The effect of sesamin on airway fibrosis in vitro and in vivo
Ching-Huei Lin1,3, Mei-Lin Shen1,3, Shung-Te Kao1, *, and Dong Chuan Wu2,3,*
1 Graduate Institute of Chinese Medicine, China Medical University, Taichung, Taiwan
2 Graduate Institute of Clinical Medical Science, China Medicine University, Taichung, Taiwan 3 Translational Medicine Research Center, China Medical University Hospital, Taichung, Taiwan
*To whom correspondence should be addressed:
Dong Chuan Wu (dongchuanwu@mail.cmu.edu.tw) and Shung-Te Kao (stkao@mail.cmu.edu.tw)
Airway fibrosis, which is a crucial pathological condition occurring in various types of pulmonary disorders, is characterized by accumulation and activation of fibroblast cells, deposition of extracellular matrix (ECM) proteins, and increase of airway basement membrane. Transforming growth factor beta 1 (TGF-β1) is the principal profibrogenic cytokine that is responsible for fibrotic responses. In the present study, we aimed to investigate the antifibrotic effects of the natural polyphenolic compound, sesamin, on TGF-β1-induced fibroblast proliferation and activation, epithelial-mesenchymal transition (EMT), and ovalbumin (OVA)-induced airway fibrosis in vivo. We found that sesamin attenuated TGF-β1-(OVA)-induced proliferation of cultured lung fibroblasts. Sesamin inhibited TGF-β1-stimulated expression of alpha smooth muscle actin (α-SMA), suggesting that sesamin plays an inhibitory role in fibroblast activation. Sesamin blocked upregulation of the mesenchymal markers (fibronectin and vimentin) and downregulation of the epithelial marker (E-cadherin), indicating an inhibitory effect on TGF-β1-induced EMT in A549 cells. TGF-β1-TGF-β1-induced Smad3 phosphorylation was also significantly reduced by sesamin in both cultured fibroblasts and A549 cells. In the airway fibrosis induced by OVA in mice, sesamin inhibited accumulation of α-SMA-positive cells and expression of collagen I in the airway. Histological studies revealed that sesamin protected against subepithelial fibrosis by reducing myofibroblast activation and collagen accumulation in the ECM. OVA-induced thickening of basement membrane was significantly alleviated in animals receiving sesamin treatments. These results suggest a therapeutic potential of sesamin as an antifibrotic agent.
Sesamin; Airway; Fibroblast; TGF-β1; α-smooth muscle actin; epithelial-mesenchymal transition (EMT).
1. Introduction
Airway fibrosis is a fatal pathological condition of the respiratory system, which occurs in many pulmonary disorders, including inflammatory (such as chronic asthma) and noninflammatory pulmonary disorders (such as idiopathic pulmonary fibrosis) [1]. Common features of airway fibrosis involve increased numbers of fibroblasts and myofibroblasts and excessive accumulation of ECM proteins [2]. These pathological alterations remodel airway structure and impair pulmonary function, causing progressive dyspnea [3]. TGF-β1 is a key mediator in induction and augmentation of fibrosis [4]. By activating its major downstream factor Smad, TGF-β1 induces loss of epithelial phenotype, proliferation of fibroblasts/myofibroblasts, upregulation of myofibroblast markers, and remodeling of basement membrane [5, 6]. TGF-β1 activates fibroblasts to express mesenchymal markers like α-SMA and secrete collagen into ECM [7]. The extent of TGF-β1 expression and myofibroblasts activation is highly associated with the disease progression [8]. Antagonism of TGF-β1 substantially suppressed collagen deposition and subepithelial fibrosis [9]. Moreover, recent studies have shown that TGF-β1 can promote epithelial cells to transform into fibroblasts and myofibroblasts through EMT, which defines a form of cell plasticity in which epithelia gain mesenchymal phenotypes and continuously contribute to fibrosis formation [10, 11]. EMT occurs under conditions of airway inflammation and injury, allowing epithelial cells to acquire mesenchymal features, including morphologically transformation into spindle or stellate-like cells, enrichment of intracytoplasmic stress fibers, and changes in the expression of epithelial and mesenchymal markers [12]. Taken together, the
TGF-β1/Smad signalling plays a crucial role in myofibroblasts activation and EMT in airway fibrosis.
Despite acting as the principal culprit of fibrogenesis, TGF-β1 is widely expressed in many cell types throughout the healthy tissue [13]. TGF-β1 stimulates the synthesis and deposition of connective tissue and inhibits connective tissue breakdown. Knockdown of TGF-β1 caused abnormal immune responses and tissue necrosis, leading to juvenile death of mice [14]. This suggests that the basal expression levels of TGF-β1 are required for normal tissue integrity in alveoli and airways [15]. Pathological processes, however, stimulate production of copious amounts of TGF-β1 not only from inflammatory cells but also from structural cells, such as fibroblasts, endothelial cells, and smooth muscle cells [16]. The overproduced TGF-β1 is secreted and accumulates in ECM to initiate fibrogenesis by overactivating downstream factors [17].
Current treatment options for airway fibrosis are still limited. Therapies like glucocorticoids exhibit protective effects against airway inflammation, however, their impact on ameliorating fibrosis is uncertain [1, 18, 19]. It is at least partially because inhibition of inflammatory cell recruitment might not be sufficient to prevent TGF-β1 overproduction from other cell types. Therefore, it is reasonable to propose that drugs targeting not only inflammation but also TGF-β1-mediated responses offer better promise of fibrosis treatments.
Sesamin is a natural polyphenolic compound found in sesame oil as well as in certain plant species, such as Asarum sieboldii [20, 21]. It has been reported that sesamin has protective effects against hypertension, thrombophilia, neuroinflammation, and apoptotic cell death
[22-24]. The sesamin-enriched diet, sesame oil, has been shown to attenuates liver fibrosis [25]. Interestingly, our recent studies have found that sesamin has anti-inflammatory effects against TH2-type allergic responses in mice airway [26]. Nevertheless, it remains unclear if sesamin has antifibrotic effects on airway fibrosis by inhibiting downstream signallings of TGF-β1 activation. In light of this, we aim to systematically study antifibrotic effects of sesamin by using both in
vitro and in vivo models of TGF-β1-activated fibrotic responses, including fibroblast activation,
EMT, and ovalbumin (OVA)-induced airway fibrosis. We found that sesamin suppressed proliferation and activation of cultured primary fibroblasts, inhibited epithelial cells transdifferentiation into mesenchymal phenotype, and protected against airway fibrosis induced by OVA in mice, suggesting the therapeutic potential of sesamin as an antifibrotic agent.
2. Materials and Methods 2.1 Chemicals and reagents
TGF-β1 was purchased from Prospec-Tany Technogene Ltd (Rehovot, Israel). Sesamin (5,5’-(1S,3aR,4S,6aR)-tetrahydro-1H,3H-furo[3,4-c]furan-1,4-diylbis (1,3-benzodioxole); CAS No. 607-80-7; molecular weight 354.4; purity 98%) was purchased from Yuanye Biological Co., Ltd. (Beijing, China). Ovalbumin and PEG400 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-collagen I, phospho-smad2/3, vimentin, smooth muscle actin, and anti-fibronectin antibodies were purchased from Santa Cruz Biotechnology; anti-gapdh antibody was purchased from GeneTex. Pierce peroxidase detection kit used for immunohistochemical staining was purchased from Thermo Scientific. Trichrome Stain Kit (modified Masson's) was purchased from ScyTek Laboratories. Hematoxylin and eosin (H&E) stain was from Merck Millipore. For in vitro experiments, sesamin was dissolved in DMSO as a stock solution and the
stock was mixed with DMEM with a final DMSO concentration <0.5% to treat the cells. DMEM containing 0.5% DMSO was used as vehicle. For in vivo experiments, sesamin was prepared in DMSO and diluted by saline plus PEG400 (final DMSO concentration <0.5%). Saline plus PEG400 (Sigma-Aldrich) with 0.5% DMSO was used as a vehicle in the “vehicle group”. OVA for intraperitoneal (i.p.) injection was adsorbed to aluminium hydroxide adjuvant (Santa Cruz, Dallas, TX, USA) at a ratio of 50 µg to 2 mg in 200 µl PBS. OVA for intratracheal (i.t.) injection was dissolved in saline at a final concentration of 2.5 mg/ml.
2.2 Animals
All animals used in this study were obtained commercially from BioLasco co., Ltd., Taiwan. Animals were housed under controlled laboratory conditions with a 12-h dark-light cycle. Experimental protocols of the present study were evaluated and approved by the Institutional Animal Care and Use Committee of China Medical University according to Care of the animals and surgical procedures of China Medical University Protocols.
2.3 Primary fibroblasts and A549 cell cultures
Primary lung fibroblasts cells were isolated from 6 to 8 week old C57BL/6N mice as previously described [27]. After sacrifice, lungs tissue was washed with PBS buffer, digested with collagenase and 0.25% trypsin, and chopped. The cell suspension was filtered through 70 and 40 μm nylon mesh, respectively, and was centrifuged at 300 g for 5 min. The pellet was resuspended and maintained in Dulbecco’s modified eagle’s medium (DMEM, Gibco, Invitrogen, USA), which was supplemented with 10% fetal bovine serum and antibiotics (100 IU/ml of penicillin and 50 g/ml of streptomycin), under conditions of humidified 5% CO2/95%
O2 at 37 °C. The purity of isolated fibroblasts was verified by positive staining of α-SMA.
The human epithelial cell line A549 cells (ATCC, CCL-185) were maintained with DMEM supplemented with 10% fetal bovine serum and antibiotics (100 IU/ml of penicillin and 50 g/ml of streptomycin) under conditions of humidified 5% CO2/95% O2 at 37 °C. Confluent cultures were passaged by trypsinization. Before experiments, primary fibroblasts or A549 cells were washed twice with phosphate buffered saline (PBS) and were starved with low-serum medium containing 0.1% fetal bovine serum for 24 hours.
2.4 Trypan blue dye exclusion assay of cell viability
Primary fibroblasts cells were seeded onto 6-well plates at a density of 1×105 cells/well and were starved for 24h with low-serum medium followed by sesamin (10μM) and/or TGF-β1 (5μM) treatments for 72h. After treated for 72 hours, cells were harvested and were resuspended in 1 ml PBS. In trypan blue dye exclusion assay, 1 part of 0.4% trypan blue and 1 part cell suspension was mixed and a drop of the trypan blue/cell mixture was applied to a hemocytometer. The unstained cells were counted as viable cells.
2.5 Immunofluorescence stain of α-SMA
The Primary fibroblasts cells were seeded on coverslips, after treatment cells were fixed by 4% paraformaldehyde in PBS and were permeated with 1% TritonX-100/PBS for 15 min followed by washing twice with PBS. The coverslips were stained by using anti-α-SMA antibody overnight at 4 °C followed by FITC-conjugated anti-mouse IgG for 1 h. The coverslips were mounted on slides and were visualized using confocal laser microscopy system (LSM
510-META; Carl Zeiss, Germany) with a water immersion objective lens for 20X resolution.
2.6 OVA-induced airway fibrosis model
The pulmonary fibrosis model was established according to Hung et al [28]. Total of 60 BALB/c mice were divided into five groups (12 mice in each group), including the sham group, the vehicle group, and three sesamin groups (with different doses of sesamin treatment). The sham group received i.p. injections or i.t. instillation of saline instead of OVA at the corresponding time points when the other four groups received OVA immunization. The vehicle group and sesamin groups were OVA-stimulated animals. They received i.p. injection of 50 μg of OVA on day 0, 7, and 14. On day 21, 22, and 23 after the first immunization of OVA, these mice were further challenged with i.t. instillation of OVA (100 μg) after anesthetized with isoflurane. The OVA-stimulated mice were subjected to subsequent i.p. injection of vehicle (the vehicle group) or of 3 different dosages of sesamin (1mg/kg, 10 mg/kg, and 20 mg/kg body weight daily) from the 24th to the 29th day after the first immunization of OVA. All mice were sacrificed on day 30.
2.7 Histological analysis
Tissue of the left lung of mice receiving sham or OVA treatments was collected and was immediately fixed with 4% paraformaldehyde/PBS (wt/vole) at 4°C for 24h. The tissue was embedded in paraffin and cut into 5 μm sections, which was subject to the following staining procedures. The peribronchial myofibroblast were determined by immunohistochemistry stain with anti-α-SMA antibody and peroxidase-conjugate and DAB substrates as described previously [29]. α-SMA-positive cells showed deep brown color stain as indicated by the arrows (Fig. 5A). The positively stained tissue was outlined by the freehand selection tool and the area was measured by ImageJ 1.45. The total region of the basement was determined as a 20 μm band
immediately beneath the epithelium, which was outlined by drawing a line along the basal border of the airway epithelium and a second line 20 μm beneath the first line. The region between the two lines was outlined and thereby calculated as the total epithelium area by using ImageJ [30].The myofibroblast accumulation was calculated by percentage of the α-SMA-positive area in the total epithelium area and was graded according to the following criteria: 0 = 0-10% myofibroblast accumulation; 1 = 10-30% myofibroblast accumulation; 3 = 60-90% myofibroblast accumulation; and 4 = 90-100% myofibroblast accumulation.
To determine subepithelial fibrosis in the airway, Masson's trichrome staining was used to differentiate collagen from other fibers, yielding black stain of nuclei, red stain of cytoplasm, muscle, and erythrocytes, and blue stain of collagen. Masson's trichrome staining was applied using the manufacturer’s standard protocol. Visual grading according to Ashcroft's score was used to determine the severity of airway fibrosis [31]. Criteria for grading lung fibrosis were as follows: Grade 0 = normal lung; Grade 1 = minimal fibrous thickening of alveolar or bronchiolar walls; Grade 3 = moderate thickening of walls without obvious damage to lung architecture; Grade 5 = increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses; Grade 7 = severe distortion of structure and large fibrous areas; Grade 8 = total fibrous obliteration of lung fields. After examination of the whole sections, the mean score of all the fields was taken as the fibrosis score for each animal [32].
Basement membranes were examined by H&E staining and imaged under the objective 40x. Basement membrane was recognized as a well-defined band beneath the epithelium. Basement membrane thickness was measured in different regions of each sample with a minimum interval of 20 μm. The averaged thickness was calculated for each sample [33, 34]. Measurements of
thickness were shown as yellow lines perpendicular to the basement membrane in Fig. 7A.
2.8 Western Blotting
Cells or airway tissues were harvested and homogenized in RIPA lysis buffer containing proteinase inhibitors. The concentration of proteins was determined by the Bio-Rad protein assay kit (Bio-Rad Laboratories, CA, USA). 50 μg protein samples were loaded and separated by 10% SDS-polyacrylamide gel electrophoresis and were transferred to PVDF membranes (Amersham, Hybond-C Extra Supported, 0.45 Micro). The blotted membranes were blocked with 5% skim milk in TBS buffer (10mM Tris-base, 150 mM NaCl, 0.1% Tween 20) at room temperature for 1 hour and then probed with vimentin, fibronectin, p-Smad2/3, α-SMA, anti-collagen Ι, or anti-GAPDH antibodies at 4 °C overnight. The blots were then washed for 3 times with TBS buffer and incubated with appropriate horseradish peroxide-conjugated secondary antibodies at room temperature for 1 hour. The membranes were finally washed 3 times, and signals were developed using enhanced chemiluminescence (ECL) reagents (Millipore, Billerica, MA, USA) according the manufacturer’s instructions.
2.9 Statistical analysis
All results were presented as mean ± standard error of mean (S.E.M.). Statistical analysis was performed using the Prism statistical analysis program (GraphPad 6.0). Two-tailed student’s t-test was used for statistical analysis between two groups. One-way ANOVA was used for statistical analysis of multiple treatment doses. P<0.05 was considered significant for all test. Statistical significance was presented as * p< 0.05; ** p< 0.01; and ***, p< 0.001. More detailed statistical analysis is described in the figure legends.
3. Results
3.1 Effects of Sesamin on TGF-β1-induced fibroblast activation and proliferation
We first used an in vitro model of fibroblast activation to examine the effect of sesamin on TGF-β1-induced fibroblasts proliferation and differentiation. Primary cultured mice fibroblast cells were treated with vehicle or sesamin (10 μM) for 30 minutes, followed by stimulation with TGF-β1 (10 ng/mL) for 72 hours in the low-serum medium with or without sesamin, respectively. Untreated cells cultured in the low-serum medium for the same period of time served as control. Fibroblasts proliferation was tested by the trypan blue dye exclusion assay. Fibroblasts with TGF-β1 stimulation showed mild enhancement in cell proliferation compared with control, whereas sesamin significantly inhibited enhancement fibroblast proliferation by TGF-β1 stimulation (Fig. 1A). Moreover, TGF-β1 treatments largely increased the expression of α-SMA, which is a cytoskeletal protein characterizing fibroblast activation into myofibroblasts [35, 36]. TGF-β1 promoted development of cell hypertrophy and stress fibers that features differentiated myofibroblasts (Fig. 1B, arrows). These morphological changes were not obviously observed in sesamin-treated fibroblasts after TGF-β1 stimulation.
3.2 Sesamin inhibited TGF-β1-induced fibroblasts transdiffereation and activation of p-Smad2/3
Fibroblast activation and its dependence of Smad2/3 was further quantified by testing the expression levels of myofibroblast marker proteins with western blot methods. TGF-β1-induced significant upregulation of myofibroblast-specific markers α-SMA, collagen I, and vimentin in cultured fibroblast cells, and changes of these proteins were inhibited by sesamin treatment. Upon TGF-β1 stimulation, phosphorylation of Smad2/3 enables the protein to enter the nucleus
to initiate gene transcription [37]. We found that sesamin significantly inhibited TGF-β-induced upregulation of phosphorylated Smad2/3 (p-Smad2/3), suggesting that inhibition of myofibroblast differentiation by sesamin was associated with suppression of Smad2/3 protein phosphorylation.
3.3 Effects of Sesamin TGF-β1-induced epithelial-to-mesenchymal morphology change inhuman alveolar epithelial A549 cells
Next, we studied the effect of sesamin on TGF-β1-induced EMT by using an in vitro EMT model of the human alveolar epithelial cell line cells, A549. A549 cells retain similar morphological characteristics and protein expression profiles as alveolar type II epithelial cells [38]. Upon explosion to low doses of TGF-β1, A549 cells gain a mesenchymal-like phenotype, such as elongated cell morphology and loose cell-cell contacts [39]. In our study, morphological changes of A549 cells were assessed by phase contrast light microscopy (Fig. 3). A549 cells in the absence of TGF-β1 kept typical epithelia-like shapes, as the pebble-like shape and clear cell-cell adhesion (Fig. 3)[39]. After treating with 5 ng/ml TGF-β1 for 48h, A549 cell-cells lost cell-cell-cell-cell contacts and transform into an elongated, fibroblast-like morphology. Per-treatment with 10 μM sesamin retained the epithelial-like morphology of A549 cells in the TGF-β1 treated cells, suggesting that sesamin may have potential inhibitory effects on TGF-β1-induced EMT in A549 cells.
3.4 Sesamin inhibited TGF-β1-induced EMT protein marker expression and p-Smad2/3 upregulation in A549 cells
same EMT model of TGF-β1 stimulation in A549 cells. Expression of EMT protein markers was examined by western blot assay. Our results showed that expression of the epithelial marker E-cadherin was remarkably decreased after TGF-β1 treatment, indicating loss of epithelial phenotypes in A549 cells (Fig. 4). In contrast, the mesenchymal markers fibronectin and vimentin were upregulated by TGF-β1. These alterations were all significantly attenuated by sesamin treatment, suggesting that sesamin effectively inhibited EMT. In addition, TGF-β1 treatment significantly increased levels p-Smad2/3, which was reduced in the sesamin-treated group. Together with the cell morphological changes in A549 cells, these data suggest that sesamin inhibited TGF-β1-induced EMT by intervention with the Smad2/3 pathway.
3.5 Sesamin inhibited OVA-induced myofibroblast activation and collagen accumulation in mice
Previous studies have established the in vivo model of OVA-induced airway fibrosis, in which inhalation of aerosolized OVA increases Smad phosphorylation in multiple types of airway cells, activates fibroblasts proliferation and differentiation, initiates EMT, and enhances collagen deposition [40]. Thus, it is important to determine whether the antifibrotic effects of sesamin from in vitro experiments can be confirmed in the in vivo animal model of airway fibrosis. Here, myofibroblasts activation was determined by immunohistological staining of the airway sections with α-SMA antibodies. We found that both the expression levels of α-SMA and the numbers of α-SMA immunopositive cells were significantly increased in the lung section of OVA-challenged mice compared with naïve mice (Fig. 5A). The upregulation of α-SMA was largely reduced in the lung tissue of OVA-treated mice receiving systematic administration of sesamin (10 and 20 mg/kg). Moreover, collagen accumulation was determined by western blot assays by
using the lung tissue from sham or OVA-challenged mice. The increase of collagen Ι protein levels was significantly reduced in animals receiving sesamin treatment (Fig. 5B). These data indicated that sesamin effectively inhibited fibroblast activation and collagen deposition in OVA-induced airway fibrosis in mice.
3.6 Sesamin suppressed OVA-induced subepithelial fibrosis in mice
In the same OVA-stimulated mice, lung sections were subjected to Masson's trichrome stain to indicate the extent of airway fibrosis (Fig. 6A). The histological results show that OVA-stimulated animals exhibited greatly thickened bronchial walls, proliferated fibroblasts, and excessive collagen deposition in the matrix in interstitial space. The severity of subepithelial fibrosis was further assessed according to Ashcroft's score (Fig. 6B). These histological alterations were largely attenuated in OVA-stimulated mice with sesamin treatments (10 or 20 mg/kg) compared with vehicle treatments, suggesting that sesamin significantly suppressed subepithelial fibrosis in the airway.
3.7 Sesamin reduced OVA-induced airway basement membrane changes in mice
Basement membrane thickness has been widely used in clinical and therapeutic studies as an indicator of airway fibrosis [33]. Thickened basement membrane results from excessive collagen deposition beneath the basement membrane, which is highly associated with subepithelial myofibroblasts and immune cell activation [41]. Here, we used H&E stain to assess changes in basement membrane thickness in the OVA model in mice (Fig. 7). Compared with the sham group, OVA treatments increased the basement membrane thicknesses, which was significantly suppressed by sesamin (10 and 20 mg/kg). These data further confirmed that sesamin has
antifibrotic effects as indicated by the clinically used parameter.
4. Discussion
Fibroblasts, the primary effector cells responsible for the collagen synthesis, maintain normal ECM equilibrium of airway tissues at quiescent state [17]. Upon activation, fibroblasts proliferate and transform into α-SMA expressing myofibroblasts, which produce large amount of collagen and promote fibrotic tissue remodeling [1]. It has been hypothesized that myofibroblast might be originated from three major resources [12]. The first proposed and likely the dominant pathway is via activation and transformation of the resident intrapulmonary fibroblasts [7]. Second, a substantial portion of the profibrotic cells arises from epithelial cells via EMT [42]. TGF-β1 is required for initiating both of these two mechanisms. The last possible origin of fibroblasts/myofibroblasts is from bone marrow-derived progenitors. However, the bone marrow-derived fibroblasts do not respond to TGF-β1 to induce α-SMA expression or myofibroblasts transformation [43]. In this regard, we focused on the first two mechanisms to elucidate effects of sesamin during pulmonary fibrotic processes by using the TGF-β1-dependent fibroblast activation and EMT models in vitro. We found that sesamin inhibited fibroblasts proliferation and differentiation and suppressed EMT in A549 cells, suggesting that sesamin effectively interfere with both major sources of myofibroblast generation.
Fibrosis is a major pathological feature of airway remodeling and it can occur early in the pathogenesis of asthma. The fibrotic aspect of asthmatic pathology involves bronchial fibroblasts activation and subepithelial fibrosis [2]. The elevated levels of TGF-β are highly correlated with airflow obstruction and airway thickening in asthma patients [44]. The OVA-induced airway
fibrosis have been widely used as experimental models in many pharmacological and toxicity studies [45, 46]. Moreover, EMT has also been demonstrated to contribute to airway fibrosis during allergic asthma [5]. TGF-β1-induced EMT of bronchial epithelial cells was largely facilitated by exposure to allergen [47]. In the murine models of chronic allergen exposure, airway epithelial cells lost tight intercellular contacts and expressed α-SMA [48], and the epithelial origin of p-Smad positive cells that migrated into the bronchial subepithelia has been further identified by cell-fate tracking [48]. Together, these findings have suggested a general consensus on the TGF-β1-dependent mechanism between the OVA model and the in vitro fibrosis models that were used in our experiments. In line with our observations from the in vitro models, sesamin also decreased myofibroblasts proliferation, collagen deposition, and basement membrane thickness in the OVA-stimulated animals. These data together indicate an antifibrotic effect of sesamin on airway fibrosis.
At present, effective therapies treating airway fibrosis are still lacking. In allergic asthma, for instance, corticosteroids are the major therapeutic choice under these conditions [36]. However, corticosteroids mainly act by suppressing inflammatory responses and might have very complex effects on tissue fibrosis. Corticosteroids could protect against fibrosis by reducing inflammatory cells, such as eosinophils [49], which is one of the major sources of TGF-β1 synthesis [50]. However, studies also showed that these drugs might cause epithelial damage and induce cell death [51]. In addition, dexamethasone increased fibroblast proliferation and simulate G1-S phase transition, suggesting that these drugs might promote fibrotic responses [52, 53]. Therefore, it is needed to develop new treatments that are effective to both inflammation and fibrotic responses. Sesamin is a natural occurring compound from diets and has very low toxicity
to human. Our recent studies have found that sesamin inhibited airway inflammation and hyperresponsiveness in OVA-induced asthma in mice [26]. Together with these results, our findings suggest that sesamin might have potential therapeutic benefits in treating asthma due to its dual effects as an anti-inflammatory and antifibrotic agent.
Smad is the primary signalling pathway downstream of TGF-β1 activation [37]. Binding of TGF-β1 with its receptors phosphorylates Smad2/3 proteins, which translocate into the nucleus to initiate transcriptional activities and regulate gene expression [54]. TGF-β1 can also interact with Smad-independent pathways, including JAK-STAT, MAPK, glucocorticoid receptor, and Wnt [55-58]. Under conditions of stress, TGF-β1 might act via the Smad-independent pathway, such as activation of MAP kinases to induce apoptosis [59]. In the present study, we showed that increased levels of p-Smad2/3, which were inhibited by sesamin treatments, accompanied both activation of fibroblasts and EMT of A549 cells. These data suggest that sesamin exerted its antifibrotic effects at least partially dependent upon the Smad-dependent pathway. In addition, we did not observe apparent apoptotic cell death in either TGF-β1 or sesamin-treated fibroblasts and A549 cells, suggesting that activation of MAP kinases pathway was not likely involved in these processes. Whether sesamin interacts with other Smad-independent pathway of TGF-β1 signalling still remains to be determined in future studies.
In conclusion, our results demonstrated that sesamin inhibited proliferation and activation of primary cultured lung fibroblast cells, suppressed EMT of airway epithelial cell line cells, and prevented airway subepithelial fibrosis, collagen deposition, and basement membrane thickening in OVA-induced airway fibrosis in mice. These effects were possibly mediated by blocking the
TGF-β1/Smad dependent pathway. The antifibrotic effect of sesamin might contribute to future development and implication of natural polyphenolic compounds in fibrotic lung diseases.
Acknowledgements:
This work was supported by grants from Taiwan National Science Council (NSC 101-2320-B-039-057, NSC 100-2632-B-039-001-MY3, NSC 2320-B-039-038-MY3, and NSC 102-2320-B-039-035) and grants from Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH102-TD-B-111-004). The funders had no role in study design, data collection, analysis and interpretation, manuscript writing, or decision to publish of the manuscript.
Figure 1. Effects of sesamin on TGF-β1-induced proliferation and activation of primary cultured fibroblast. (A) TGF-β1 (10 ng/mL, n=4) facilitated cell proliferation compared with
vehicle-treated fibroblasts (n=4). Sesamin treatment (10 μM) alone (n=4) did not cause apparent changes in either cell numbers or cell morphologies (B) over 72 hours. However, sesamin (10 μM) significantly inhibited fibroblast proliferation induced by TGF-β1 (n=4). Magnification: 20×. (B) TGF-β1 (10 ng/mL) enhanced α-SMA fiber expression, which was inhibited by sesamin (10 μM). The arrows indicate examples of intracytoplasmic stress fibers. Data were represented as the mean ± S.E.M. Statistical significance was determined by paired student’s t test and was defined as # p<0.05, ## p<0.01, ### p<0.001 compared with the vehicle group, and * p< 0.05, ** p< 0.01, *** p<0.001 compared with the TGF-β1 group.
Figure 2. Effects of sesamin on TGF-β1-induced expression changes of myofibroblast marker proteins. (A) The expression levels of α-SMA, collagen I, vimentin, and p-Smad2/3
were tested by western blot in primary cultured fibroblasts. TGF-β1 (10 ng/mL) increased the expression levels of α-SMA (n=4), collagen I (n=5), vimentin (n=6), and p-Smad2/3 (n=4). These changes were significantly inhibited by sesamin (10 μM). Quantified results were shown in (B)-(E). All results were presented as the relative levels normalized to the GAPDH internal control. Data were represented as the mean ± S.E.M. Statistical significance was determined by paired student’s t test and was defined as * p< 0.05 and ** p< 0.01 compared with the TGF-β1 group.
Figure 3. Effects of sesamin on TGF-β1-induced morphological changes in human alveolar epithelial A549 cells. A549 cells were incubated with 5 ng/ml TGF-β1 for 48 h in the absence or
presence of 10 μM sesamin. TGF-β1 stimulated A549 cells transform into an elongated, fibroblast-like morphology. The morphological changes were not observed in sesamin-treated cells. Magnification: 20×.
Figure 4. Effects of sesamin on TGF-β1 induced EMT marker expression and p-Smad2/3 levels in A549 cells. (A) The expression levels of fibronectin, E-cadherin, vimentin, and
p-Smad2/3 were tested by western blot in A549 cells. TGF-β1 (5ng/mL) decreased the levels of E-cadherin (n=6) and increased the expression levels of fibronectin (n=7) and vimentin (n=7). Phosphorylation levels of Smad2/3 were also increased by TGF-β1 (n=4). These changes were significantly attenuated by sesamin (10 μM). Quantified results were shown in (B)-(E). All results were presented as the relative levels normalized to the GAPDH internal control. Data
were represented as the mean ± S.E.M. Statistical significance was determined by paired student’s t test and was defined as * p< 0.05 and ** p< 0.01compared with the TGF-β1 group.
Figure 5. Effects of sesamin on induced α-SMA and collagen I expression in OVA-challenged mice. (A) Representative α-SMA staining of lung tissue sections from mice of sham
group, OVA-sensitized and challenged mice treated with vehicle (saline, i.p.), or with sesamin (SE, 1, 10, or 20 mg/kg,i.p.). α-SMA was characterized by intense staining in the subepithelial regions as indicated by red arrows as an example. Magnification: 63×. The number of α-SMA positive cells was upregulated in mice receiving OVA challenges (n=10) compared with the sham group (n=10). In OVA-sensitized and challenged animals, the number of α-SMA positive cells was significantly reduced in mice treated with 10 or 20 mg/kg sesamin (n = 10 in each group) but not with 1 mg/kg sesamin (n = 10), compared with mice treated with vehicles. (B) Immunoblotting detection of collagen I expression in lung tissue from mice of sham group, OVA-challenged mice treated with vehicle, or sesamin (1, 10, or 20 mg/kg, i.p.). Increased collagen I levels were observed in mice receiving OVA treatment compared with mice receiving sham treatment. Upregulation of collagen I were in the OVA-stimulated mice were reduced in mice treated with 10 mg/kg but not with 1 or 20 mg/kg sesamin, compared with mice treated with vehicles. The quantitative results were from three independent experiments. Each bar represents the mean ± S.E.M. Statistical significance between the sham and vehicle groups was determined by student’s t test and was defined as # p<0.05, ### p<0.001. Statistical significance of the sesamin groups compared with the vehicle group was determined by one-way ANOVA and was defined as * p<0.05, ** p<0.01.
Figure 6. Effects of sesamin on OVA-induced airway fibrosis. (A) Representative images
showing Masson's trichrome staining of lung tissue sections from mice of sham group, OVA-sensitized and challenged mice treated with vehicle (saline, i.p.), or with sesamin (1, 10, or 20 mg/kg,i.p.). Magnification: 40×. (B) Grade of fibrosis was enhanced in mice receiving OVA challenges (n=11) compared with the sham group (n=10). In OVA-sensitized and challenged animals, the grade of fibrosis was significantly reduced in mice treated with 10 or 20 mg/kg sesamin (n = 10 in each group) but not with 1 mg/kg sesamin (n = 11), compared with mice treated with vehicles. Statistical significance between the sham and vehicle groups was determined by student’s t test and was defined as ### p<0.001. Statistical significance of the sesamin groups compared with the vehicle group was determined by one-way ANOVA and was defined as * p<0.05.
Figure 7. Effects of sesamin on OVA-induced airway basement membrane change. (A)
Representative images showing H&E staining of lung tissue sections from mice of sham group, OVA-sensitized and challenged mice treated with vehicle (saline, i.p.), or with sesamin (1, 10, or 20 mg/kg,i.p.). Black arrows indicate the location of the basement membrane band beneath the epithelium. Yellow lines perpendicular to the basement membrane show the measurements of thickness. Magnification: 40×. (B) Basement membrane thickness was increased in mice receiving OVA challenges (n=11) compared with the sham group (n=10). In OVA-sensitized and challenged animals, the increased thickness of basement membrane was significantly reduced in mice treated with 10 or 20 mg/kg sesamin (n = 10 in each group) but not with 1 mg/kg sesamin (n = 11), compared with mice treated with vehicles. Statistical significance between the sham and vehicle groups was determined by student’s t test and was defined as ### p<0.001. Statistical
significance of the sesamin groups compared with the vehicle group was determined by one-way ANOVA and was defined as * p<0.05.
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