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Anti-inflammatory effects of trans-anethole in a mouse model of chronic obstructive pulmonary disease
Biomedicine & Pharmacotherapy 91 (2017) 925–930
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Anti-inflammatory effects of trans-anethole in a mouse model of chronic obstructive pulmonary disease Ka Young Kima,b,1, Hui Su Leea,1, Geun Hee Seola,* a b
Department of Basic Nursing Science, School of Nursing, Korea University, Seoul 02841, Republic of Korea Department of Nursing, College of Nursing, Gachon University, Incheon 21936, Republic of Korea
A R T I C L E I N F O
Article history: Received 13 March 2017 Received in revised form 6 May 2017 Accepted 6 May 2017 Keywords: Trans-anethole Chronic obstructive pulmonary disease (COPD) Interleukin-6 (IL-6) Blood pressure Porcine pancreatic elastase (PPE) Lipopolysaccharide (LPS)
A B S T R A C T
Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease that is generally characterized by progressive and irreversible airflow obstruction and alveolar destruction. Long-term treatment with current medications has been associated with various adverse effects, indicating a need for alternative approaches for the prevention and treatment of COPD. This study investigated the mechanism underlying the effects of trans-anethole in a mouse model of COPD induced by porcine pancreatic elastase (PPE) and lipopolysaccharide (LPS). BALB/c mice were orally administered transanethole (62.5, 125, 250, or 500 mg/kg) 2 h before intranasal challenge with 1.2 units of PPE and 7 mg of LPS. Lactate dehydrogenase (LDH) activity, cell counts, lung histology, cytokine production, and blood pressure were analyzed. Trans-anethole reduced LDH activity and inflammatory cell counts, including macrophage, neutrophil, and lymphocyte counts. trans-anethole 125 mg/kg restored the histopathological changes induced in mouse lungs by PPE and LPS. trans-anethole reduced the serum concentrations of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a), as well as significantly reducing blood pressure during chronic inflammation. Trans-anethole ameliorated chronic lung inflammation in a mouse model of COPD by reducing the serum concentrations of proinflammatory cytokines such as TNF-a and IL-6, and by reducing blood pressure. The present results indicate that trans-anethole may be a potential therapeutic agent for prophylaxis and treatment in patients with chronic lung inflammation. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Trans-anethole, the major constituent obtained from anise, star anise, and fennel, has been reported to have anti-inflammatory [1,2], antioxidant [3,4], anticarcinogenic [5,6], neuroprotective [7] and vasoactive effects [8]. Anethole showed nitric oxide (NO)independent vasodilation in KCl-induced vasospasm of isolated rat aorta rings [9]. Investigations in a mouse model of lipopolysaccharide (LPS)-induced acute lung injury have indicated that the anti-inflammatory properties of anethole are mediated by its reduction of pro-inflammatory mediators, including tumor necrosis factor-alpha (TNF-a), metalloproteinase-9 (MMP-9), and NO, and its inhibition of nuclear factor-kappa B (NF-kB) [1]. Moreover, a randomized clinical trial showed that anethole dithiolethione
* Corresponding author at: Department of Basic Nursing Science, School of Nursing, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea. E-mail address: [email protected] (G.H. Seol). 1 KYK and HSL contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2017.05.032 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
decreased new dysplastic lesions in smokers with bronchial dysplasia [10]. To date, however, the mechanisms underlying the anti-inflammatory effects of anethole in chronic obstructive pulmonary disease (COPD) remain undetermined. COPD is a chronic inflammatory lung disease that is generally characterized by progressive and irreversible airflow obstruction and alveolar destruction [11]. Inflammation in COPD causes an accumulation of inflammatory cells, including macrophages, neutrophils, and lymphocytes, and the release of reactive oxygen species (ROS), cytokines, chemokines, and elastase [11]. COPD can develop in patients with cardiovascular diseases, including pulmonary hypertension, as damage to the lungs can reduce the amount of oxygen to the blood [12,13]. Although COPD is associated with early mortality, high rates of morbidity and mortality, and high treatment costs, clinical studies are limited [14]. Mouse models can effectively reflect the pathophysiology of human COPD [15]. The tuber of Alismataceae Alisma orientale Juzepzuk, a constituent of herbal medicine, has been reported to induce histological changes in a COPD mouse model [16]. Longterm treatment with currently available medications has been
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associated with serious side-effects in patients with COPD, indicating a need for alternative approaches to prevent and treat this chronic condition. Treatment of mice with LPS and the endopeptidase porcine pancreatic elastase (PPE) has been found to induce COPD [17,18]. Pathological changes observed in these mice include increased airway inflammation, activation of immune cells, and alveolar wall destruction [19–21]. This model mimics many of the crucial pathological features of human COPD [22]. Inhalation of the traditional Chinese medicine chung-pae, which is composed of four herbs, was found to effectively reduce the chronic inflammation and pathological changes in LPS/PPE-induced COPD in mice [18]. Similarly, the medicinal plant Gamijinhae-gang showed antiinflammatory activity in a mouse model of COPD induced by PPE and LPS [20]. Trans-anethole is a major constituent of these traditional medicines. This study therefore analyzed the antiinflammatory effects of trans-anethole in a mouse model of LPS/ PPE-induced COPD.
anesthetized by intraperitoneal injection of a mixture of 0.3 mg/ kg tiletamine-zolazepam (Zoletil 50, Virbac Laboratories, Carros, France) and 0.2 mg/kg xylazine (Rompun, Bayer Korea, Ansan, Korea). Bronchoalveolar lavage (BAL) fluid was collected three times from each mouse through a tracheal cannula with sterile PBS.
2. Materials and methods
2.4. Inflammatory cell counts
2.1. Reagents
BAL fluid samples were centrifuged at 500g for 10 min at 4 C, and the sedimented cells were resuspended in PBS. Total cell numbers and types of inflammatory cells were determined on a Countess automated cell counter (Invitrogen Life Technologies, Carlsbad. CA, USA) using Diff-Quik stain (International Reagents Co., Kobe, Japan). The results are expressed as the number of each cell type per milliliter of BAL fluid.
LPS from Escherichia coli 055:B5, trans-anethole, and dexamethasone were purchased form Sigma Aldrich Co. (St Louis, MO, USA). PPE was obtained from Elastin Products Company (Owensville, MO, USA).
2.3. Lactate dehydrogenase assay (LDH) The activity of lactate dehydrogenase (LDH), a marker for cytotoxicity, in acellular BAL fluid was measured using a cytotoxicity detection kit according to the manufacturer’s instructions (Roche Applied Science, Mannheim, Germany). BAL fluid samples were mixed 1:1 with freshly prepared reaction mixture and incubated in the dark for 30 min at room temperature. The absorbance of each reaction mixture was measured at 490 nm and at a reference wavelength of 620 nm using a microplate reader (BMG Labtech, Ortenberg, Germany).
2.2. Animals All animals were treated according to National Institutes of Health guidelines for the use of experimental animals. The study protocol was approved by the institutional animal care and use committee of Korea University (KUIACUC-2016-153), and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23; revised 1996). Specific pathogen-free male BALB/c mice (5 weeks old), weighing 19–21 g were obtained from Orient Bio Inc. (Seongnam, Korea). A total of 80 mice were randomly divided into 9 groups of 5–12 mice each, and treated with PPE and LPS (Fig. 1). Briefly, mice were orally administered vehicle (1% Tween 80saline), trans-anethole (62.5, 125, 250, or 500 mg/kg), or dexamethasone (3 mg/kg) 2 h before anesthetization with isoflurane and intranasal administration of PPE (1.2 units on day 1 of each of 4 weeks) or LPS (7 mg of LPS on day 4 of each of 4 weeks); control mice were intranasally challenged with phosphate-buffered saline (PBS) [21,23]. On day 32, systolic (sBP) and diastolic blood pressure (dBP) were measured using a tail cuff and pulse transducer (AD Instruments, Sydney, Australia), with the results reported as the means of five data points. The mice were subsequently
BP, BAL fluid 3d
1d
4d
3d
1 week
4d
3d
2 weeks
4d
3d
4d
3 weeks
2.5. Histopathological analysis Lung tissue from 5–6 mice per group were fixed overnight in 10% paraformaldehyde and embedded in paraffin. Paraffin blocks were sectioned at a thickness of 4 mm. Sections were stained with hematoxylin and eosin (H&E) and four sections from each mouse examined using an Axioplan 2 microscope (Carl Zeiss, Göttingen, Germany). The alveolar diameter of each airspace was calculated using Image J software. 2.6. Enzyme-linked immunosorbent assay (ELISA) The concentrations of the cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) in BAL fluid were measured using ELISA kits, according to the manufacturer’s instructions (Koma Biotech, Seoul, Korea). 2.7. Statistical analysis All statistical analyses were performed using IBM SPSS Statistics 23.0 software (SPSS Inc., Chicago, IL, USA). Normality was analyzed using the Shapiro-Wilk test and the data were compared using one-way analysis of variance (ANOVA), followed by the Tukey’s LSD post hoc test. The results were considered significant at p < 0.05.
4 weeks 32d
3. Results ANE ANE ANE ANE ANE ANE ANE ANE PPE PPE PPE PPE LPS LPS LPS LPS Fig. 1. Schematic diagram of the study protocol. Mice were administered PPE on day 1 and LPS on day 4 each week for 4 weeks, with inhaled trans-anethole administered 2 h before the administration PPE or LPS. On day 32, BP was measured and BAL fluid was collected. Abbreviations: ANE, transanethole; PPE, porcine pancreatic elastase; LPS, lipopolysaccharide; BP, blood pressure; BAL fluid, bronchoalveolar lavage fluid.
3.1. Effect of trans-anethole on LDH activity in BAL fluid of PPE/LPSinduced COPD mice LDH activity in BAL fluid was 3.10-fold higher in PPE/LPStreated than in control mice (p < 0.001) (Fig. 2). Compared with PPE/LPS-treated animals, significant decreases in LDH activity were observed in animals pretreated with 62.5 mg/kg (2.03-fold,
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p = 0.006), 125 mg/kg (2.72-fold, p = 0.001), 250 mg/kg (2.18-fold, p = 0.001), and 500 mg/kg (2.68-fold, p < 0.001) trans-anethole, as well as dexamethasone (1.99-fold, p = 0.004). 3.2. Effect of trans-anethole on inflammatory cell counts in PPE/LPStreated mice
Fig. 2. Effect of trans-anethole on LDH activity in BAL fluid of PPE/LPS-induced COPD mice. LDH activity was analyzed in BAL fluid of PPE/LPS-induced COPD mice. Data represent mean SEM (n = 5–12 per group). ### P < 0.001 compared with the control group; ** P < 0.01, *** P < 0.001 compared with the COPD group. Abbreviations: ANE, trans-anethole; DEXA, dexamethasone.
The pathogenesis of COPD involves the recruitment of inflammatory cells, including macrophages, neutrophils, and lymphocytes, which are associated with chronic inflammation of the airways. The numbers of total cells in BAL fluid were 2.23-fold higher in PPE/LPStreated than in control mice (p < 0.001; Fig. 3D), as were the numbers of macrophages (2.55-fold, p < 0.001; Fig. 3A), neutrophils (2.59fold, p < 0.001; Fig. 3B), and lymphocytes (1.94-fold, p = 0.006; Fig. 3C). Pretreatment of PPE/LPS-treated mice with 62.5, 125, 250, and 500 mg/kg trans-anethole reduced macrophages by 1.59-fold (p = 0.001), 1.37-fold (p = 0.014), 1.88-fold (p < 0.001), and 1.19-fold (p = 0.173), respectively; neutrophils by 1.50-fold (p = 0.003), 2.33fold (p < 0.001), 3.03-fold (p < 0.001), and 1.36-fold (p = 0.034), respectively; lymphocytes by 1.45-fold (p = 0.086), 3.06-fold (p = 0.001), 2.63-fold (p < 0.001), and 1.66-fold (p = 0.051), respectively; and total cells by 1.56-fold (p < 0.001), 1.69-fold (p < 0.001), 2.21-fold (p < 0.001), and 1.28-fold (p = 0.042), respectively.
Fig. 3. Effect of trans-anethole on inflammatory cell counts in PPE/LPS-treated mice. The numbers of (A) macrophages, (B) neutrophils, (C) lymphocytes, and (D) total cells were measured in BAL fluid. Data represent means SEMs (n = 5–12 per group). # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the control group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the COPD group. Abbreviations: ANE, trans-anethole; DEXA, dexamethasone.
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Fig. 4. Effect of trans-anethole on lung histopathology of PPE/LPS-induced mouse model. Mouse lung tissues were stained with (A-D) hematoxylin and eosin (H&E) and alveolar diameters were analyzed by Image J software (E) (200). Data represent means SEMs (n = 5 6 per group). # P < 0.05, ### P < 0.001 compared with the control group; *** P < 0.001 compared with the COPD group. Abbreviations: ANE, trans-anethole; DEXA, dexamethasone.
3.3. Effect of trans-anethole on lung histopathology of PPE/LPSinduced mouse model Histologic examination of the lungs of PPE/LPS-treated mice (Fig. 4B) showed alveolar destruction and airspace enlargement when compared with control mice (Fig. 4A). By contrast, treatment with trans-anethole 125 mg/kg (Fig. 4C) and dexamethasone (Fig. 4D) resulted in recovery from PPE/LPS-induced lung inflammation. Quantified alveolar diameter was analyzed statistically to confirm the chronic inflammation (Fig. 4E). 3.4. Effect of trans-anethole on IL-6 and TNF-a in BAL fluid of mice with PPE/LPS-induced chronic inflammation Proinflammatory cytokines such as IL-6 (Fig. 5A) and TNF-a (Fig. 5B) are secreted by inflammatory cells. Compared with control mice, mice treated with PPE/LPS showed increased expression of IL-6 (2.76-fold, p < 0.001) and TNF-a (2.44-fold, p < 0.001). Pretreatment of PPE/LPS-treated mice with 62.5, 125,
and 250 mg/kg trans-anethole significantly reduced IL-6 concentrations in BAL fluid by 1.74-fold (p = 0.004), 1.65-fold (p = 0.019), and 1.87-fold (p = 0.001), respectively, whereas 500 mg/kg transanethole had no effect on IL-6 concentration. Pretreatment of PPE/ LPS-treated mice with 62.5, 125, 250, and 500 mg/kg transanethole significantly reduced TNF-a concentrations by 1.34-fold (p = 0.036), 2.75-fold (p < 0.001), 2.14-fold (p < 0.001), and 2.76fold (p < 0.001), respectively. 3.5. Effect of trans-anethole on BP in PPE/LPS-exposed mice Both sBP (1.17-fold, p = 0.002; Fig. 6A) and dBP (1.20-fold, p = 0.001; Fig. 6B) were significantly higher in PPE/LPS-treated than in control mice. Pretreatment of PPE/LPS-treated mice with 62.5, 125, 250, and 500 mg/kg trans-anethole significantly reduced sBP by 1.22-fold (p < 0.001), 1.16-fold (p = 0.003), 1.10-fold (p = 0.033), and 1.23-fold (p < 0.001), respectively; and dBP by 1.19-fold (p = 0.003), 1.15-fold (p = 0.009), 1.12-fold (p = 0.030), and 1.22-fold (p = 0.001), respectively.
Fig. 5. Effect of trans-anethole on IL-6 and TNF-a in BAL fluid of mice with PPE/LPS-induced chronic inflammation. The concentrations of (A) IL-6 and (B) TNF-a in BAL fluid were analyzed by ELISA. Data represent means SEMs (n = 5 12 per group). ## P < 0.01, ### P < 0.001 compared with the control group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the COPD group. Abbreviations: ANE, trans-anethole; DEXA, dexamethasone.
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Fig. 6. Effect of trans-anethole on BP in PPE/LPS-exposed mice. (A) Systolic blood pressure (sBP) and (B) diastolic blood pressure (dBP) were measured using a tail cuff and pulse transducer. ## P < 0.01 compared with the control group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the COPD group. Abbreviations: ANE, trans-anethole; DEXA, dexamethasone.
4. Discussion The present results show that trans-anethole ameliorated chronic inflammation, including cytotoxicity, increases in proinflammatory cells, and lung damage, induced by PPE and LPS in a mouse model of COPD by reducing the concentrations of proinflammatory cytokines such as TNF-a and IL-6. Lung histopathology showed alveolar destruction and airspace enlargement. Furthermore, trans-anethole significantly decreased the increased blood pressure associated with chronic inflammation. COPD, a major cause of morbidity and mortality worldwide, is usually associated with an abnormal and progressive inflammatory response of lungs to harmful gases and particles, including cigarette smoke and environmental pollutants [12,15]. Analysis of lung tissue and airway sections from patients with COPD has shown increases in both the numbers and activation of inflammatory cells, with an increased number of macrophages particularly associated with COPD severity [24]. Most patients with COPD have a reduced health-related quality of life due to various signs and symptoms, including cough, dyspnea, wheezing, sputum production, and alteration in mental status [25]. Furthermore, COPD is associated with increased cardiovascular risk including pulmonary hypertension [13,26], with 5–10% of patients with advanced COPD reported to have severe pulmonary hypertension [26]. PPE-treated mice were found to have increased right ventricular pressure and norepinephrine concentrations, indicators of pulmonary hypertension [27]. This mouse model of PPE- and LPS-induced COPD may be useful for further study of both inflammation and cardiovascular sequelae associated with COPD. In particular, the present study found that trans-anethole effectively attenuated the increases in sBP and dBP induced by challenge with PPE and LPS. Pulmonary hypertension is a complication of advanced lung disease [13], as well as being negatively prognostic in patients with COPD [28]. The mouse model of PPE/LPS-induced COPD may be useful in assessing the pathology of COPD and the antiinflammatory and anti-hypertensive properties of various alternative therapies [15]. Trans-anethole has been reported to have anti-inflammatory properties [2]. In particular, it has been reported to be effective in the treatment of lung diseases, including lung inflammation [1] and lung cancer [10,29]. Anethole dithiolethione, a derivative of anethole, has been reported to reduce homocysteine levels associated with the pathogenesis of cardiovascular disease [30].
In our study, trans-anethole attenuated chronic inflammation by reducing the concentrations of IL-6 and TNF-a in BAL fluid. Inflammatory markers, such as IL-6, IL-8, and TNF-a, are increased in patients with COPD [31]. TNF-a level has been reported to increase in acute and chronic inflammatory diseases [32]. In contrast, IL-6 may be more important in chronic lung diseases [33], as increased IL-6 levels have been associated with COPD exacerbation [31,34]. The anti-inflammatory activity of transanethole has been reported to involve its effects on proinflammatory mediators including TNF-a, MMP-9, and NO, along with IL-6 in acute lung inflammation [1]. Therefore, its reduction in IL-6 may be essential for the anti-inflammatory effects of transanethole in chronic lung inflammation. We also found that trans-anethole decreased blood pressure in this COPD model. Trans-anethole has been reported to suppress the increase of store-operated Ca2+ entry-induced Ca2+ levels in vascular endothelium [35]. Foeniculum vulgare essential oil and its major constituent, anethole, have been reported to have vasorelaxant effects [9]. Despite having antihypertensive effects, it remains unclear whether trans-anethole has direct vasoactive effects or whether its vasoactive properties are secondary to its anti-inflammatory action. 5. Conclusions trans-anethole recovered chronic lung inflammation by reducing the concentrations of the pro-inflammatory cytokines TNF-a and IL-6 in BAL fluid, as well as reducing blood pressure, in a mouse model of LPS/PPE-induced COPD. The present results suggest that trans-anethole may be a potential therapeutic agent for prophylaxis and treatment in patients with chronic lung inflammation. Conflict of interest The authors declare they have no conflicts of interest. Acknowledgements This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korea government (MEST) (2016R1D1A1B03931081).
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Anti-inflammatory effects of trans-anethole in a mouse model of chronic obstructive pulmonary disease Ka Young Kima,b,1, Hui Su Leea,1, Geun Hee Seola,* a b
Department of Basic Nursing Science, School of Nursing, Korea University, Seoul 02841, Republic of Korea Department of Nursing, College of Nursing, Gachon University, Incheon 21936, Republic of Korea
A R T I C L E I N F O
Article history: Received 13 March 2017 Received in revised form 6 May 2017 Accepted 6 May 2017 Keywords: Trans-anethole Chronic obstructive pulmonary disease (COPD) Interleukin-6 (IL-6) Blood pressure Porcine pancreatic elastase (PPE) Lipopolysaccharide (LPS)
A B S T R A C T
Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease that is generally characterized by progressive and irreversible airflow obstruction and alveolar destruction. Long-term treatment with current medications has been associated with various adverse effects, indicating a need for alternative approaches for the prevention and treatment of COPD. This study investigated the mechanism underlying the effects of trans-anethole in a mouse model of COPD induced by porcine pancreatic elastase (PPE) and lipopolysaccharide (LPS). BALB/c mice were orally administered transanethole (62.5, 125, 250, or 500 mg/kg) 2 h before intranasal challenge with 1.2 units of PPE and 7 mg of LPS. Lactate dehydrogenase (LDH) activity, cell counts, lung histology, cytokine production, and blood pressure were analyzed. Trans-anethole reduced LDH activity and inflammatory cell counts, including macrophage, neutrophil, and lymphocyte counts. trans-anethole 125 mg/kg restored the histopathological changes induced in mouse lungs by PPE and LPS. trans-anethole reduced the serum concentrations of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a), as well as significantly reducing blood pressure during chronic inflammation. Trans-anethole ameliorated chronic lung inflammation in a mouse model of COPD by reducing the serum concentrations of proinflammatory cytokines such as TNF-a and IL-6, and by reducing blood pressure. The present results indicate that trans-anethole may be a potential therapeutic agent for prophylaxis and treatment in patients with chronic lung inflammation. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Trans-anethole, the major constituent obtained from anise, star anise, and fennel, has been reported to have anti-inflammatory [1,2], antioxidant [3,4], anticarcinogenic [5,6], neuroprotective [7] and vasoactive effects [8]. Anethole showed nitric oxide (NO)independent vasodilation in KCl-induced vasospasm of isolated rat aorta rings [9]. Investigations in a mouse model of lipopolysaccharide (LPS)-induced acute lung injury have indicated that the anti-inflammatory properties of anethole are mediated by its reduction of pro-inflammatory mediators, including tumor necrosis factor-alpha (TNF-a), metalloproteinase-9 (MMP-9), and NO, and its inhibition of nuclear factor-kappa B (NF-kB) [1]. Moreover, a randomized clinical trial showed that anethole dithiolethione
* Corresponding author at: Department of Basic Nursing Science, School of Nursing, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea. E-mail address: [email protected] (G.H. Seol). 1 KYK and HSL contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2017.05.032 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
decreased new dysplastic lesions in smokers with bronchial dysplasia [10]. To date, however, the mechanisms underlying the anti-inflammatory effects of anethole in chronic obstructive pulmonary disease (COPD) remain undetermined. COPD is a chronic inflammatory lung disease that is generally characterized by progressive and irreversible airflow obstruction and alveolar destruction [11]. Inflammation in COPD causes an accumulation of inflammatory cells, including macrophages, neutrophils, and lymphocytes, and the release of reactive oxygen species (ROS), cytokines, chemokines, and elastase [11]. COPD can develop in patients with cardiovascular diseases, including pulmonary hypertension, as damage to the lungs can reduce the amount of oxygen to the blood [12,13]. Although COPD is associated with early mortality, high rates of morbidity and mortality, and high treatment costs, clinical studies are limited [14]. Mouse models can effectively reflect the pathophysiology of human COPD [15]. The tuber of Alismataceae Alisma orientale Juzepzuk, a constituent of herbal medicine, has been reported to induce histological changes in a COPD mouse model [16]. Longterm treatment with currently available medications has been
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associated with serious side-effects in patients with COPD, indicating a need for alternative approaches to prevent and treat this chronic condition. Treatment of mice with LPS and the endopeptidase porcine pancreatic elastase (PPE) has been found to induce COPD [17,18]. Pathological changes observed in these mice include increased airway inflammation, activation of immune cells, and alveolar wall destruction [19–21]. This model mimics many of the crucial pathological features of human COPD [22]. Inhalation of the traditional Chinese medicine chung-pae, which is composed of four herbs, was found to effectively reduce the chronic inflammation and pathological changes in LPS/PPE-induced COPD in mice [18]. Similarly, the medicinal plant Gamijinhae-gang showed antiinflammatory activity in a mouse model of COPD induced by PPE and LPS [20]. Trans-anethole is a major constituent of these traditional medicines. This study therefore analyzed the antiinflammatory effects of trans-anethole in a mouse model of LPS/ PPE-induced COPD.
anesthetized by intraperitoneal injection of a mixture of 0.3 mg/ kg tiletamine-zolazepam (Zoletil 50, Virbac Laboratories, Carros, France) and 0.2 mg/kg xylazine (Rompun, Bayer Korea, Ansan, Korea). Bronchoalveolar lavage (BAL) fluid was collected three times from each mouse through a tracheal cannula with sterile PBS.
2. Materials and methods
2.4. Inflammatory cell counts
2.1. Reagents
BAL fluid samples were centrifuged at 500g for 10 min at 4 C, and the sedimented cells were resuspended in PBS. Total cell numbers and types of inflammatory cells were determined on a Countess automated cell counter (Invitrogen Life Technologies, Carlsbad. CA, USA) using Diff-Quik stain (International Reagents Co., Kobe, Japan). The results are expressed as the number of each cell type per milliliter of BAL fluid.
LPS from Escherichia coli 055:B5, trans-anethole, and dexamethasone were purchased form Sigma Aldrich Co. (St Louis, MO, USA). PPE was obtained from Elastin Products Company (Owensville, MO, USA).
2.3. Lactate dehydrogenase assay (LDH) The activity of lactate dehydrogenase (LDH), a marker for cytotoxicity, in acellular BAL fluid was measured using a cytotoxicity detection kit according to the manufacturer’s instructions (Roche Applied Science, Mannheim, Germany). BAL fluid samples were mixed 1:1 with freshly prepared reaction mixture and incubated in the dark for 30 min at room temperature. The absorbance of each reaction mixture was measured at 490 nm and at a reference wavelength of 620 nm using a microplate reader (BMG Labtech, Ortenberg, Germany).
2.2. Animals All animals were treated according to National Institutes of Health guidelines for the use of experimental animals. The study protocol was approved by the institutional animal care and use committee of Korea University (KUIACUC-2016-153), and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23; revised 1996). Specific pathogen-free male BALB/c mice (5 weeks old), weighing 19–21 g were obtained from Orient Bio Inc. (Seongnam, Korea). A total of 80 mice were randomly divided into 9 groups of 5–12 mice each, and treated with PPE and LPS (Fig. 1). Briefly, mice were orally administered vehicle (1% Tween 80saline), trans-anethole (62.5, 125, 250, or 500 mg/kg), or dexamethasone (3 mg/kg) 2 h before anesthetization with isoflurane and intranasal administration of PPE (1.2 units on day 1 of each of 4 weeks) or LPS (7 mg of LPS on day 4 of each of 4 weeks); control mice were intranasally challenged with phosphate-buffered saline (PBS) [21,23]. On day 32, systolic (sBP) and diastolic blood pressure (dBP) were measured using a tail cuff and pulse transducer (AD Instruments, Sydney, Australia), with the results reported as the means of five data points. The mice were subsequently
BP, BAL fluid 3d
1d
4d
3d
1 week
4d
3d
2 weeks
4d
3d
4d
3 weeks
2.5. Histopathological analysis Lung tissue from 5–6 mice per group were fixed overnight in 10% paraformaldehyde and embedded in paraffin. Paraffin blocks were sectioned at a thickness of 4 mm. Sections were stained with hematoxylin and eosin (H&E) and four sections from each mouse examined using an Axioplan 2 microscope (Carl Zeiss, Göttingen, Germany). The alveolar diameter of each airspace was calculated using Image J software. 2.6. Enzyme-linked immunosorbent assay (ELISA) The concentrations of the cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) in BAL fluid were measured using ELISA kits, according to the manufacturer’s instructions (Koma Biotech, Seoul, Korea). 2.7. Statistical analysis All statistical analyses were performed using IBM SPSS Statistics 23.0 software (SPSS Inc., Chicago, IL, USA). Normality was analyzed using the Shapiro-Wilk test and the data were compared using one-way analysis of variance (ANOVA), followed by the Tukey’s LSD post hoc test. The results were considered significant at p < 0.05.
4 weeks 32d
3. Results ANE ANE ANE ANE ANE ANE ANE ANE PPE PPE PPE PPE LPS LPS LPS LPS Fig. 1. Schematic diagram of the study protocol. Mice were administered PPE on day 1 and LPS on day 4 each week for 4 weeks, with inhaled trans-anethole administered 2 h before the administration PPE or LPS. On day 32, BP was measured and BAL fluid was collected. Abbreviations: ANE, transanethole; PPE, porcine pancreatic elastase; LPS, lipopolysaccharide; BP, blood pressure; BAL fluid, bronchoalveolar lavage fluid.
3.1. Effect of trans-anethole on LDH activity in BAL fluid of PPE/LPSinduced COPD mice LDH activity in BAL fluid was 3.10-fold higher in PPE/LPStreated than in control mice (p < 0.001) (Fig. 2). Compared with PPE/LPS-treated animals, significant decreases in LDH activity were observed in animals pretreated with 62.5 mg/kg (2.03-fold,
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p = 0.006), 125 mg/kg (2.72-fold, p = 0.001), 250 mg/kg (2.18-fold, p = 0.001), and 500 mg/kg (2.68-fold, p < 0.001) trans-anethole, as well as dexamethasone (1.99-fold, p = 0.004). 3.2. Effect of trans-anethole on inflammatory cell counts in PPE/LPStreated mice
Fig. 2. Effect of trans-anethole on LDH activity in BAL fluid of PPE/LPS-induced COPD mice. LDH activity was analyzed in BAL fluid of PPE/LPS-induced COPD mice. Data represent mean SEM (n = 5–12 per group). ### P < 0.001 compared with the control group; ** P < 0.01, *** P < 0.001 compared with the COPD group. Abbreviations: ANE, trans-anethole; DEXA, dexamethasone.
The pathogenesis of COPD involves the recruitment of inflammatory cells, including macrophages, neutrophils, and lymphocytes, which are associated with chronic inflammation of the airways. The numbers of total cells in BAL fluid were 2.23-fold higher in PPE/LPStreated than in control mice (p < 0.001; Fig. 3D), as were the numbers of macrophages (2.55-fold, p < 0.001; Fig. 3A), neutrophils (2.59fold, p < 0.001; Fig. 3B), and lymphocytes (1.94-fold, p = 0.006; Fig. 3C). Pretreatment of PPE/LPS-treated mice with 62.5, 125, 250, and 500 mg/kg trans-anethole reduced macrophages by 1.59-fold (p = 0.001), 1.37-fold (p = 0.014), 1.88-fold (p < 0.001), and 1.19-fold (p = 0.173), respectively; neutrophils by 1.50-fold (p = 0.003), 2.33fold (p < 0.001), 3.03-fold (p < 0.001), and 1.36-fold (p = 0.034), respectively; lymphocytes by 1.45-fold (p = 0.086), 3.06-fold (p = 0.001), 2.63-fold (p < 0.001), and 1.66-fold (p = 0.051), respectively; and total cells by 1.56-fold (p < 0.001), 1.69-fold (p < 0.001), 2.21-fold (p < 0.001), and 1.28-fold (p = 0.042), respectively.
Fig. 3. Effect of trans-anethole on inflammatory cell counts in PPE/LPS-treated mice. The numbers of (A) macrophages, (B) neutrophils, (C) lymphocytes, and (D) total cells were measured in BAL fluid. Data represent means SEMs (n = 5–12 per group). # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the control group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the COPD group. Abbreviations: ANE, trans-anethole; DEXA, dexamethasone.
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Fig. 4. Effect of trans-anethole on lung histopathology of PPE/LPS-induced mouse model. Mouse lung tissues were stained with (A-D) hematoxylin and eosin (H&E) and alveolar diameters were analyzed by Image J software (E) (200). Data represent means SEMs (n = 5 6 per group). # P < 0.05, ### P < 0.001 compared with the control group; *** P < 0.001 compared with the COPD group. Abbreviations: ANE, trans-anethole; DEXA, dexamethasone.
3.3. Effect of trans-anethole on lung histopathology of PPE/LPSinduced mouse model Histologic examination of the lungs of PPE/LPS-treated mice (Fig. 4B) showed alveolar destruction and airspace enlargement when compared with control mice (Fig. 4A). By contrast, treatment with trans-anethole 125 mg/kg (Fig. 4C) and dexamethasone (Fig. 4D) resulted in recovery from PPE/LPS-induced lung inflammation. Quantified alveolar diameter was analyzed statistically to confirm the chronic inflammation (Fig. 4E). 3.4. Effect of trans-anethole on IL-6 and TNF-a in BAL fluid of mice with PPE/LPS-induced chronic inflammation Proinflammatory cytokines such as IL-6 (Fig. 5A) and TNF-a (Fig. 5B) are secreted by inflammatory cells. Compared with control mice, mice treated with PPE/LPS showed increased expression of IL-6 (2.76-fold, p < 0.001) and TNF-a (2.44-fold, p < 0.001). Pretreatment of PPE/LPS-treated mice with 62.5, 125,
and 250 mg/kg trans-anethole significantly reduced IL-6 concentrations in BAL fluid by 1.74-fold (p = 0.004), 1.65-fold (p = 0.019), and 1.87-fold (p = 0.001), respectively, whereas 500 mg/kg transanethole had no effect on IL-6 concentration. Pretreatment of PPE/ LPS-treated mice with 62.5, 125, 250, and 500 mg/kg transanethole significantly reduced TNF-a concentrations by 1.34-fold (p = 0.036), 2.75-fold (p < 0.001), 2.14-fold (p < 0.001), and 2.76fold (p < 0.001), respectively. 3.5. Effect of trans-anethole on BP in PPE/LPS-exposed mice Both sBP (1.17-fold, p = 0.002; Fig. 6A) and dBP (1.20-fold, p = 0.001; Fig. 6B) were significantly higher in PPE/LPS-treated than in control mice. Pretreatment of PPE/LPS-treated mice with 62.5, 125, 250, and 500 mg/kg trans-anethole significantly reduced sBP by 1.22-fold (p < 0.001), 1.16-fold (p = 0.003), 1.10-fold (p = 0.033), and 1.23-fold (p < 0.001), respectively; and dBP by 1.19-fold (p = 0.003), 1.15-fold (p = 0.009), 1.12-fold (p = 0.030), and 1.22-fold (p = 0.001), respectively.
Fig. 5. Effect of trans-anethole on IL-6 and TNF-a in BAL fluid of mice with PPE/LPS-induced chronic inflammation. The concentrations of (A) IL-6 and (B) TNF-a in BAL fluid were analyzed by ELISA. Data represent means SEMs (n = 5 12 per group). ## P < 0.01, ### P < 0.001 compared with the control group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the COPD group. Abbreviations: ANE, trans-anethole; DEXA, dexamethasone.
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Fig. 6. Effect of trans-anethole on BP in PPE/LPS-exposed mice. (A) Systolic blood pressure (sBP) and (B) diastolic blood pressure (dBP) were measured using a tail cuff and pulse transducer. ## P < 0.01 compared with the control group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the COPD group. Abbreviations: ANE, trans-anethole; DEXA, dexamethasone.
4. Discussion The present results show that trans-anethole ameliorated chronic inflammation, including cytotoxicity, increases in proinflammatory cells, and lung damage, induced by PPE and LPS in a mouse model of COPD by reducing the concentrations of proinflammatory cytokines such as TNF-a and IL-6. Lung histopathology showed alveolar destruction and airspace enlargement. Furthermore, trans-anethole significantly decreased the increased blood pressure associated with chronic inflammation. COPD, a major cause of morbidity and mortality worldwide, is usually associated with an abnormal and progressive inflammatory response of lungs to harmful gases and particles, including cigarette smoke and environmental pollutants [12,15]. Analysis of lung tissue and airway sections from patients with COPD has shown increases in both the numbers and activation of inflammatory cells, with an increased number of macrophages particularly associated with COPD severity [24]. Most patients with COPD have a reduced health-related quality of life due to various signs and symptoms, including cough, dyspnea, wheezing, sputum production, and alteration in mental status [25]. Furthermore, COPD is associated with increased cardiovascular risk including pulmonary hypertension [13,26], with 5–10% of patients with advanced COPD reported to have severe pulmonary hypertension [26]. PPE-treated mice were found to have increased right ventricular pressure and norepinephrine concentrations, indicators of pulmonary hypertension [27]. This mouse model of PPE- and LPS-induced COPD may be useful for further study of both inflammation and cardiovascular sequelae associated with COPD. In particular, the present study found that trans-anethole effectively attenuated the increases in sBP and dBP induced by challenge with PPE and LPS. Pulmonary hypertension is a complication of advanced lung disease [13], as well as being negatively prognostic in patients with COPD [28]. The mouse model of PPE/LPS-induced COPD may be useful in assessing the pathology of COPD and the antiinflammatory and anti-hypertensive properties of various alternative therapies [15]. Trans-anethole has been reported to have anti-inflammatory properties [2]. In particular, it has been reported to be effective in the treatment of lung diseases, including lung inflammation [1] and lung cancer [10,29]. Anethole dithiolethione, a derivative of anethole, has been reported to reduce homocysteine levels associated with the pathogenesis of cardiovascular disease [30].
In our study, trans-anethole attenuated chronic inflammation by reducing the concentrations of IL-6 and TNF-a in BAL fluid. Inflammatory markers, such as IL-6, IL-8, and TNF-a, are increased in patients with COPD [31]. TNF-a level has been reported to increase in acute and chronic inflammatory diseases [32]. In contrast, IL-6 may be more important in chronic lung diseases [33], as increased IL-6 levels have been associated with COPD exacerbation [31,34]. The anti-inflammatory activity of transanethole has been reported to involve its effects on proinflammatory mediators including TNF-a, MMP-9, and NO, along with IL-6 in acute lung inflammation [1]. Therefore, its reduction in IL-6 may be essential for the anti-inflammatory effects of transanethole in chronic lung inflammation. We also found that trans-anethole decreased blood pressure in this COPD model. Trans-anethole has been reported to suppress the increase of store-operated Ca2+ entry-induced Ca2+ levels in vascular endothelium [35]. Foeniculum vulgare essential oil and its major constituent, anethole, have been reported to have vasorelaxant effects [9]. Despite having antihypertensive effects, it remains unclear whether trans-anethole has direct vasoactive effects or whether its vasoactive properties are secondary to its anti-inflammatory action. 5. Conclusions trans-anethole recovered chronic lung inflammation by reducing the concentrations of the pro-inflammatory cytokines TNF-a and IL-6 in BAL fluid, as well as reducing blood pressure, in a mouse model of LPS/PPE-induced COPD. The present results suggest that trans-anethole may be a potential therapeutic agent for prophylaxis and treatment in patients with chronic lung inflammation. Conflict of interest The authors declare they have no conflicts of interest. Acknowledgements This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korea government (MEST) (2016R1D1A1B03931081).
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