A total of 80 male Wistar rats (age, 8 weeks; weight, 200–240 g) were obtained from Vital River Laboratories (Beijing, China). The animals were fed a standard rat chow and water ad libitum and housed in a temperature-controlled environment (20–22°C) with an alternating cycle of 12-h light and dark. All rats were allowed to acclimatize for one week prior to the initiation of any experiments. The experimental design and procedures were approved by the Ethical Committee for Animal Care and Use at Pingmei Shenma Medical Group General Hospital (Pingdingshan, China).

Rats were randomized into 5 groups, each containing 16 animals. Group 1 consisted of saline-treated control rats and group 2 comprised bleomycin-treated rats. Animals in groups 3, 4 and 5 were treated with 10, 15 and 20 mg/kg/day apigenin, respectively, starting concurrently with the induction of lung fibrosis by the administration of bleomycin and continued until the end of the experiment. Single doses of bleomycin (5.0 mg/kg in 1.0 ml phosphate-buffered saline; Tianjin Taihe Pharmaceutical Co., Ltd., Tianjin, China) were injected into the rat lung intratracheally. Control animals received an identical volume of intratracheal saline instead of bleomycin. Apigenin (dissolved in olive oil; Sigma-Aldrich, St. Louis, MO, USA) was administered orally via a feeding tube. The day of bleomycin injection was defined as day 0 and the weight of the animals was recorded every 3–4 days.

Following the treatment, on days 7 and 28, the rats were anesthetized using 3% pentobarbital sodium and sacrificed by exsanguination at the cephalic artery. The lungs were removed and weighed and washed twice with cold saline. Then each lung was divided into two sections: The right section was fixed in 10% formalin solution for histological examination and the left section was prepared for biochemical assay and cytokine detection as described below.

The lung samples were prepared as 10% homogenates in 0.9% saline by homogenizer on ice according to their respective weight. The resulting homogenate was centrifuged, and the supernatant was collected and stored at −20°C until assayed. The assays of superoxide dismutase (SOD; A001-3), myeloperoxidase (MPO; A044) and hydroxyproline (A030-2) levels were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and performed according to the manufacturer's instructions.

Lung specimens were fixed in 10% formalin solution for 24 h, dehydrated in ethyl alcohol and embedded in paraffin. Subsequently, 5-µm sections were stained with hematoxylin and eosin (H&E) and Masson trichrome for histological evaluation of lung injury and fibrosis using an Olympus CKX41 light microscope (Olympus Corporation, Tokyo, Japan).

Lung homogenate (10%) was centrifuged at 12,000 × g for 30 min at 4°C to remove cell debris, and the supernatants were used for TNF-α and TGF-β determination. The levels of TNF-α were determined using an ELISA kit from Assaypro, LLC (St. Charles, MO, USA). TGF-β levels were assayed using a commercially available TGF-β ELISA kit (TGF-β E max ImmunoAssay System; Promega Corporation, Madison, WI, USA).

Statistical analysis was performed using SPSS software, version 19.0 (IBM SPSS, Armonk, NY, USA). Data are presented as the mean ± standard deviation. Statistical differences were analyzed using one-way analysis of variance, followed by post-hoc multiple comparison tests (least significant difference). P<0.05 was considered to indicate a statistically significant difference.

By contrast to the control group, the body weight of the bleomycin-treated group decreased gradually and reached the lowest level at day 7 after bleomycin injection (Fig. 1A). Apigenin treatment provided a dose-dependent protective effect on the body weight loss caused by bleomycin (Fig. 1A).

Lung index [weight of wet lung (mg)/body weight] is a parameter used for evaluating lung edema. In contrast with body weight loss, the lung index of the bleomycin-treated rats increased due to lung weight gain at days 7 and 28 after bleomycin injection compared with that of the normal control rats (Fig. 1B). Apigenin treatment inhibited the increase of the lung index of the bleomycin-treated rats in a concentration-dependent manner (Fig. 1B).

In order to assess the extent of fibrosis, the lung hydroxyproline levels were determined. As shown in Fig. 2A, at day 28 after bleomycin the lung hydroxyproline level in the bleomycin-treated rats was increased markedly compared with that of the normal control rats. Administration of apigenin reduced the hydroxyproline level in the lung homogenates in a dose-dependent manner (Fig. 2A).

The depletion of SOD indirectly indicates the production of oxygen free radicals following bleomycin injection. As shown in Fig. 2B, administration of bleomycin significantly decreased the SOD activity in the lung homogenates at day 28 after bleomycin injection. Treatment with apigenin concentration-dependently inhibited the decline of SOD activity caused by bleomycin administration (Fig. 2B).

MPO activity has been used as an index of leukocyte adhesion and accumulation in a range of tissues (10). Treatment with bleomycin elicited a profound increase in MPO activity in the lung homogenates at days 7 and 28 after bleomycin injection compared with that in the control group (Fig. 2C). Administration of apigenin led to a significant reduction in MPO activity, in a dose-dependent manner (Fig. 2C).

In order to identify the function of cytokines in lung fibrosis, TNF-α and TGF-β levels in lung homogenates were determined. As shown in Fig. 3A, at day 7 after bleomycin administration the level of TNF-α increased markedly compared with those in normal control rats. Apigenin suppressed the increase of TNF-α level dose-dependently (Fig. 3A). In addition, treatment of rats with bleomycin elicited a notable increase in TGF-β levels in the lung homogenates at days 7 and 28 after bleomycin injection compared with that in normal control rats (Fig. 3B). Administration of apigenin inhibited the increase of TGF-β level caused by bleomycin treatment in a concentration-dependent manner (Fig. 3B).

Rat lungs were examined histologically on day 7 after bleomycin injection (Fig. 4). The lung architecture in the control group appeared unaffected (Fig. 4A), while the bleomycin-treated rats exhibited collapsed and narrowed alveoli, marked thickening of the interalveolar septa and dense interstitial infiltration by inflammatory cells (Fig. 4B). In addition, Masson trichrome staining results demonstrated that there was excessive collagen deposition in the lung tissues of the bleomycin-treated rats (Fig. 4E) compared with the control rats (Fig. 4D). In contrast to the bleomycin-treated rats (Fig. 4B), the rats treated with bleomycin and 20 mg/kg apigenin presented with a significant suppression of inflammatory cell infiltration, reduced thickening of the interalveolar septa and increased inflation of the alveoli (Fig. 4C). Furthermore, 20 mg/kg apigenin treatment markedly reduced collagen deposition in the lung tissues of the bleomycin-treated rats, as indicated by Masson trichrome staining (Fig. 4F).