sinensis treatment. A total of 50 male Wistar rats (body weight, 200±20 g; age, 8–10 weeks) were purchased from the Shandong University Experimental Animal Center (Jinan, China), and the animal experiments were performed in accordance with and approved by the Institutional Animal Care and Use Committee of Shandong University (Jinan, China). The rats were housed in 24±1°C with humidity of 50±10%, a 12 h light/dark cycle and had access to a standard diet and water ad libitum. The rats were randomly divided into five groups as follows: Control group (CON); COPD group (COPD); COPD + low dose of C. sinensis group (LOW; 2.5 g/kg/day C. sinensis); COPD + moderate dosage of C. sinensis group (MOD; 5 g/kg/day C. sinensis); and COPD + high dose of C. sinensis group (HIG; 7.5 g/kg/day C. sinensis) (n=10/group).

The rat model of COPD was established by cigarette smoking and an intratracheal injection of lipopolysaccharide (LPS), according to a previous report (28). Briefly, intratracheal injection of 1 mg/ml LPS (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was conducted on day 1 (0.2 ml) and 14 (0.1 ml). Except for days 1 and 14, rats were exposed to smoke generated by 8 cigarettes (Hademen^®Filter tip cigarette; Jinan Cigarette Factory of China Tobacco Shandong Industrial Co., Ltd., Jinan, China; tar, 10 mg; nicotine content, 1.0 mg; carbon monoxide, 12 mg) in a sealed box connected to the smoke source for 30 min, twice daily for the first 2 weeks; then 15 cigarettes per treatment, three times daily for 10 weeks. Artificially cultured C. sinensis powder was obtained from Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd. (Hangzhou, China). C. sinensis powder was dissolved in normal saline (500 mg/ml) to prepare the turbid liquid suspension, and applied to the LOW, MOD and HIG groups (2.5, 5 or 7.5 g/kg/day, respectively) intragastrically following cigarette smoke exposure once a day for 12 weeks. Rats in the CON group were exposed to air and treated with PBS. After 12 weeks, rats were sacrificed with an intraperitoneal injection of pentobarbital sodium (150 mg/kg; Sigma-Aldrich; Merck KGaA).

BAL was performed in the left lung through a tracheal cannula under anesthesia using 1 ml sterile isotonic saline three times in each rat. The BALF was immediately centrifuged at 200 × g for 10 min at 4°C. The supernatant was stored at −80°C for cytokine measurements. Total cell counts in BALF were performed using a hemocytometer. Differential cell counts were performed on cytospin preparations with Wright-Giemsa stain. Briefly, the cells were evenly coated on clean glass slides and fixed with absolute methanol after drying at room temperature. The slides were stained with Wright's-Giemsa solution for 10 min at room temperature, then washed, dried and observed by light microscope (original magnification, ×1,000; Olympus Corporation, Tokyo, Japan). At least 200 cells/sample were scored.

The levels of interleukin (IL)-8 (cat. no. MBS7606869; MyBioSource, Inc., San Diego, CA, USA), tumor necrosis factor (TNF)-α (cat. no. RTA00; R&D Systems, Inc., Minneapolis, MN, USA) and TGF-β1 (cat. no. MB100B; R&D Systems, Inc.) were determined using a sandwich ELISA method, according to the manufacturer's protocol.

Tissue samples from the left lung of euthanized rats were fixed with 4% paraformaldehyde at room temperature for 24 h and processed for paraffin embedding. Lung paraffin sections were sliced to 5-µm-thick sections and then stained with hematoxylin (3 min) and eosin (1 min) at room temperature using a staining kit (cat. no. G1120; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) under a light microscope (original magnification, ×200; Olympus Corporation).

The lung sections were also examined with Masson's trichrome stain to assess the deposition of peribronchial collagen. Following dewaxing and hydration with xylene and a gradient concentration of ethanol (100, 95, 80 and 70%). The slides were stained with Weigert iron hematoxylin for 8 min and then differentiated with 1% hydrochloric acid alcohol. Subsequently, slides were stained with Ponceau fuchsin for 8 min. Following staining with phosphomolybdic acid solution for ~5 min, the slides were stained with aniline blue for 5 min, differentiated in 1% acetic acid for 1 min, dehydrated in an ascending series of ethanol (95 and 100%), washed in xylene and sealed with neutral gum. All steps were conducted at room temperature. Masson's trichrome staining was used to differentiate collagen from other fibers, staining nuclei in black, cytoplasm and muscles in red and collagen in blue. The sections were visualized using a light microscope at a magnification of ×200.

Additionally, three random microscope fields in which the bronchiole diameters were <100 µm (shortest path/lumen diameter, cope fields in which the brox200 were observed to determine the changes of thickness and area of the tube wall. The wall area/total bronchiole area (MA%) and the wall thickness/bronchiole diameter (MT%) were then calculated.

In the present study, it was found that the moderate (5 g/kg/day) and high (7.5 g/kg/day) dose of C. sinensis group had a marked inhibitory effect on airway remodeling parameters compared with the low (2.5 g/kg/day) dose group, but there was no significance between 5 and 7.5 g/kg/day treatments. Therefore, the 5 g/kg/day dose of C. sinensis was selected for pathological evaluation in following experiments.

Immunohistochemistry was performed by a two-step immunoperoxidase protocol. Lung tissue was fixed at room temperature in 4% paraformaldehyde for 24 h and embedded in paraffin an sliced into a 4-µm-thick sections. Slides were deparaffinized, rehydrated with xylene and ethanol of gradient concentration (100, 95, 80 and 70%) and antigen retrieval was performed by microwave (95°C, 15–20 min). Endogenous peroxidases were blocked by soaking slides in 3% H₂O₂ for 30 min at room temperature. Subsequently, the slides were incubated with anti-α-SMA (1:200; cat. no. sc-53142), anti-collagen I (1:100; cat. no. sc-59772; both Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-TGF-β1 (1:100; cat. no. ab92486), anti-TGF-β receptor (TβR) I (1:100; cat. no. ab31013) and anti-TβR II (1:100; cat. no. ab186838; all Abcam, Cambridge, MA, USA) antibodies overnight at 4°C in a humidified chamber. Following this, the sections were incubated with the secondary horseradish peroxidase-conjugated goat anti-rabbit (cat. no. ZB-2301) and goat anti-mouse (cat. no. ZB-2305) antibodies (both 1:500; OriGene Technologies, Inc., Beijing, China) for 1 h at room temperature. Sections were stained with diaminobenzidine for 2–5 min and counterstained with 10% hematoxylin for 3 min at room temperature. Stained areas of the sections were visualized using a light microscope at magnification of ×200. Positive areas were quantified by densitometry using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA).

Total proteins in right lung tissue samples from each group were extracted prior to western blotting. Lung tissue was mixed with RIPA lysate buffer (P0013K; Beyotime Institute of Biotechnology, Haimen, China) with a ratio of 100 mg/ml. The mixture was centrifuged (12,000 × g) at 4°C for 5 min to collect the supernatant. The total protein was quantitated using a Pierce BCA Protein Assay Kit. The protein (20 µg/lane) was loaded and separated using 10% SDS-PAGE, and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon; EMD Millipore, Billerica, MA, USA) at 4°C. The PVDF membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) for 60 min at room temperature. The membranes were incubated with the following antibodies: Anti-TGF-β1 (1:500), anti-TβR I (1:500), anti-TβR II (1:500), anti-collagen I (1:1,000) anti-α-SMA (1:1,000), anti-phosphorylated (p)-Smad2 (1:1,000; cat. no. 3104, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p-Smad3 (1:1,000; cat. no. 9520, Cell Signaling Technology, Inc.) anti-Smad7 (1:1,000; cat. no. sc-365846) and GAPDH (1:1,000; cat. no. sc-47724; both Santa Cruz Biotechnology, Inc.) overnight at 4°C. The membrane was washed three times for 5 min each with 1X TBS containing 0.15 Tween-20 followed by incubation with secondary horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies (both 1:5,000) at 37°C for 1 h. The immunoreactive bands were visualized by a enhanced chemiluminescence kit (cat. no. K820-500; BioVision, Inc. Milpitas, CA, USA). GAPDH was used for normalization. The quantification of protein expression was analyzed by densitometry using ImageJ software (version 1.46; National Institutes of Health, Bethesda, MD, USA).

In the present study, it was found that C. sinensis at a dose of 5 g/kg/day efficiently decreased the protein and mRNA expression levels of TGF-β1, TβR I and TβR II, but there was no significant difference between 5 and 7.5 g/kg/day treatments. Therefore, the optimal dose was 5 g/kg/day of C. sinensis, which was selected for following experiments.

Total RNA was extracted from the right lung tissue of rats using TRIzol reagent (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. Total RNA was extracted from the right lung tissue of rats using TRIzol reagent (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. A total of 1 µg total RNA was used for reverse transcription with PrimeScript RT Master Mix (TaKaRa, Bio, Inc) according to the manufacturer's protocol. qPCR was performed by using the SYBR^® Premix Ex Taq^™ II (Takara Bio, Inc.) according to the manufacturer's protocol on a LightCycler 480 System (Roche Diagnostics, Basel, Switzerland). The qPCR reaction mixture consisted of 2 µl cDNA, 10 µl SYBR Premix Ex Taq II (2X), 0.8 µl of 10 uM forward primer, 0.8 µl of 10 uM reverse primer, and 6.4 µl double distilled H₂O to a final volume 20 µl. The thermocycling conditions were as follows: 95°C for 30 sec (initial denaturation), followed by 40 cycles of 95°C for 5 sec (denaturation) and 60°C for 30 sec (annealing). Next, a melting curve was performed at 95°C for 5 sec, 60°C for 1 min and then continuously at 95°C, followed by 50°C for 30 sec (cooling). Relative quantification of mRNA was calculated using the 2^−ΔΔCq method (29). The sequences of primers used for RT-qPCR in the present study are demonstrated in Table I. GAPDH gene was used as an internal control.

All data are presented as the mean ± standard error of the mean. All experiments in the present study were repeated at least three times. One-way analysis of variance followed by Newman-Keuls post hoc tests were used to compare measurements of individual groups. The statistical analysis was conducted using SPSS software (version 19.0; IBM Corp, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

To detect the airway structural changes in rats with COPD, the slides from each group were stained with H&E. As demonstrated in Fig. 1A, airway structural changes, including airway wall thickening, numerous inflammatory cell infiltration, subepithelial fibrosis, increased smooth muscle mass and epithelial metaplasia, were observed in the COPD group. However, C. sinensis treatment markedly ameliorated the aforementioned changes. In addition, Masson's trichrome staining was used to reveal the deposition of collagen and proliferation of smooth muscle hypertrophy in airway areas of the COPD group, while these changes were effectively decreased in rats treated with C. sinensis (Fig. 1B). Additionally, a significantly larger bronchiole tube area, thicker tube wall surrounded by proliferative connective tissue and increased MA% and MT% were detected in the COPD compared with that observed in the control group (P<0.01). These characteristics were significantly improved by C. sinensis treatment (P<0.05; Fig. 1C-F).

As inflammation serves an important role in the development of airway remodeling in COPD, the effect of C. sinensis treatment on lung inflammation was evaluated. BALF cell counts were performed to examine the effect of C. sinensis on the inflammatory response induced by cigarette smoke. Smoking treatment induced extensive infiltration of inflammatory cells, as demonstrated by the significant increase in the numbers of total cells, macrophages, neutrophils and lymphocytes in the COPD group compared with the numbers in the CON group (P<0.01). C. sinensis significantly reversed all types of cell accumulation in BALF of the COPD rats (P<0.05; Fig. 2A-D). In addition, the inflammatory cytokines, including TNF-α, IL-8 and TGF-β1, in BALF were significantly increased in the rats with COPD compared with the levels observed in the CON group (P<0.01; Fig. 2E-G), and the production of TNF-α, IL-8 and TGF-β1 were significantly reduced in rats treated with C. sinensis at all treatment doses (P<0.01; Fig. 2E-G).

To detect whether C. sinensis could inhibit airway remodeling, the effect of C. sinensis on the expression of collagen I and α-SMA in rats with COPD was examined by immunohistochemistry, western blotting and RT-qPCR. The present study revealed that the protein and mRNA expression levels of collagen I and α-SMA were significantly elevated in rats with COPD compared with the levels observed in the CON group (P<0.05; Fig. 3A-C). However, the collagen I and α-SMA protein and mRNA expression levels were significantly decreased in rats treated with C. sinensis (P<0.05; Fig. 3A-C).

A role for TGF-β1 has been proposed in airway remodeling of COPD (6,9,10). To explore the mechanisms by which C. sinensis inhibits airway remodeling, the expression levels of TGF-β1, TβR I and TβR II were assessed by immunohistochemistry, western blotting and RT-qPCR. As demonstrated in Fig. 4, the protein and mRNA expression levels of TGF-β1 were significantly increased in rats with COPD compared with the levels observed in the CON group (P<0.01). Meanwhile, the protein expression levels of TβR I and TβR II were also significantly increased in the COPD group compared with the levels observed in the CON group (P<0.01). The administration of C. sinensis significantly attenuated TGF-β1, TβR I and TβR II expression (P<0.05; Fig. 4A-C).

The TGF-β1/Smad signaling pathway is crucial in the regulation of the transcription of genes involved in airway remodeling in COPD (13). As demonstrated in the present study, the expression levels of signal proteins of p-Smad2 and p-Smad3 were significantly upregulated in the COPD group compared with the levels observed in the CON group (P<0.01; Fig. 5A-C). The expression of Smad7 was significantly decreased in the COPD group compared with the level observed in the CON group (P<0.01). Administration of C. sinensis for 12 weeks significantly decreased the high expression of p-Smad2 and p-Smad3 (P<0.05), and increased the protein expression level of Smad7 (P<0.05) in rats with COPD to different degrees (Fig. 5A-C).