BLM and melatonin were from Sigma Chemical Co. (St. Louis, MO). XBP-1, α-SMA, GAPDH and phosphor-JNK antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). GRP78, phosphor-IRE1α, ATF6, and phosphor-eIF2α antibodies were from Cell Signaling Technology (Beverley, MA). Chemiluminescence (ECL) detection kit was from Pierce Biotechnology (Rockford, IL). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO) if not otherwise stated.

Adult male CD-1 mice (8 week-old, 28–32 g) were purchased from Beijing Vital River whose foundation colonies were all introduced from Charles River Laboratories, Inc. The animals were allowed free access to food and water at all times and maintained on a 12-h light/dark cycle in a controlled temperature (20–25°C) and humidity (50±5%) environment. This study was approved by the Association of Laboratory AnimalSciences and the Center for Laboratory Animal Sciences at Anhui Medical University (Permit Number: 12-0046). All procedures on animals followed the guidelines for humane treatment set by the Association of Laboratory Animal Sciences and the Center for Laboratory Animal Sciences at Anhui Medical University.

For the induction of pulmonary fibrosis, mice were intratracheally injected with BLM (5.0 mg/kg body weight in 50 µL phosphate buffered saline). To investigate the effects of melatonin on BLM-induced pulmonary fibrosis, mice were intraperitoneally (i.p.) injected with melatonin (5 mg/kg) daily, beginning at 30 min before BLM. Melatonin was dissolved in 10% ethanol and further diluted in saline (0.09% NaCl w/v) to give a final concentration of 1% ethanol. Thus, control mice received an i.p. injection of 1% ethanol daily as a control for the melatonin injections. All mice were euthanized by exsanguination during pentobarbital anesthesia (75 mg/kg,i.p.) 21 days after BLM injection. Lungs were weighed and relative lung weights were calculated. Lung fibrosis was assessed by pulmonary hydroxyproline content as well as lung histology. Some lung samples were collected and kept at –80°C for subsequent immunoblots.

Pulmonary collagen content was determined by the measurement of hydroxyproline content. In brief, lung lobes were homogenized in 1 mL of phosphate buffered saline (PBS, pH = 7.4) and then hydrolyzed in 1 mL of 6 N hydrochloric acid for 16 hours at 110°C, and neutralized to pH 7.0 with NaOH. Chloramines T reagent (1 mL of 0.5 mol/L) was then added and the samples were left at room temperature for 20 minutes. Then 20% p-Dimethylaminobenzaldehyde solution (dissolved in 3.15 N perchloric acid) was added to each sample, and the mixture was incubated at 60°C for 15 minutes. Absorbance was measured at 550 nm. Pulmonary hydroxyproline content was expressed as mg/lung.

Lung tissues were fixed in 4% formalin and embedded in paraffin according to the standard procedure. Paraffin-embedded lung tissues were serially sectioned. At least five consecutive longitudinal sections were stained with hematoxylin and eosin (H&E) and scored for the extent of pathology on a scale of 0 to 5, where 0 was defined as no lung abnormality, and 1, 2, 3, 4, and 5 were defined as the presence of inflammation involving 10%, 10–30%, 30–50%, 50–80%, or >80% of the lungs, respectively. Lung fibrosis was evaluated by Sirius red staining for collagen accumulation. The percentages of collagen deposition areas were quantified using NIH ImageJ software (http://rsb.info.nih.gov/ij/).

Total pulmonary lysate was prepared by homogenizing 50 mg lung tissue in 300 µl lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecylsylphate, 1 mM phenylmethylsulfonyl fluoride) supplemented with a cocktail of protease inhibitors (Roche). For nuclear protein extraction, total pulmonary lysate was suspended in hypotonic buffer and then kept on ice for 15 min. The suspension was then mixed with detergent and centrifuged for 30 s at 14,000×g. The nuclear pellet obtained was resuspended in complete lysis buffer in the presence of the protease inhibitor cocktail, incubated for 30 min on ice, and centrifuged for 10 min at 14,000 × g. Protein concentrations were determined with the bicinchoninic acid (BCA) protein assay reagents (Pierce, Rockford, IL) according to manufacturer's instructions. For immunoblots, same amount of protein (30–60 µg) was separated electrophoretically by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membranes were incubated for 2 h with the following antibodies: XBP-1, α-SMA, GAPDH, phosphor-JNK, GRP78, phosphor-IRE1α, ATF6, and phosphor-eIF2α. For total proteins, GAPDH was used as a loading control. For nuclear protein, lamin A/C was used as a loading control. After washes in DPBS containing 0.05% Tween-20 four times for 10 min each, the membranes were incubated with goat anti–rabbit IgG or goat anti–mouse antibody for 2 h. The membranes were then washed for four times in DPBS containing 0.05% Tween-20 for 10 min each, followed by signal development using an ECL detection kit. The density of the specific bands was quantified using NIH ImageJ software (http://rsb.info.nih.gov/ij/).

For immunohistochemistry, paraffin-embedded lung sections were deparaffinized and rehydrated in a graded ethanol series. After antigen retrieval and quenching of endogenous peroxidase, sections were incubated with α-SMA monoclonal antibodies (1∶200 dilution) at 4°C overnight. The color reaction was developed with HRP-linked polymer detection system and counterstaining with hematoxylin.

Normally distributed data were expressed as means ± SEM. ANOVA and the Student-Newmann-Keuls post hoc test were used to determine differences among different groups. Data that were not normally distributed were assessed for significance using non-parametric tests techniques (Kruskal-Wallis test and Mann–Whitney U test). P <0.05 was considered to indicate statistical significance.

An obvious pulmonary edema was observed in BLM-treated mice. Consistent with pulmonary edema, the absolute and relative weights of the lungs were significantly increased in BLM-treated mice (Figures 1A and 1B). Of interest, melatonin significantly attenuated BLM-induced pulmonary edema. Correspondingly, melatonin significantly alleviated BLM-induced elevation of the absolute and relative lung weight (Figure 1A and 1B). Histological examination showed that melatonin significantly alleviated BLM-induced infiltration of inflammatory cells in the lungs (Figure 1C and 1D). The hallmark characteristic of BLM-induced pulmonary fibrosis is the excessive deposition of an extracellular matrix, such as collagen. As shown in Figures 2A and 2B, an obvious matrix protein deposition, as evidenced by Sirius red staining, was observed in the lungs of BLM-treated mice. As expected, melatonin significantly attenuated BLM-induced matrix protein deposition in the lungs. Moreover, melatonin significantly attenuated BLM-induced elevation of hydroxyproline content in the lungs (Figure 2C).

Alpha-SMA is a hallmark of myofibroblasts and is also accepted as a marker of pulmonary fibrosis. As shown in Figures 3A and 3B, α-SMA was up-regulated in the lungs of BLM-treated mice. Immunohistochemistry showed that α-SMA was expressed in the area of pulmonary fibrosis in BLM-treated mice (Figure 3C). Of interest, melatonin significantly attenuated BLM-induced up-regulation of α-SMA in the lungs (Figures 3A, 3B and 3C).

The effects of melatonin on BLM-induced pulmonary ER stress were analyzed. As shown in Figure 4A, the cleaved ATF6 in the nuclei was significantly increased in the lungs of mice treated with BLM. Correspondingly, pulmonary GRP78, an ER chaperone and the target of ATF6 pathway, was up-regulated in BLM-treated mice (Figure 4B). Of interest, melatonin markedly attenuated BLM-induced elevation of the cleaved ATF6 in the lungs. Moreover, melatonin inhibited BLM-induced up-regulation of pulmonary GRP78. Eukaryotic initiation factor 2α (eIF2α) is a downstream target of the PERK pathway. As shown in Figure 5, phosphorylated eIF2α in the lung was increased in BLM-treated mice. Of interest, melatonin significantly attenuated BLM-induced eIF2α phosphorylation in the lungs. The effects of melatonin on pulmonary IRE1 pathway were then analyzed. As shown in Figure 6A, the level of phosphorylated IRE1α in the lungs was significantly increased in BLM-treated mice. Consistent with IRE1α phosphorylation, nuclear XBP-1, a downstream target of the IRE1α pathway, was elevated in the lungs of BLM-treated mice (Figure 6B). JNK, another downstream target of the IRE1 signaling, was activated in the lungs of BLM-treated mice (Figure 6C). As expected, melatonin significantly attenuated BLM-induced pulmonary IRE1α phosphorylation (Figure 6A). Moreover, melatonin significantly alleviated BLM-evoked activation of XBP-1 and JNK in the lungs (Figure 6B and 6C).