., 2000. Inducible nitric oxide synthase and inflammatory diseases. Molecular Medicine 6, 347–373. Zhou, X., Leung, P.H., Li, N., Ye, Y., Zhang, L., Zuo, Z., Lin, G., 2009. Oral absorption and antitussive activity of tuberostemonine alkaloids from the roots of Stemona tuberosa. Planta Medica 75, 575–580. Zhou, Y., Jiang, R.-W., Hon, P.-M., Xu, Y.-T., Chan, Y.-M., Chan, T.-W.D., Xu, H.-X., Ding, L.-S., But, P.P.-H., Shaw, P.-C., 2006. Analyses of Stemona alkaloids in Stemona tuberosa by liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry 20, 1030–1038.