Submicron emulsion of cinnamaldehyde ameliorates ...

Biomedicine & Pharmacotherapy 102 (2018) 765–771

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Submicron emulsion of cinnamaldehyde ameliorates bleomycin-induced idiopathic pulmonary fibrosis via inhibition of inflammation, oxidative stress and epithelial-mesenchymal transition

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Li Yana, Fan Songa, Hua Lia, Yao Lic, Jie Lia, Qiao-Yan Hea, Di Zhanga, Fang Wanga, Meng Zhanga, ⁎ ⁎ Hang Zhaoa, Tian Fenga, Ying-Yong Zhaob, , Si-Wang Wanga, a

Department of Natural Medicine, School of Pharmacy, The Fourth Military Medical University, 169 Changle West Road, Xi’an, 710032, China Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, School of Life Science, Northwest University, No. 229 Taibai North Road, Xi’an, 710069, China c School of Pharmacy, Shaanxi University of Chinese Medicine, Century Road, Xianyang, 712000, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Cinnamaldehyde Pulmonary fibrosis Bleomycin Anti-inflammatory Epithelial-mesenchymal transition

Aims: Idiopathic pulmonary fibrosis (IPF) is the most frequent and severe form of idiopathic interstitial pneumonias. The pathogenesis is associated with inflammation and oxidative stress and epithelial-mesenchymal transition (EMT). Cinnamaldehyde exhibits antiinflammatory and antioxidant properties, but its effect on IPF is unknown. The present study is to investigate the anti-fibrotic effect and action mechanism of cinnamaldehyde on IPF. Materials and methods: IPF was induced by intratracheal bleomycin in mice. Submicron emulsion of cinnamaldehyde was given by intraperitoneal injection once everyday for 7 or 21 continuous days after bleomycin administration. Lung histological and injury indexes were analyzed. The protein expressions of inflammation and oxidative stress as well as EMT markers alpha-smooth muscle actin (α-SMA) and E-cadherin in mice and cultured A549 cells were measured. Results: Cinnamaldehyde attenuated the bleomycin-induced histological injury, reduced hydroxyproline level and improved pulmonary function by the inhibiting inflammatory cytokines and reactive oxygen species production as well as enhancing total superoxide dismutase activity in bleomycin-induced mice. Cinnamaldehyde also inhibited EMT in both bleomycin-induced mice and TGF-β1-stimulated A549 cells. Conclusions: Cinnamaldehyde ameliorated bleomycin-induced IPF via inhibition of inflammation and oxidative stress and EMT.

1. Introduction Fibrosis can be defined as the excessive accumulation of extracellular matrix (ECM) particularly fibrillar collagens. Fibrosis is a vital factor of progressive organ dysfunction and damage in various inflammatory and metabolic-associated diseases, such as pulmonary fibrosis, advanced kidney disease and advanced liver disease [1–8]. Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal interstitial lung disease. The pathogenesis of IPF is still unknown but thought to be associated with excessive inflammation, oxidative stress and abnormal epithelial-mesenchymal transition (EMT) [9,10]. There have been no fundamental breakthroughs in drug treatment for IPF. Lung transplantation remains the viable therapeutic option for patients with IPF [11]. Despite many drugs have been applied to treat IPF,

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accumulated evidence demonstrated their intervention is not completely effective [12]. The anti-fibrotic agents of pirfenidone and nintedanib were approved for treatment of IPF in 2014, but they received only a conditional recommendation for use and neither one had a clear advantage on mortality outcomes [12]. Methylprednisolone could partly reduce IPF induced by bleomycin in animal model, but the main disadvantage is its strong immunity inhibition. Hence, there is a pressing need to develop novel therapeutic agents with minimal side effect for prevention and treatment of IPF. Cinnamaldehyde is a plant secondary metabolite isolated from the stem bark of Cinnamomum trees [13]. Previous studies indicated that cinnamaldehyde exhibited a wide range of biological activities including anti-tumor, anti-bacterial and anti-mutagenic properties [14,15]. Our previous studies demonstrated that cinnamaldehyde

Corresponding authors. E-mail addresses: [email protected] (Y.-Y. Zhao), [email protected] (S.-W. Wang).

https://doi.org/10.1016/j.biopha.2018.03.145 Received 22 December 2017; Received in revised form 21 March 2018; Accepted 23 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.


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Fig. 1. Effect of SME-CA on the bleomycin-induced pulmonary fibrosis. Histopathological evaluation of the doses of SME-CA in bleomycin-induced pulmonary fibrosis mice. Sections of pulmonary tissue were subjected to H&E staining on day 7 (A and D) and Masson trichrome staining on day 21 (B and E). Hydroxyproline (HYP) levels in lungs on day 21 (C and F).*P < 0.05 vs Control; #P < 0.05 vs BLM. Table 1 Lung-to-body weight ratio in the bleomycin-induced mice.

7d 21d

Control

SME-CA

BLM

BLM + emulsion

BLM+SME-CA

BLM + methylprednisolone

0.69 ± 0.03 0.70 ± 0.04

0.68 ± 0.03# 0.73 ± 0.03#

1.53 ± 0.10* 1.17 ± 0.09*

1.49 ± 0.11* 1.15 ± 0.11*

1.10 ± 0.12# 0.90 ± 0.14#

1.06 ± 0.09# 1.12 ± 0.12

The wet lung-to-body weight ratio is an indicator of lung inflammation. We found that the bleomycin-treated mice increased lung-to-body weight ratios, which could be ameliorated by SME-CA. Each value represents the Mean ± SD. N = 6 mice per group. * P < 0.05 vs Control. # P < 0.05 vs BLM.

horseradish peroxidase and GAPDH were purchased from Solarbio Biological Technology Company. Tumor necrosis factor α (TNF-α), Interleukin-1β (IL-1β), Malondialdehyde (MDA) and total superoxide dismutase (T-SOD) test kits were purchased from Wuhan Boshide Biological Technology Company. A549 cells were purchased from American Type Culture Collection. Transforming growth factor-β1 (TGF-β1) recombinant was obtained from Peprotech EC. Rabbit polyclonal anti-TGF-β1,α-SMA and E-cadherin antibody was obtained from Abcam (Cambridge, UK). ECL-Kit was purchased from Millipore. SMECA was prepared as described previously [20].

showed protective effects on viral myocarditis, myocardial ischemia, cardiac inflammation and fibrosis [16–19]. However, the therapeutic effect of cinnamaldehyde on IPF is not completely clear. Because cinnamaldehyde is easily oxidized to cinnamic acid and unstable in blood, recently we developed the submicron emulsion of cinnamaldehyde to promote its application. The present study is to investigate the effect of submicron emulsion of cinnamaldehyde (SME-CA) against IPF in bleomycin-induced mice and its underlying mechanism.

2. Materials and methods 2.1. Materials

2.2. Animal experiments

Bleomycin was purchased from Nippon Kayaku. Cinnamaldehyde (99.0% purity) was purchased from Yuancheng Pharmaceutical Co., Ltd. Diff-Quick reagents, anti-rabbit or anti-rat conjugated to

Mice were randomly divided into eight groups (n = 12/group): 1) saline control (Control); 2) saline + SME-CA (SME-CA); 3) bleomycin model (BLM); 4) bleomycin + blank emulsion (BLM + emulsion); 5) 766


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Fig. 2. Effect of SME-CA on the lung mechanic indexes and inflammatory cells in BALF. The lung mechanics of methylprednisolone or SME-CA treatment in bleomycin-induced pulmonary fibrosis mice. Airway elastic resistance (A), respiratory system dynamic compliance (B), static compliance (C), the number of inflammatory cells including total cells (D), alveolar macrophages (E) and neutrophils (F) in BALF were determined.*P < 0.05 vs Control; #P < 0.05 vs BLM.

bleomycin + 70 mg/kg SME-CA (70 mg/kg SME-CA); 6) bleomycin + 35 mg/kg SME-CA (BLM + SME-CA, also called 35 mg/kg SME-CA); 7) bleomycin + 17.5 mg/kg SME-CA (17.5 mg/kg SME-CA) and 8) bleomycin + methylprednisolone (BLM + methylprednisolone). The mice were treated with 4 mg/kg bleomycin under anesthesia with sodium pentobarbital as described previously [21]. Normal saline (20 mL/kg), blank emulsion (20 mL/kg), SME-CA (35 mg/kg) or methylprednisolone (10 mg/kg) was given by intraperitoneal injection once everyday for 7 or 21 continuous days after bleomycin administration.

hydroxyproline assay according to the manufacturer’s instructions. The levels of MDA and T-SOD were measured using an RT-9600 Semi-automatic Biochemical Analyzer. The levels of TNF-α and IL-1β were measured by enzyme linked immunosorbent assay. Reactive oxygen reagent kit was used to detect ROS content as instruction described. 2,7Dichlorofluorescin diacetate was used as a probe for the detection of ROS. The fluorescence intensity reflecting the level of ROS was detected by fluorescence microplate reader.

2.3. Measurement of lung mechanic indexes

Immunofluorescence staining was performed as described previously [27,28]. A549 cells were cultured on coverslip and treated as indicated. The cells were fixed with fresh 4% paraformaldehyde solution, and then blocked by normal goat serum. The cells were stained with anti-α-SMA and E-cadhrein antibody. Cell images were obtained by the laser-scanning confocal microscope (FV1000, Olympus, Japan).

2.7. Immunofluorescence staining

Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg/kg) to suppress spontaneous breathing. After a tracheostomy, the mice were connected to the flexiVent system. The computer-controlled small animal ventilator was used to substitute for autonomous respiration [22]. Lung mechanic indexes including airway resistance, airway elastic resistance, respiratory system dynamic compliance and static compliance were measured at the end of nebulization.

2.8. Western blot analysis The protein concentration of each sample was assayed by using Bicinchoninic Acid protein assay. Protein expression was performed by Western blot analysis as described previously [29,30]. The blot was developed using the enhanced chemiluminescence method by ECL-Kit according to the manufacturer’s instructions. Each band density was normalized by GAPDH expression level.

2.4. Histopathological analysis Hematoxylin and eosin (H&E) and Masson's trichrome staining were performed as described previously [23–26]. The lung tissues were fixed in 4% paraformaldehyde for 24 h. Sections (5 μm) were stained with H& E and Masson’s trichrome staining. All pathological sections were examined by light microscopy at 200× magnification.

2.9. Electron microscopic analysis For ultrastructural studies, on day 21 the lung samples were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 18 h. The lungs were dissected into small pieces and fixed for 1.5 h in 1% osmium tetroxide dissolved in 0.1 M phosphate buffer (pH 7.4), then dehydrated through a series of graded ethanol solutions and embedded in araldite. Ultrathin sections were cut, stained with uranyl acetate and lead nitrate, mounted on copper grids and examined under a transmission electron microscope.

2.5. Counting of cells in bronchoalveolar lavage fluid (BALF) At day 7, BALF was collected as described previously [21]. The left lung rather than the entire lung was irrigated. Total cell number was counted using a hemocytometer. Cells were stained with Diff-Quick reagents, and the number of alveolar macrophages, neutrophils, and lymphocytes were counted. 2.6. Quantitative analyses of hydroxyproline, IL-1β, TNF-α, ROS, MDA and T-SOD

2.10. Statistical analysis Data were expressed as mean ± SD. Statistical comparisons were performed by using one-way ANOVA with a Tukey’s multiple

The same region of the right lung lobes were collected for 767


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Fig. 3. Effect of SME-CA on the inflammatory cytokines and oxidative stress in lung. The indicators reflecting inflammation and oxidative stress including IL-1β (A), TNF-α (B), ROS (C), MDA (D) and T-SOD (E) in lung tissue on day 7.*P < 0.05 vs Control; #P < 0.05 vs BLM.

comparison test. Analyses were conducted with GraphPad Prism 5. A value of P < 0.05 was considered to be statistically significant.

Bleomycin significantly elevated hydroxyproline content and SME-CA significantly reduced the bleomycin-induced IPF and hydroxyproline content in lung tissues (Fig. 1C and F). Bleomycin significantly elevated the lung-to-body weight ratios on day 7 and day 21. The effect of bleomycin could be reduced by treatment with SME-CA and methylprednisolone. There was still significantly difference between the SMECA-treated mice and the IPF mice on day 21. However, there was no significant difference between the methylprednisolone-treated mice and the IPF mice on day 21 (Table 1).

3. Results 3.1. SME-CA prevented bleomycin-induced IPF in mice The lung tissue of H&E staining (Fig. 1A and D) and Masson’s trichrome staining (Fig. 1B and E) exhibited bleomycin-induced the prominent distortion of the alveolar architecture and thickening of interalveolar septa. Treatment with SME-CA significantly ameliorated lung injury, especially for 35 mg/kg dosage. Methylprednisolone showed weak effects on the bleomycin-induced histopathological changes.

3.2. SME-CA improved the lung mechanic indexes To explore the effect of SME-CA on the respiratory function of 768


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Fig. 4. Effect of SME-CA on the TGF-β1-induced EMT in the A549 cells. (A) Immunofluorescence staining of E-cadherin and α-SMA in the TGF-β1-stimulated A549 cells for 6, 12 and 24 h. Representative Western blot and quantitative analyses for E-cadherin (B) and α-SMA (C) in the A549 cells.*P < 0.05 vs Control; #P < 0.05 vs BLM.

3.4. Effects of SME-CA on the expressions of EMT in the bleomycin-induced IPF mice and cultured A549 cells

bleomycin-induced IPF mice, we measured the lung mechanic indexes. As shown in Fig. 2A–C, Bleomycin significantly elevated the airway elastic resistance but reduced the respiratory system dynamic adaptation and the static compliance. SME-CA partly improved bleomycininduced lung mechanic indexes, which indicated improvement of the respiratory function. Methylprednisolone had no significant effects on these indexes.

We determined the effects of SME-CA on the expressions of EMTassociated protein expression in both bleomycin-induced IPF mice and TGF-β1-induced A549 cells. TGF-β1 induced EMT in A549 cells (Fig. 4A). After TGF-β1 stimulation in the A549 cells, α-SMA expression was significantly increased in the epithelial cells. In cultured A549 cells, SEM-CA reversed the decreased E-cadherin expression and increased αSMA expression (Fig. 4B, C). The expressions of TGF-β1 and α-SMA were significantly up-regulated in the bleomycin-induced IPF mice. SEM-CA almost reversed the bleomycin-induced TGF-β1 and α-SMA expressions (Fig. 5A, B).

3.3. Effects of SME-CA on the inflammation and oxidative stress in BALF and bleomycin-induced IPF mice We measured the inflammatory cells in BALF and lung tissue. Bleomycin induced a significant increase in total cells (Fig. 2D), alveolar macrophages (Fig. 2E) and neutrophils (Fig. 2F) on day 7 in the BALF. Both SME-CA and methylprednisolone significantly reduced the total cells, alveolar macrophages and neutrophils in BALF. Bleomycin significantly elevated the inflammatory cytokines IL-1β and TNF-α, the oxidation products MDA and ROS, but reduced the antioxidant enzyme T-SOD in lung tissue. However, SME-CA significantly reduced these changes. The methylprednisolone effect was similar to those of SME-CA (Fig. 3).

3.5. Tracheal organ culture IL-1β must combine with IL-1RI to exert its effect. As shown in Fig. 5C, the level of IL-1RI in BLM group was significantly higher than that in the control group. Compared with the model group, the levels of IL-1RI in SME-CA and methylprednisolone group were significantly decreased. 769


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Fig. 5. Effect of SME-CA on the expressions of TGF-β1, α-SMA, IL-1RI and electron microscopic analysis in mice. Representative western blot and quantitative analyses for TGF-β1 (A), α-SMA (B) and IL-1RI (C) in tissue samples. GAPDH was used as control.*P < 0.05 vs Control; #P < 0.05 vs BLM. Sections of pulmonary tissue were subjected to fiber deposits (D) and destroyed mitochondria inspection (E).

mediated TLR 4/6-IRAK 4/1 signaling to suppress inflammasome activation [19]. In cultured murine J774 A.1 macrophages, cinnamaldehyde inhibited the lipopolysaccharide-stimulated production of proinflammatory cytokines via reduction of ROS release and JNK1/2 and ERK1/2 activation [39]. Compared with the bleomycin-induced mice, SME-CA reduced the total cells, the neutrophils and alveolar macrophages in BALF as well as reduced the inflammation cytokine production including IL-1β, TNF-α, ROS and MDA levels, and elevated T-SOD activity in lung. These results are consistent with the previous studies. Oscillatory mechanics of lung is correlated with the ECM in lung parenchyma of bleomycin-induced fibrosis [40]. In the present study, increased airway elastic resistance, decreased respiratory system dynamic compliance and static compliance indicated that the lungs became "hard" in bleomycin-induced fibrotic mice. SME-CA significantly inhibited these bleomycin-induced alternations of lung mechanic indexes, which are consistent with the results of histopathological staining and hydroxyproline measurement. In IPF, the aberrant EMT is one of the determinants of fibroitc formation. The trans-differentiated myofibroblasts share the characters with both fibroblasts and smooth muscle cells [41]. Excessive EMT ultimately leads to the formation of fibroblastic foci, the abnormal remodeling of the ECM and the subsequent destruction of the lung architecture. TGF-β1 is a major mediator of EMT in many organ fibrosis [42–45]. It has been reported that cinnamaldehyde reversed CoCl2-induced EMT and decreased matrix metalloprotease family through inhibition of Wnt/β-catenin pathway in non-small cell lung cancer cell line [46]. SME-CA significantly reduced the bleomycin-induced overexpressions of TGF-β1 and α-SMA in lungs. Furthermore, in vitro experiment, we confirmed these results in the A549 cells. Therefore, SMECA protects against bleomycin-induced IPF through inhibition of EMT. In summary, the present study firstly demonstrated that SME-CA

3.6. Electron microscopic analysis A large number of fiber depositions are an important feature of IPF. Fiber deposition in bleomycin-induced IPF mice was higher than that of the control group, and the fiber deposition in the SME-CA group was significantly reduced compared with the model group (Fig. 5D). In addition, our study showed that the number of damaged mitochondria in bleomycin-induced IPF mice was higher than that of the control group. SME-CA treatment could ameliorate damaged mitochondria (Fig. 5E). 4. Discussion Accumulated evidence has shown that inflammation and oxidative stress play a key role in the pathogenesis of tissue fibrosis particularly for the fibrosis of pulmonary, liver and kidney tissues [31–36]. Excessive ROS generation activates cytokines and transcription factors. This causes the excessive ECM production. Therefore, inhibiting inflammation and oxidative stress could be considered as a potential therapeutic target in the treatment of fibrotic-associated diseases. The present study firstly demonstrated that SME-CA ameliorated bleomycin-induced IPF. The intervention effect of SME-CA was associated with the inhibition of inflammation and oxidative stress as well as EMT in lung. Both excessive inflammation and ROS play a key role in the onset or progression of IPF [37]. Study in patients with IPF demonstrated that the generation of ROS from alveolar inflammatory cells, such as neutrophils and macrophages, was enhanced and this may promote alveolar epithelial cell injury and induces chronic inflammation, thus initiates the development of pulmonary fibrosis [38]. Kang et al showed that cinnamaldehyde reduced fructose-induced cardiac inflammation and fibrosis by attenuating scavenger receptor CD36770


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ameliorated bleomycin-induced IPF in mice. SEM-CA protects against bleomycin-induced IPF via the inhibition of inflammation and oxidative stress as well as EMT. SME-CA may be a new therapeutic agent for IPF.

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