The pericarp of Citrus reticulata Blanco (Rutaceae) was obtained from the Department of Medicinal Materials, Affiliated Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing University of Chinese Medicine (Nanjing, China), and identified by Professor Z.N. Gong from the School of Life Science in Nanjing Normal University (Nanjing, China). The voucher specimen (no. TCM130917) was deposited in the hospital.

The chemicals and reagents were purchased from the following sources: bleomycin A5 hydrochloride from Nippon Kayaku Co., Ltd. (Tokyo, Japan); prednisone from Hubei HolleyPharm Co., Ltd. (Hubei, China); hydroxyproline test kits from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China); transforming growth factor (TGF)-β₁ from Strept Avidin-Biotin Complex (SABC); immunohistochemical test kit from Roche Diagnostics (Indianapolis, IN, USA); penicillin and streptomycin from Wuhan Boster Biological Technology, Ltd. (Wuhan, China); DMEM from HyClone (Logan, UT, USA); fetal bovine serum (FBS) from Hangzhou Ever Green Organism Engineering Materials Co., Ltd. (Hangzhou, China).

The alkaline extract of C. reticulata was prepared according to our previously reported procedure (23). A portion of the alkaline extract (10 g) was added to a silica gel (125 g) column, and eluted with CHCl₃-triethylamine-MeOH (99.2:0.4:0.4, 98.6:0.4:1 and 97.6:0.4:2) successively to yield 5 fractions: fr-1 (0.4 g), fr-2 (0.6 g), fr-3 (3.9 g), fr-4 (2.5 g) and fr-5 (2.2 g). Fr-3 was further chromatographed on a medium-pressure column with CHCl₃-triethylamine-MeOH (99.5:0.4:0.1), to obtain amines 1 (22 mg) and 2 (40 mg). Fr-4 was applied to the same medium-pressure column with CHCl₃-triethylamine-MeOH (99.5:0.4:0.2) to produce compounds 2 (43 mg) and 3 (45 mg). Repeated medium-pressure column chromatography of fr-5 with CHCl₃-triethylamine-MeOH (99.5:0.4:0.2) produced amines 4 (18 mg) and 5 (20 mg). The structures of the isolated amines (4-methoxy-phenethylamine, synephrine, para-tyramine, N-methyltyramine and N-methyl-4-methoxyphenethylamine) were determined based on detailed comparisons of proton NMR (¹H-NMR) spectra and MS data with those of commercially available samples. For the animal experiments, the commercially available amine (Tokyo Chemical Industry, Tokyo, Japan) was used.

To improve the solubility of the amines, each of the above-mentioned amines (amines 1–5) was dissolved in methanol (5 mg/ml), and 10% HCl methanol solution was added (1:2, v/v). Subsequently, the solution was completely evaporated under reduced pressure to provide each amine hydrochloride for bioassays.

The hELFs used in the present study were obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The procedure for cell culture followed our previously reported method (21). In brief, the hELFs were grown in DMEM containing 10% FBS and 1% penicillin-streptomycin. The logarithmically growing cells were detached with 0.25% trypsin PBS and centrifuged (100 × g, 5 min, 4°C), and the cell concentration was then adjusted to 5×10⁴/ml using culture medium. Each amine salt was dissolved in the medium and then diluted to the desired concentrations. The amine and hELF cell solutions (each 100 μl) were added to 96-well culture plates and incubated for 48 h. Subsequently, an MTT assay was used to evaluate cell viability.

To evaluate the cytotoxicity of the amine hydrochlorides, an LDH release assay was performed using an LDH cytotoxicity detection kit from Nanjing Keygen Biotech Co., Ltd. (Nanjing, China). hELF cell culture was conducted according to the same protocol as described above, except that the culture period was 24 h. Following culture, the LDH concentration was measured following the manufacturer's instructions. The relative LDH release was calculated as the ratio of the LDH release over the positive control, and the positive control was treated with only 1% NP-40 and set as 100% LDH release, as previously reported (23). All cultures were kept in a CO₂ incubator under moist conditions with 5% CO₂, at 37°C.

The rats used in the present study were purchased from the Laboratory Animal Center, Nanjing University of Chinese Medicine (Nanjing, China). Pathogen-free mature male Sprague-Dawley (SD) rats (weighing 180–200 g) were used in our experiments. The rats were housed in a conventional animal facility with a 12-h light/dark cycle, and had access to standard pelleted food and water ad libitum. The rats were allowed to acclimatize for 7 days prior to their use in our experiments.

All of the procedures involving animals and their care were approved by the Jiangsu Animal Care and Use Committee and followed the national and institutional rules regarding animal experiments.

The SD rats were randomly divided into 6 groups (n=6 per group). The rat model of bleomycin-induced pulmonary fibrosis was established following our previously reported method (23). Briefly, the rats were anesthetized by an intraperitoneal injection of pentobarbital, followed by a single intratracheal instillation of 5 mg/kg of bleomycin in 2.0 ml/kg PBS. The animals in the normal group received an intratracheal injection of an equal volume of the PBS instead of bleomycin. There were 4 groups for drug treatment: the rats were orally administered 4-methoxyphenethylamine hydrochloride (designated as amine hydrochloride 1) once daily at doses of 5 (treatment group 1; 1–5), 10 (treatment group 2; 1–10) and 20 (treatment group 3; 1–20) mg/kg (in distilled water) and prednisone (treatment group 4) at a dose of 5 mg/kg once per day. The animals in the normal (group 5) and control (group 6) groups received an equal volume of distilled water only. The experimental period lasted for 4 weeks. At the end of the experiment, the rats were euthanized by pentobarbital administration, and the serum and lung tissues were collected for bioassays. The blood was centrifuged at 2,500 rpm for 20 min, and then kept at −20°C until use. The lung vasculature was perfused to free the blood. The right lung was fixed in neutral buffered 10% formalin (pH 7.4) for histopathological analysis and analysis of TGF-β1 expression. The left lung was frozen in liquid nitrogen and stored at −80°C for hydroxyproline level measurements.

The right lung tissue was fixed by using 10% neutralized buffered formalin on the trachea, and embedded in paraffin. Serial sections (4-μm-thick) were acquired and stained with hematoxylin and eosin (H&E) and Masson's trichrome staining, following the manufacturer's instructions for light microscopic evaluation. The histological grading of alveolitis and fibrosis in the lung specimens was performed by experienced pathologists in a blinded manner, and recorded in grades from − to +++ and corresponding scores from 0 to 3, as previously described (24).

The hydroxyproline content was determined as an index of the collagen content in the serum and left lung tissues as previously described (20). Briefly, 0.5 ml of serum or 30–100 mg lung tissue samples were respectively hydrolyzed in 1 ml lysis buffer solution (pH 7.4, 10 mM Tris-HCl, 0.1 mM EDTA-2Na, 10 mM saccharose, 0.8% sodium chloride solution) at 100°C for 20 min. Hydroxyproline was then measured using the test kit according to the manufacturer's instructions. The absorbance of colored products was measured at 550 nm.

The semi-quantification of the TGF-β₁ protein levels in the right lungs of rats was assessed with an SABC immunohistochemical test kit following the manufacturer's instructions as previously described (20). Briefly, paraffin-embedded lung sections were dewaxed with xylene and dehydrated with a series of ethanol. Endogenous peroxidase was inactivated by treatment with 3% hydrogen peroxide (H₂O₂) for 10 min at room temperature. Non-specific binding was blocked with normal goat serum for 20 min. The sections were then incubated with primary antibody rabbit anti-TGF-β1 for 2 h at 37°C, and successively with biotinylated goat anti-rabbit secondary antibody for 20 min at 25°C, followed by SABC for 20 min at 25°C. Immunoreactivity was detected by the addition of diaminobenzidine (DAB). The sections were counterstained with hematoxylin, dehydrated and mounted with permont. As the negative controls, each primary antibody was substituted with PBS. The protein expression of TGF-β1 was assessed using a quantitative image analysis system.

Data are presented as the means ± standard deviation (SD). Statistical analysis was undertaken by analysis of variance (ANOVA) followed by appropriate post hoc tests, including multiple comparison tests (LSD and t-test). The alveolitis and fibrosis scores of lung tissues were evaluated using the Mann-Whitney test. The SPSS 13.0 software package (SPSS, Inc., Chicago, IL, USA) was used for the analysis. A p-value <0.05 was considered to indicate a statistically significant difference.

The alkaline extract of C. reticulata (10 g) was first subjected to normal- and medium-pressure silica gel column chromatographies repeatedly, and subsequently 5 amines, 4-methoxyphenethylamine, synephrine, para-tyramine, N-methyltyramine and N-methyl-4-methoxyphenethylamine were isolated. The structures of the isolated amines were then determined, based on detailed comparisons of ¹H-NMR spectra and MS data with those of commercially available samples (data not shown).

To improve the aqueous solubility of the isolated amines, the amine hydrochlorides, 4-methoxyphenethylamine hydrochloride (amine hydrochloride 1), synephrine hydrochloride (amine hydrochloride 2), para-tyramine hydrochloride (amine hydrochloride 3), N-methyltyramine hydrochloride (amine hydrochloride 4) and N-methyl-4-methoxyphenethylamine hydrochloride (amine hydrochloride 5) were prepared with 10% HCl methanol solution (Fig. 1), and used for all of the following bioassays.

To screen the inhibitory activity of the amine hydrochlorides, hELFs were used. All amine hydrochlorides were dissolved in medium, and their inhibitory activity on the proliferation of hELFs was firstly assayed at 10 μM. As shown in Fig. 2, amine hydrochlorides 1 and 5 significantly inhibited hELF proliferation following 48 h of incubation, and it should be noted that amine hydrochloride 1 caused a 51.63% inhibition of cell viability. Amine hydrochloride 3 exerted no inhibitory effect, whereas amine hydrochlorides 2 and 4 exerted a weak inhibitory effect. Due to the fact that compound 1 displayed the most potent effect, further detailed tests on its minimal half inhibitory concentration (IC₅₀) were performed, and the IC₅₀ of compound 1 was noted as 12.7 μM.

The cytotoxicity of amine hydrochlorides 1 and 5 on hELF viability was evaluated by an LDH release assay. The results of the assay revealed that at concentrations up to 20 μM, amine hydrochlorides 1 and 5 did not cause cytotoxicity to the cells (data not shown).

As shown in Table I, the body weight of the rats decreased significantly on the 7th day following the administration of bleomycin and decreased gradually thereafter throughout the whole experiment period compared to the normal rats (p<0.01). Even though the rats in the control group gained weight slightly throughout the experimental period, their weight was lower than the rats in the normal group. Prednisone, a clinically available drug used to treat IPF was used as a positive control, did not have much of a positive impact on weight loss. However, body weight was significantly increased following treatment with amine hydrochloride 1 at doses of 10 and 20 mg/kg/day from the 14th to 28th day compared to the bleomycin-treated group.

The effects of amine hydrochloride 1 on collagen deposition in the rats with IPF were first evaluated by light microscopy using lung tissue sections stained with Masson's trichrome staining. As shown in Fig. 3, in the normal group, only a small amount of collagen fibers in the alveolar septum were observed. In the lungs of the bleomycin-treated rats, we noted an extended web of collagen-positive stained areas in an irregular pattern (control group). However, the lungs of the rats treated with amine hydrochloride 1 displayed less collagen accumulation compared with those of the rats in the control group, particularly in the rats treated with amine hydrochloride 1 at 20 mg/kg/day, where only a mild deposition of collagen fibers in the alveolar septum was observed.

Subsequently, the hydroxyproline content in the lungs and serum was determined. The rats treated with bleomycin alone (the control group) showed a significant increase in hydroxyproline content, by 1.8-fold in the lungs (μg/g, p<0.01) and 1.36-fold in the serum (μg/ml, p<0.01), compared with those of normal rats (Fig. 4). Treatment with amine hydrochloride 1 at all doses significantly decreased the hydroxyproline contents in both the lung and serum compared with the control group. These results clearly indicated that amine hydrochloride 1 inhibited collagen deposition.

Lung fibrosis was further scored by histopathological observations of the lung sections on the 28th day. H&E staining of the lung tissues from the normal rats revealed a normal histological appearance in terms of bronchi, bronchioles and alveoli (Fig. 5, Nor). The administration of bleomycin caused increased thickness of the alveolar wall, alveolar and vascular congestion, and severe infiltration of inflammatory cells in the alveolar septa and interstitium in lung tissues were observed (Fig. 5, Con). The prednisone group displayed only slightly thickened alveolar walls with some inflammatory cells. Treatment with amine hydrochloride 1 significantly reduced bleomycin-induced inflammatory cell infiltration, ameliorated the thickening of the interalveolar septum with edema and restored the alveolar architecture (Fig. 5, 1–5, 1–10 and 1–20).

As shown in Fig. 6, the semi-quantitative assessment of the lung sections revealed that an approximately 3-fold score increase in alveolitis, and 4.4-fold in fibrosis, was observed in the control groups compared to the normal group. Both the alveolitis and fibrosis scores were significantly decreased in the prednisone-treated groups. The lungs of the rats treated with amine hydrochloride 1 only at 20 mg/kg/day exhibited reduced pathological alveolitis scores, and the fibrosis scores were markedly attenuated at all doses.

Since TGF-β₁ appears to be closely associated with fibroblast-to-myofibroblast activation and drives pulmonary fibrosis in the current rat model (21), TGF-β₁ protein expression was assessed using immunohistochemical staining. The results of the protein expression of TGF-β₁ in the lungs is shown in Table II. As expected, the administration of bleomycin markedly induced TGF-β₁ protein expression, while the positive control, prednisone, significantly suppressed its expression. Treatment of the rats with amine hydrochloride 1 significantly repressed TGF-β₁ protein overexpression at all doses, particularly the dose of 20 mg/kg/day, which decreased the expression to values close to those of the normal group.