Ardisia japonica inhibits airway remodeling in chronic obstructive pulmonary disease in rats through inhibiting Wnt signaling pathway

Background The effects of Ardisia japonica (AJ) on airway remodeling in chronic obstructive pulmonary disease (COPD) rats, and the mechanisms have not been veried. This study aimed to investigate the effects of a Miao medicine, AJ on airway remodeling in COPD rats, and to assess the mechanisms. Methods COPD model was produced by cigarette smoke and intratracheal injection of lipopolysaccharide (LPS). The experiments were divided into a normal group, a COPD group, different doses of AJ treatment groups and a positive control group. After treatments, matrix metalloprotein (MMP)-9, platelet derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) levels in inferior lobes of the right lung tissues were detected by ELISA. Expression of Wnt signaling pathway was detected by immunohistochemistry and Western blotting. To verify the function of Wnt signaling pathway in airway remodeling, airway broblasts obtained from COPD rats were treated with Wnt signaling pathway agonists. The cell proliferation, apoptosis, and MMP-9, PDGF, TGF-β1 levels were determined. were by eosin (H&E) staining. MMP-9, PDGF and levels were promoted in COPD rats, which were signicantly reduced by different doses of AJ treatment. Importantly, components of Wnt signaling pathway, Wnt5a, β-catenin and were up-regulated in COPD which were also signicantly reduced by with different AJ. The obtained from and based promoted promoted PDGF and TGF-β1 levels.

airway remodeling of COPD (7,8). Therefore, the control of airway remodeling could be a new and useful treatment for COPD patients.
The Wnt signaling pathways affect physiological and pathological processes such as stress, immunity, cell differentiation, apoptosis, etc. (9). The Wnt signaling pathway is closely related to the mechanisms of pulmonary brosis, idiopathic pulmonary hypertension, pulmonary interstitial diseases and other diseases (10,11). Abnormal activation of Wnt signaling pathway is also closely associated with chronic obstructive pulmonary disease, bronchial asthma and interstitial lung disease (12). TPA (12-O-tetradecanoylphorbol-13-acetate) can up-regulate Wnt signaling pathway by enhancing casein kinase 1 (CK1) activity (13). Wnt signaling pathway also interacts with mitogen-activated protein kinases (MAPKs) signaling pathway (14), which is a signaling pathway regulating cell growth and proliferation, affecting lung development (15). In addition, MAPK signaling pathway has also been reported to correlate with lung cancer and pneumonia (16)(17)(18).
Ardisia japonica (AJ) is a signi cant and unique Guizhou Miao medicine in China. Clinically, AJ has obvious antitussive and expectorant effects (19). COPD belongs to "cough", "lung distention" etc. based upon the theory of traditional Chinese medicine (TCM). Especially, "cough", "phlegm", "asthma", "fullness" and "stu ness" are the main symptoms of "lung distention", while AJ has a good effect on the treatment of lung distention (20). The active components consist of gallic acid, bergenin, epicatechin, epicatechin gallate, isoquercitrin, and quercetin-3-rhamnoside (21). Modern pharmacological studies further suggest that the effective components of AJ has certain antiasthmatic, anti-fungal, hypolipidemic and anti-tumor effect (22,23), and can effectively treat chronic bronchitis (19). Additionally, AJ also has antitussive and antiasthmatic, antibacterial, anti-in ammatory and antiviral activities (22,24,25). However, the effects of AJ on airway remodeling in COPD rats, and the mechanisms have not been veri ed. In this study, we aimed to investigate the effects of AJ on airway remodeling in COPD rats, and to assess the potential signaling pathway involved in the protection.

Materials And Methods
Preparation of Ardisia japonica Ardisia japonica (500 g), purchased from Anguo TCM market (Hebei, China) was added into 500 mL of water and soaked for 20 min. Thereafter, the mixture was boiled and then another 500 mL of water was added. Then, the medicine was decocted into 100 ml using slow re. Finally, a decoction (100 g/100 mL) was prepared and kept at 4 ℃ for use. Animals and COPD model 120 healthy male SD rats (120 ± 20 g, 6 weeks) were purchased from Tengxin Biotechnology Co., Ltd. in Chongqing [Certi cate No. SCXK (Chongqing) 2012-0005] and housed in a SPF condition with a standard 12-h light/12-h dark cycle and ad libitum access to food and water. The average humidity was 40%-60%, and the average temperature was 20-25 ℃. The experiments were approved by the Ethics Committee of Guiyang College of Traditional Chinese Medicine.
The COPD model was established as previously described (26). Brie y, SD rats were put in a smoking box (40 x 50 x 60 cm). In each smoking box, 10 rats were placed and exposed to 8 cigarettes each time and the smoking process lasted about 30 min. The passive smoking was delivered for twice each day for consecutive 28 days. On the rst and the 14 st days, lipopolysaccharide (LPS) (0.2 mL, 200 μg/200 μL, SIGMA) was administrated through intratracheal injection in the anesthetized rats (10% chloral hydrate, 0.37 mL/100 g). After anesthesia, the rats were xed on the table in supine position. Longitudinal incision along the midline neck (about 1 cm) was prepared to deliver LPS. The rats were erected to keep the LPS owing along the wall of the trachea to the alveoli. The wound was sutured layer by layer, and penicillin powder was applied to the incision. Eight days after modeling, animals were anesthetized with 5% iso urane and airway tissues were xed in 4% paraformaldehyde (PFA). The lung injury was con rmed by H&E staining.
The experiments were divided into normal group (Group A), COPD group (Group B), COPD + low dose of AJ group (0.75 mL/kg, Group C); COPD + medium dose of AJ group (1.5 mL/kg, group D); COPD + high dose of AJ group (3 mL/kg, group E); COPD + dexamethasone group (Group F). AJ was delivered through intragastric administration once daily for 30 days. In COPD + dexamethasone group, the rats were treated with 1 mg/kg dexamethasone (intramuscular injection, once daily) for consecutive 30 days. After that, inferior lobes of the right lung tissues were collected for molecular biochemical experiments or xed in 4% for pathological staining (Fig.1).

H&E staining
Inferior lobes of the right lung tissues were xed in 4% paraformaldehyde for ~1 week at 4˚C. Thereafter, the tissues were dehydrated, embedded, and sliced. The para n section is dewaxed and hydrated. The tissues were rinsed for several hours, thereafter dehydrated, embedded, and sliced. The para n section is dewaxed and hydrated. The sections were stained with hematoxylin for 5 min and with eosin for 3 min.
The images were taken by a light microscopy.

Preparation of airway broblasts
Under the anesthesia with iso urane (5%), the rats were decapitated and thoracotomized in aseptic conditions. Fresh airway tissue of COPD rats was washed repeatedly with PBS in aseptic worktable for 5 times. Sterile scalpel was used to collect the tissue around the airway. The tissue was cut into 2 mm x 2 mm with sterilized ophthalmic scissors and pasted into the culture plate, and placed in a 5% CO 2 incubator 37˚C for 4 h. The cells completely adhered to the wall and the newly prepared DMEM (Gibco) + 20% FBS (Hyclone) medium was added to the culture plate, and the cells were further cultured in a 37˚C, 5% CO 2 incubator. The growth of the cells was observed and then treated routinely. The cultured dishes were xed in 4% paraformaldehyde for 15 min, and permeated with 0.5% Triton X-100 (PBS) for 20 min at room temperature. 5% BSA was dripped into the dish and sealed at 37 ˚C for 30 minutes. A su cient amount of diluted vimentin (1:250, ab92547, Abcam) was dripped into the dish and incubated at 37 ˚C for 3 h. The diluted uorescent antibody Cy3 (1:200) was added and incubated at 37˚C for 30 min. DAPI (4',6diamidino-2-phenylindole) was used to stain the nuclei. The images were observed under uorescence microscope.

ELISA
Matrix metalloprotein (MMP)-9, platelet derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) levels were detected by ELISA following the instructions of the assay kits (QIYI Biotech, Shanghai, China). The reagents in the kit were kept at room temperature for 30 minutes. A standard curve was established to calculate the levels of target proteins. The airway tissues were homogenated and centrifuged at 11,000 g for 10 min at 4 ℃. The supernatants were used in the experiments. All standard samples and test samples required 3 duplicates. A blank control was set without sample and enzyme reagent. Absorbance was detected at the wavelength of 450 nm by a Microplate Reader (RT-6100, Rayto).
Inferior lobes of the right lung tissues were xed in 4% paraformaldehyde for ~1 week at 4˚C. Thereafter, the tissues were dehydrated, embedded, and sliced. The para n section is dewaxed and hydrated.
Immunostaining of histological sections was performed using monoclonal antibodies against RhoA Chemiluminescent substrate detection reagent was applied to show the staining.

Data analysis
The data were expressed by mean ± standard deviation and statistically analyzed by SPSS 19 (SPSS, Inc., Chicago, IL, USA). One-way ANOVA with Newman-Keuls as the post-hoc test was applied to determine statistical signi cance. A value of P<0.05 was considered to be signi cant.

AJ reduced COPD-induced pathological changes
Lung injury in COPD model was con rmed by H&E staining (Fig.1). In control group, the airway tissue showed regular alveolar structure. Pathological expansion and fusion of alveolar cavity were not observable. Bronchial tube wall was normal; the airway mucosa epithelium was smooth; cilia were arranged neatly; obvious in ammatory exudation was not found in the tracheal cavity. By contrast, emphysema, alveolar dilatation, and alveolar wall gradually broken and fused into lung ulcers and the numbers of alveolar decreased signi cantly in COPD model group. The goblet cells of bronchial epithelium proliferated and a large number of in ammatory cells such as neutrophils and giant cells in ltrated the lumen, accompanied by the proliferation of brous connective tissue AJ reduced COPD-induced MMP-9, PDGF and TGF-β1 levels The expression of MMP-9, PDGF and TGF-β1 in inferior lobes of the right lung tissues were shown in Fig.2. Compared with the control group, COPD group showed increased MMP-9, PDGF and TGF-β1 expression in inferior lobes of the right lung tissues. By contrast, different doses of AJ and dexamethasone signi cantly reduced MMP-9, PDGF and TGF-β1 expression compared with those in COPD group (P < 0.05).

AJ reduced COPD-induced RhoA expression
The expression of RhoA in inferior lobes of the right lung tissues was detected by immunohistochemistry and Western blotting (Fig.3). Compared with the control group, the expression of RhoA in COPD group increased signi cantly. Compared with the COPD group, RhoA expression was signi cantly reduced in different doses of AJ groups and dexamethasone group (P < 0.05).

AJ reduced COPD-induced β-catenin expression
The expression of β-catenin in inferior lobes of the right lung tissues was detected by immunohistochemistry and Western blotting (Fig.4). Compared with the control group, the expression of β-catenin in COPD group increased signi cantly. Compared with the COPD group, RhoA expression was signi cantly reduced in different doses of AJ groups and dexamethasone group (P < 0.05).

AJ reduced COPD-induced Wnt5a expression
The expression of Wnt5a in inferior lobes of the right lung tissues was detected by both of immunohistochemistry and Western blotting (Fig.5). Compared with the control group, the expression of Wnt5a in COPD group increased signi cantly. Compared with the COPD group, Wnt5a expression was signi cantly reduced in different doses of AJ groups and dexamethasone group (P < 0.05).

Wnt signaling pathway agonists promoted cell proliferation of airway broblasts
As shown in Fig.6A, immuno uorescence staining for vimentin of the cultured rat alveolar cells identi ed the presence of broblasts. Cell proliferation was detected based on the ratio of CCK8 24 h to CCK8 0 h. The results showed that HLY78, LiCl, TPA and EGF promoted the ratio (Fig.6B), which indicates that Wnt signaling pathway agonists promoted cell proliferation.

Wnt signaling pathway agonists reduced apoptosis of airway broblasts
Apoptosis was also detected in the airway broblasts. The results showed that HLY78, LiCl, TPA and EGF reduced apoptosis signi cantly (Fig.7).

Discussion
In this study, we revealed a novel effect of AJ on airway remodeling in COPD rats. AJ reduced MMP-9, PDGF and TGF-β1 levels in COPD model, which implicated the protection of AJ in COPD model. Moreover, we demonstrated that AJ antagonized the airway remodeling likely through inactivating Wnt5a pathway, as Wnt agonists promoted cell proliferation, reduced apoptosis and promoted MMP-9, PDGF and TGF-β1 levels of airway broblasts.
COPD is a group of lung diseases characterized by air ow restriction and incomplete reversibility. Although the exact cause is still unknown, COPD is related to smoking, air pollution, dust and other factors (27). Oxidative stress, in ammation, protease activation are the main pathogenesis of COPD (28,29). The pathological changes in COPD patients include chronic bronchitis, emphysema, airway reconstruction and pulmonary remodeling (30). LPS has a complex of polysaccharide and protein structure of the outer membrane with gram negative bacteria and could cause release of proin ammatory cytokines, directly impairing airway epithelial layer (31). In this study, COPD model was established in SD rats by cigarette smoking and LPS injection, because SD rats are sensitive to the smoke, dust, sulfureted hydrogen and other stimuli in the air, and were susceptible to respiratory diseases (32). The results of H&E staining con rmed that COPD model was successfully established in our study.
TGF-β1 is a multifunctional cytokine that could induce epithelial cell layer destruction, airway in ammation, smooth muscle cell proliferation, goblet cell proliferation and vascular remodeling (33). TGF-β1 inhibits enzymes that degrade matrix protein and regulate the expression of matrix protein on the cell surface (34). Animal experiments further demonstrated that TGF-β1 could stimulate the pathological aggregation of extracellular matrix (34). Overexpression of TGF-β1 was found in asthmatic and COPD patients, as well as in epithelial cells and smooth muscle cells of acute, subacute and chronic asthmatic mice (35). Therefore, TGF-β1 played an important role in airway remodeling in smooth muscle cells and broblasts (35). In this present study, we found TGF-β1 was promoted in COPD rats, which was reduced by different doses of AJ or dexamethasone treatments. As evidenced by previous ngerprint study, Ardisia japonica is comprised of gallic acid, bergenin, chlorogenic acid and quercitrin (36). Moreover, these components possess airway protection in pathological conditions (37). PDGF is mainly released by mononuclear cells, endothelial cells, bronchial epithelial cells and smooth muscle cells (38) and can stimulate the proliferation and chemotaxis of broblasts and the synthesis of extracellular matrix, which is one of the important mechanisms underlying airway reconstruction (39).
PDGF can stimulate bronchial intermediate muscle broblasts to produce FK506 binding protein, motorrelated protein 3 and heat shock protein. It is considered that PDGF and its signal transduction pathway may play important roles in airway remodeling and brosis (40). In this present study, we found PDGF was promoted in COPD rats, which was reduced by different doses of AJ or dexamethasone treatments. These data further supported the protection of AJ.
Extracellular matrix (ECM) is required to maintain alveolar structure. The degradation and synthesis of ECM should maintain a dynamic balance, which is required to ensure normal function of lung (41). MMPs are mainly involved in the renewal of ECM, the healing of damage and the response to injury (35).
MMP-9 is produced by lung structural cells and in ammatory cells, which can degrade proteoglycan, promote airway brosis and activate potential binding growth factors, and induce smooth muscle proliferation to participate in airway remodeling (42). In this study, we found that the contents of MMP-9 increased signi cantly after COPD modeling. These results revealed that MMP-9 played important roles in the process of airway remodeling, and were also consistent with those studies mentioned above (34,35).
Critically, AJ reduced MMP-9 level in COPD model.
Wnt5a is one of the important members of the Wnt family. Like other Wnt members, Wnt5a could function through an autocrine or paracrine forms, after binding to the transmembrane receptor Frz receptor (43). β-catenin mainly locates at the cell membrane. Its function mainly mediates intercellular adhesion and participates in regulating gene expression (44). The down-regulation of β-catenin would destroy the adhesion between cells and promote cell invasion ability (45). β-catenin also functions as the downstream of the classical Wnt signaling pathway to regulate cell growth and differentiation (46). Rho GTP enzyme plays an important role in the regulation of cytoskeleton recombination. As evidenced, Rho GTP enzyme is highly expressed in a variety of malignant tumors and is closely related to tumor occurrence, invasion and metastasis (47). Our present study demonstrated that the expression of Wnt5a, β-catenin and RhoA increased signi cantly after the COPD modeling. These results implicated that the COPD model may affect airway remodeling by up-regulating the expression of Wnt signaling pathway.
Wnt signaling pathway and MAPK signaling pathway have similarities in functions, and the two signaling pathways also have a mutually reinforcing role (48,49). Components of the MAPK signaling pathway, such as ERK1/2, p38 and JNK, can promote the phosphorylation of LDL Receptor Related Protein 6, thus up-regulating Wnt/beta-catenin signaling pathway (50). Wnt5a can promote ERK1/2 protein phosphorylation (14). In this study, we found that both signaling pathways were involved in the remodeling of broblasts from COPD rats.
We have previously reported that hyperplasia suppressor gene (HSG) overexpression inactivated airway broblasts from COPD by inhibiting the Wnt signaling pathway (51). In this study, we also provided in vivo data showing that AJ in different doses ameliorated airway remodeling and antagonized Wnt signaling pathway. Using the cultured airway broblasts obtained from COPD model, we further veri ed that Wnt agonists LiCl and HLY78 promoted the cell proliferation and reduced apoptosis. We could speculate that the antagonists of Wnt signaling pathway would block the cell proliferation and promote apoptosis, like the effect of HSG overexpression (51). Additionally, MAPK pathway agonists also exhibited the similar functions as Wnt signaling pathway agonists. These results implications a complex crosstalk involved in the remodeling of airway broblasts. The mechanisms still require deep clari cation in future.