Tanshinone IIA sulfonate protects against cigarette smoke-induced COPD and down-regulation of CFTR in mice

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by abnormal inflammation, persistent and progressive lung function decline. The anti-inflammatory actions of tanshinone IIA, which is the most important active component from Chinese herbal medicine Danshen, have been well studied. However, it remains unknown whether sodium tanshinone IIA sulfonate (STS) protects against the development of COPD. Here we found that STS inhalation (5 mg/kg, 30 min per session, twice a day) significantly attenuated lung function decline, airspace enlargement, mucus production, bronchial collagen deposition, inflammatory responses and oxidative stress caused by cigarette smoke (CS) and lipopolysaccharide (LPS) exposures in mice. Moreover, treatment with STS (10 μg/ml) reduced CS extract (CSE)-induced IL-6 and IL-8 secretion in human bronchial epithelial (16HBE) cells. The anti-inflammatory actions of STS were associated with inhibition of ERK1/2 and NF-κB activations. Interestingly, STS inhibited CS-induced reduction of cystic fibrosis transmembrane conductance regulator (CFTR) in mouse lungs and in 16HBE cells. Treatment with a specific CFTR inhibitor CFTR-Inh172 augmented CSE-induced ERK1/2 and NF-κB-dependent inflammatory responses, but abolished the inhibitory action of STS on IL-6 and IL-8 secretion in 16HBE cells. These results demonstrate that CS-induced COPD and down-regulation of CFTR are prevented by STS.

Sodium tanshinone IIA sulfonate (STS), a water-soluble substance, derives from tanshinone IIA (TIIA). TIIA is the main pharmacological component from Chinese herbal medicine Danshen, the dried root of Saviamiltiorrhiza. Danshen has been widely used in many countries including China, Japan and the United States to treat cardiovascular and cerebrovascular diseases with few adverse effects 9 . In recent years, STS and TIIA have been found to be anti-inflammatory in experimental lung and cardiac diseases [10][11][12] . In these experiments, STS or TIIA was administered via intraperitoneal injection. However, intraperitoneal injection may bring poor treatment compliance. Furthermore, excess STS will be administered intraperitoneally so as to reach effective concentration in lungs, which inevitably limits its clinical use. More importantly, it is not known whether STS attenuates CS-induced inflammatory responses and subsequent COPD.
In this study, we evaluated effects of STS inhalation on the development of COPD in mice exposed to CS. We also investigated the mechanisms underlying the protective roles of STS on lung inflammatory and injurious responses to CS in vivo mouse lungs and in vitro lung epithelial cells. To the best of our knowledge, this is the first report regarding the effects of STS inhalation on CS-induced COPD and underlying mechanisms.

STS protects against CS-induced reduction of CFTR in 16HBE cells and mouse lungs. Sustained
exposure to CS or CSE has been shown to reduce CFTR expression. As expected, CSE exposure reduced CFTR protein in 16HBE cells (Fig. 7A). STS treatment attenuated the reduction of CFTR protein caused by CSE incubation (CSE group: 48.93 ± 4.35%, CSE + STS group: 84.18 ± 9.55%, percentage of the CTL group, Fig. 7A). Similarly, STS administration attenuated the inhibitory effect of CS on CFTR protein abundance in mouse lungs, determined by Western blotting (CS group: 39.88 ± 9.85%, CS + STS group: 71.20 ± 8.53%, percentage of the CTL group, Fig. 7B) and immunohistochemistry (Fig. 7C). These results indicate STS protects against CS-induced reduction of CFTR level.

Discussion
Danshen and its derivative products including STS have been applied to treat cardiovascular and cerebrovascular diseases both in Asian and Western countries 9 . Our study provided evidence that a long-term aerosol inhalation of STS reduced lung physiological and pathological manifestations in mice exposed to CS. This was associated with the reduction of CS-induced oxidative stress and inflammatory responses in vivo in mice and in vitro in cells. This is corroborated by another finding showing that STS is effective in clinical treatment of pulmonary hypertension, a common complication of COPD 13 . LPS helps to induce emphysema in shorter time than pure CS exposure (couples of months vs 6-8 months) 14 . We used both LPS and CS exposures in mice, and treated them with STS. STS was described as an antioxidant to reduce oxidative stress and inflammatory responses, which is associated with its function such as scavenging oxygen free radicals, preventing lipid peroxidation 15 , inhibiting low density lipoprotein oxidation 16 , increasing Zn superoxide dismutase (SOD) activity as well as mRNA and protein expression 17 , activating the Nrf2 pathway 18 and inhibiting NF-κB and MAPK signaling pathways 19 . In the present study, we found that STS treatment significantly reduced lung inflammatory responses to CS exposure. This may be due to inhibition of NF-κB and MAPK signaling pathway, which is shown in our and previous studies 19 . A broad spectrum of anti-inflammatory drugs, including inhibitors of the pro-inflammatory enzymes PDE4, Janus kinases, NF-κB kinase, p38 mitogen-activated protein kinase, and PI3 kinase-γ and -δ, were developed for COPD treatment, but side effects after oral administration limited their clinical application 20 . Unlike significant advances in the development of long-acting bronchodilators, it has been proved difficult to find safe and effective anti-inflammatory treatments for COPD 20 . As a medicine used in traditional Chinese medicine, side effects and safety of Danshen and Danshen products have been fully evaluated in clinical practice 9 . Indeed, we found that increases of organ and body indices were attenuated by STS treatment, suggesting the dose of STS (5 mg/kg, 30 min per session, twice per day) here to treat COPD did not cause a detectable toxic effect. Based on our findings, STS is a potential drug for preventing COPD, which could be conveniently delivered by aerosol inhalation.
Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial anion channel, is expressed mainly in exocrine tissues. In lung, loss of CFTR function results in mucociliary clearance defect, airway surface liquid depletion, chronic bacterial infection and airway chronic inflammation 21 . CS-mediated CFTR dysfunction results in inflammation and oxidative stress, and both are involved in the pathogenesis of COPD emphysema 22 . Therefore, pharmacological approaches aimed at enhancing CFTR expression and/or function could be potentially beneficial to COPD.
In response to CS exposure, level of CFTR was reduced in mouse lungs and in lung epithelial cells. Recent studies showed CFTR gene expression in human airway epithelial cells was regulated by oxidative stress 23,24 . Current findings and others showed that STS protected against oxidative stress 25 . These findings suggest that STS protects against CFTR reduction through reducing oxidative stress. GSH, one of the most important antioxidants against free radicals in lungs, affords protection against oxidative damage on the alveolar epithelial fluid interface 26 . Compared with mice without STS treatment, higher ratio of free GSH to GSSH was found in lung tissue from COPD model mice treated with STS, which means a better cellular stability against oxidative damage 27 . These findings suggest that there is an interaction between CFTR and oxidative stress, which is disrupted by CS exposure. Mucus hypersecretion, an important factor of predicting the morbidity and mortality in COPD, is contributed by CS-induced acquired CFTR dysfunction, while it can be markedly improved by the CFTR potentiator in vitro 28 . Our preliminary studies have shown that STS inhalation down-regulates Muc5AC and Muc5B levels in BALF of mice with COPD. This was in agreement with the finding that STS inhalation reduced CS-induced goblet cell hypertrophy and hyperplasia of the airway epithelium in COPD mice. The pathogenesis of airway mucus hypersecretion induced by CS, which is complicated and incompletely understood, may involve inflammation, oxidative stress, signal transduction and decreased expression of CFTR 29 . Treatments should counteract each factor contributing to airway mucus hypersecretion. Our study shows that treatment with aerosolized STS twice daily (5 mg/kg) is associated with significant improvements in mucus clearance. The mechanism of benefit is thought to be attributed to inflammation and oxidative stress inhibition, as well as CFTR expression maintenance.
MAPK signaling pathways regulate pro-inflammatory mediator IL-8 expression in cystic fibrosis lung epithelial cell lines 30 . Inhibition of CFTR function enhanced inflammatory responses to CS by activation of p38/ ERK MAPK or NF-κB pathway in airway bronchial epithelial cells 31,32 . This is consistent with our findings that pharmacological inhibition of CFTR augments CS-induced IL-6 and IL-8 expression as well as MAPK and NF-κB activation, despite CFTR inhibition by CFTR-Inh172 (10 µg/ml) alone did not cause inflammatory responses in lung epithelial cells. The mechanisms underlying the discrepancies between these findings are not clear. Interestingly, protective role of STS on CS-induced inflammatory responses was abolished in response to CFTR inhibition. All these findings suggest that STS reduces CS-induced inflammatory responses by attenuating CFTR reduction, although there is a need to use CFTR knockout cells to further unravel its role in CS-induced inflammatory and injurious responses.
In summary, our current study demonstrates that STS inhalation effectively attenuates CS-induced clinical or pathological presentations of COPD, such as emphysema, collagen deposition in the small airway, goblet cell hyperplasia of airway epithelium and lung function decline. This was associated with protective effects of STS on CS-induced oxidative stress, NF-κB-dependent inflammatory responses. Furthermore, CS-induced down-regulation of CFTR is also prevented by STS. STS could be a potential anti-inflammatory drug for COPD.
Nevertheless, our study has some limitations, such as not figuring out the specific molecular mechanism underlying that STS inhibited CS-induced CFTR reduction in COPD. However, the study has already provided evidence to support that STS inhalation has clinical efficiency, feasibility and relatively lower cost compared with  classic COPD medication as a potential drug for COPD. STS inhalation is a valuable candidate worthy of further investigation for future CS-induced COPD therapy.

Methods
Animal model. Wide-type C57BL/6 J male mice (6-8 weeks old) were purchased from the Nanjing BioMedical Research Institute of Nanjing University (Nanjing, China). Animals were housed in the specific pathogen-free facilities and the Animal Care and Use Committee of Guangzhou Medical University approved all experimental protocols. All methods were performed in accordance with the guidelines and regulations approved by the Animal Care and Use Committee of Guangzhou Medical University. The cigarettes used in the mouse model establishment were Plum brand filtered cigarettes (Guangdong Tobacco Industry, China) and each cigarette yields 11 mg tar, 1.0 mg nicotine and 13 mg carbon monoxide. A COPD model was established using CS exposure plus intranasal inhalation of Lipopolysaccharide (LPS) (Fig. 1A). Briefly, mice received LPS (7.5 μg/ Figure 8. CFTR inhibition abolishes inhibitory effect of STS on CSE-induced release in IL-6 and IL-8, but increases CSE-induced activation in ERK/NF-κB pathway. 16HBE cells were treated with 2% CSE for 48 h with 2 h of STS (10 μg/ml) pre-incubation. In order to inhibit CFTR function, cells were treated with CFTR specific inhibitor CFTR-Inh172 (10 μM) 1 h prior to STS treatment. (A) cells were treated with CFTR-Inh172 (0-50 μM) for 48 hours and cell viability was measured by CCK8 assay. (B) IL-6 and IL-8 expression in cell culture medium was measured. (C) The levels of phosphorylated ERK1/2 in cytosol protein were determined with ERK1/2 as internal control (n = 3). The level of p65/NF-κB in nuclear extraction was determined with histone H3 as internal control (n = 6). Full-length blots are presented in supplementary Figure 3. *P < 0.05 and **P < 0.01, CFTR, cystic fibrosis transmembrane conductance regulator; CSE, cigarette smoke extract; CS, cigarette smoke; STS, Sodium tanshinone IIA sulfonate. mouse in 50 μl saline; L8643, Sigma-Aldrich) or saline (vehicle control) by intranasal inhalation at the 1 st and 14 th day. Meanwhile, mice were exposed to CS (9 cigarettes/h, 2 hours/session, twice/day and 6 days/week in a whole-body exposure chamber) from day 0 to 60 except for the days giving LPS. For STS (Jiangsu Carefree Pharmaceutical, Nanjing, China) treatment, mice were given saline or STS (5 mg/kg, 30 min per session, twice per day) by airway inhalation with a PARI Nebuliser in a whole-body exposure chamber for 30 min daily before being exposed to CS. Finally, all mice were subjected to lung function analysis, and sacrificed at day 61.

Lung function measurement. Lung function was evaluated using the Forced Pulmonary Maneuver
System (Buxco Research Systems, Wilmington, North Carolina, USA) according to the manufacturer's protocol. Briefly, mice anesthetized with pentobarbital (50 mg/kg body weight) were tracheostomized, intubated and put in the body chamber of the system. The average breathing frequency of anesthetized animals was forcibly set at 150 breaths/min. Three semiautomatic maneuvers were carried out: Boyle's law functional residual capacity (FRC), quasi-static pressure volume (PV) and fast flow volume (FV) maneuver. The FRC was determined with Boyle's law FRC. To measure total lung capacity (TLC) and chord compliance (Cchord), the quasi-static PV maneuver was performed. With the fast FV maneuver, forced expiration volume in 50 milliseconds (FEV50) and forced vital capacity (FVC) were measured.
Hematocrit measurement. Briefly, at the terminal of chronic CS exposure, whole blood was collected into capillary tubes (0.5 mm outside diameter, VWR Scientific, Radnor, PA) via right ventricle puncture with K 2 EDTA as an anticoagulant and centrifuged at 7,000 rpm for 5 min, and read on a hematocrit chart (VWR Scientific). BALF analysis. The left lung was injected with three sequential 0.6 ml of ice cold saline intra-tracheally and slowly. A total of 1 ml BALF was collected and centrifuged at 500 g for 10 minutes at 4 °C to pellet the cells. The cell pellets were re-suspended in 1 ml saline for total cell counts using haemacytomer. The cells were subjected to Giemsa staining for differential counting of neutrophils, macrophages and lymphocytes. The supernatant was stored at −80 °C for pro-inflammatory cytokine detection.
Histopathology and immunohistochemistry. The left lung specimens of mice were fixed in 10% neutral buffered formalin for 24 h, embedded in paraffin wax, and cut into 4 mm thick slices stained with hematoxylin and eosin (H&E) to evaluate morphological changes. The Image Analysis Software Image-Pro Plus 6.0 was used to assess the average alveolar intercept, which means the ratio of total alveolar length to the number of alveoli per field under microscopy. For immunohistochemistry, the slices were immunostained with anti-CFTR antibody (1:100 ACL006AN0602, Alomone Labs, Jerusalem, Israel) and developed by diaminobenzidine (DAB) solution according to manufacturer's instructions. Periodic Acid-Schiff (PAS) stain (Shanghai Sun Biotechnology, Shanghai, China) was utilized to detect goblet cells in the airway epithelium following manufacturer's instructions. Masson Trichrome stain (KGMST-8003) was utilized to detect collagen deposition of small airway according to manufacturer's instructions. Semi-quantitative analyses of positive area in lungs were completed by two independent investigators using the Image-Pro Plus 6.0. At least three fields per mouse were taken.
Measurement of liver, spleen, and kidney indices. The liver, spleen, or kidneys were weighed and each was calibrated with whole body weight.
Preparation of nuclear protein extract. Lung tissue or 16HBE cells were extracted with NE-PER nuclear and cytoplasmic reagents (Thermo Scientific) containing protease inhibitor cocktail according to the manufacturer's instructions. Histone H3 served as the loading control in Western blotting of nuclear proteins.
Preparation of CSE. CSE was freshly prepared from Plum brand filtered cigarettes within 30 min prior to treatments according to a previously described protocol 33 . The acquired CSE suspension in yellowish color with optical density (OD) at 405 nm 0.506 ± 0.008 was adjusted to pH7.4, passed through a 0.22 μm filter to remove bacteria and particles and considered as concentration 100% in cell treatments.
Cell viability assay. The cell viability of 16HBE cells was determined by using a CCK8 assay kit (Sigma-Aldrich) according to the manufacturer's instruction. Briefly, the cells (5,000 cells/well) were seeded in 96-well plates, cultured overnight, treated with CSE (0-20% in medium containing 1% FBS) or STS (0-60 μg/ ml) or CFTR-Inh172 (0-50 μM) for 48 h. Subsequently, CCK8 solution (10 µl) was added to each well and incubated for 2 h. Finally, the absorbance at 450 nm was read in a microplate absorbance reader (Thermo Scientific, Waltham, MA). Cell viability was presented as percentage of normal control cells, i.e. cells without CSE or STS or CFTR-Inh172 treatment.
Cell culture. Human bronchial epithelial cells 16HBE were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured with DMEM medium added with 10% FBS, 100 ug/ml penicillin and 100 μg/ml streptomycin in a humidified incubator at 37 °C with 95% (v/v) air and 5% (v/v) CO 2 . The cells were treated with CSE (2%) and STS (10 μg/ml) with or without CFTR inhibitor CFTR-Inh172 (10 μM, Sigma) in serum starved medium.
Glutathione fluorometric assay. The ratio of GSH/GSSG was measured in lung using the GSH/GSSG ratio detection assay kit (BioVision, Milpitas, CA) according to manufacturer's instruction. Briefly, lung tissue was homogenized using cold glutathione assay buffer. To every 60 μl of homogenate was added 20 μl of prechilled perchloric acid (PCA, 6 N). The solution was mixed, centrifuged and the supernatant was kept at −80 °C. The PCA-preserved samples were assayed for glutathione after addition of ice cold KOH (6 N, 20 μl to 40 μl per sample) to precipitate PCA and centrifuged. For GSH measurement, 10 μl of sample was added to 90 μl of assay buffer. For GSSH measurement, 10 μl of sample was added to 70 μl of assay buffer, followed by addition of a quencher buffer to quench GSH and a reducing agent to destroy excessive GSH quencher and transform GSSG to GSH. Finally, o-phthalaldehyde (OPA) probe was added to each sample and the relative fluorescence units (RFU) were read in a fluorescence plate reader with λ ex = 340 nm and λ ex = 420 nm. A standard curve with known concentrations of GSH supplied with the kit was used to calculate GSH concentration. The results were expressed as molar ratio of GSH/GSSH. Statistical analysis. Data were analyzed with ANOVA or two-tailed Student's t-test. If significant F-ratios were acquired with ANOVA, pair-wise comparison of mean was carried out with Boferroni analysis. Data are presented as mean ± SEM. "n" means the number of repeats in cell experiments or the number of animals. P < 0.05 and P < 0.01 were considered significant.