The present study was approved by the ethics committee of the Harbin Medical University (Harbin, China). All procedures were performed according to the Harbin Medical University Institutional Animal Care and Use Committee guidelines and national guidelines for the treatment of animals. The adult male Sprague Dawley rats weighing 240–280 g used in the present study were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The rats were maintained under a controlled temperature (24±2ºC) on a 12 h light/12 h dark cycle, and were given ad libitum acces to standard laboratory chow and tap water. Rats were assigned to receive continuous intraperitoneal infusions of α-CGRP or 0.9% normal saline. They subsequently underwent either LPS or 0.9% normal saline intratracheal instillation. Rats were divided into four groups: i) Saline-saline (S-S) group, n=10; ii) saline-α-CGRP (S-C) group, n=10; iii) LPS-saline (L-S) group, n=14 and iv) LPS-α-CGRP (L-C) group, n=14. Four animals from each group were used for the evaluation of pulmonary permeability with Evans blue (EB) dye.

Rats were anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg; Shanghai Xitang Biotechnology Co., Ltd., Shanghai, China). Subsequently, a tracheostomy was performed, the rats were mechanically ventilated [fraction of insprired oxygen (FiO₂), 0.6; rate, 65 breaths/min; tidal volume, 8 ml/kg and positive end-expiratory pressure, 3 cm H₂O; Harvard Inspira ASV, Harvard Apparatus, Holliston, MA, USA] and femoral arterial and vein cannulas were inserted. Anesthesia and muscle relaxation were maintained by intravenous pentobarbital (30 mg/kg/h) and pancuronium bromide (0.2 mg/h; Gedeon Richter Ltd., Budapest, Hungary) injection. Warming pads were used to maintain the animals’ body temperature at 36.5–37.5ºC. Electrocardiography (ECG), arterial pressure and rectal temperatures were continuously monitored (AcqKnowledge and MP150 version 2.7.2; BIOPAC Systems, Inc., Goleta, CA, USA), and femoral arterial blood was used for blood gas analysis. In the S-C and L-C groups, the animals were intraperitoneally pre-treated with α-CGRP (C0292; Sigma-Aldrich, St. Louis, MO, USA) at 0.4 μg/kg/min for 30 min. This dose was determined with pilot experiments and did not result in a significant decrease in the mean arterial pressure (MAP), while the oxygenation index was significantly improved. In the S-S and L-S groups, an equal volume of normal saline was infused over 30 min. Thereafter, in the L-S and L-C groups, 0.5 mg/kg LPS (Escherichia coli, O55:B5; Sigma-Aldrich) was administered intratracheally in 0.3 ml normal saline. In the S-S and S-C groups, 0.3 ml normal saline was administered. MAP, temperature, heart rate and arterial blood gases (pH, PaO₂, PaCO₂) were recorded hourly throughout the experiments. Following 4 h, animals were sacrificed by exsanguination, and the heart-lung block was dissected from the thorax. Additionally, bronchoalveolar lavage fluid (BALF) was obtained from the left lung, the upper right lobe was harvested for the wet-dry (W/D) weight ratio and the middle right lobe was harvested for histology. All remaining lung tissue was frozen in liquid nitrogen and kept at −80ºC for further analyses.

The left lung was lavaged three times with 3 ml cold normal saline through a tracheal cannula. BALF was centrifuged at 1,500 rpm for 10 min at 4ºC. The supernatant was frozen at −20ºC for later analysis. The cells were resuspended in 1 ml 0.9% normal saline, subjected to Wright-Giemsa staining (Nanjing Jiancheng Biotechnology Institute, Nanjing, China) and counted using a Neubauer chamber (Anxin Optical Instrument Manufacture Co., Ltd., Shanghai, China).

The upper right lobe was weighed immediately following excision, and the dry weight was determined after heating the lungs at 80ºC for 48 h. The W/D ratio was calculated by dividing the wet weight by the dry weight.

To assess pulmonary permeability, EB extravasation was measured as previously described (20). Animals received EB dye (30 mg/kg; Sigma-Aldrich) intravenously 2 h prior to sacrification (n=4 per group). The lungs were perfused free of blood with saline via a thoracotomy, excised en bloc, blotted dry and weighed. Pulmonary tissue was homogenized in saline (0.1 ml/100 μg tissue) and incubated with two volumes of formamide (Merck Millipore, Billerica, MA, USA) for dye extraction (24 h, 37ºC). The supernatant was separated from the lung tissue by centrifugation at 5,000 × g for 30 min, and the optical supernatant density was determined spectrophotometrically at 620 nm. The extravasated EB concentration was calculated against a standard curve and expressed as micrograms of EB dye per gram of lung tissue.

The middle right lobe was fixed in 4% paraformaldehyde (Sigma-Aldrich) for 48 h, embedded in paraffin and then cut into 5-μm sections. The sections were stained with hematoxylin and eosin (H&E; Sigma-Aldrich) and examined by light microscopy (Eclipse 80i; Nikon Corporation, Tokyo, Japan).

BALF tumor necrosis factor (TNF)-α, IL-1β, macrophage-inflammatory protein-2 (MIP2) and intracellular cell adhesion molecule 1 (ICAM-1) levels were measured using Rat TNF-α Boster™ ELISA kit, Rat IL-1β Boster™ ELISA kit, Rat MIP2 Boster™ ELISA kit and Rat ICAM-1 Boster™ ELISA kit, according to the manufacturer’s instructions (Boster Systems, Inc., Wuhan, China).

α-CGRP concentration in lung tissue from the S-S and L-S groups was analyzed at each time-point (n=4 per point) with a commercial ELISA kit (CGRP kit; Uscn Life Science, Inc., Wuhan, China).

Immunohistochemistry was performed on paraffin-embedded sections. Following deparaffinization and rehydration in two changes of xylene and six different concentrations of alcohol (Sigma-Aldrich), sections were retrieved in a retrieval solution (9 mM sodium citrate, 2 mM citric acid) at 120ºC for 3 min and blocked with 2% bovine serum albumin (Boster Systems, Inc.) in phosphate-buffered saline (PBS) for 30 min once cool. Sections were immunostained with purified polyclonal goat anti-rat CRLR (1:20 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4ºC. Sections were incubated with a rabbit anti-rat secondary immunoglobulin G antibody (1:400; Boster Systems, Inc.) for 30 min. Specific labeling was detected with 3,3′-diaminobenzidine substrate (Sigma-Aldrich). All sections were counterstained with hematoxylin. A sample treated analogously but with omission of the primary antibody served as a negative control.

Lung tissue total RNA was isolated using an RNA pure kit (BioTeke Corp., Beijing, China) according to the manufacturer’s instructions; 1 μg total RNA was used in a 20-μl reaction to synthesize cDNA with a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Life Technologies, Foster City, CA, USA). qRT-PCR was performed using an Accupower 2xGreenstar qPCR Master mix (Bioneer, Daejeon, Korea). Rat β-actin mRNA was used as the internal control. The primer sequences used were as follows (5′-3′): β-actin forward, GTC AGG TCA TCA CTA TCG GCA AT and reverse, AGA GGT CTT TAC GGA TGT CAA CGT; CRLR forward, CAA ACA GAC TTG GGA GTC ACT AG and reverse, CTG CAA CGT CAT TCC AGC AT; RAMP1 forward, CAC CAA ACT CGT GGC AAA C and reverse, GGG GGA GCA CAA TGA AAG G; ICER forward, GCT CCT ACT ACT GCT TTG C and reverse, GCT TTC GAG TTG TTG CTT CTT C; TNF-α forward, CTT CTC ATT CCT GCT CGT GG and reverse, TCC TCC GCT TGG TGG TTT. All primers were designed and synthesized by Settlebio, Co. (Harbin, China). The reaction was performed as follows: Denaturation at 94ºC for 2 min, 30 cycles at 94ºC for 30 sec, 55ºC for 60 sec and 72ºC for 50 sec, followed by 10 min at 75ºC. Each sample was evaluated in duplicate. The amplification specificity of PCR products was confirmed by melting curve analysis, agarose gel electrophoresis and sequencing. Relative messenger RNA (mRNA) levels were calculated using the 2^−Δ(ΔCT) method and normalized to levels of β-actin as a reference (21).

Total protein was obtained from lung tissue homogenates in a membrane lysis buffer containing a detergent [16 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 20 mM Tris-HCl, pH 7.5, 1 mM Na₂EDTA and 1 mM dithiothreitol] (Sigma-Aldrich) with protease inhibitors (1 mM benzamidine, 1 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor and 0.5 mM PMSF; Sigma-Aldrich), as previously described (22). Protein concentrations were determined using a Bradford reagent assay (23). CRLR and RAMP1 western blot analysis was performed using 30 μg protein. Proteins were separated by 10–15% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked for 2 h in Tris-buffered saline containing 0.1% Tween and 5% non-fat dried milk (Nanjing Jiancheng Biotechnology Institute) and incubated with primary antibody at 4ºC overnight. The primary antibodies used in this experiment were goat polyclonal anti-CRLR (sc-18007; Santa Cruz Biotechnology, Inc.) at a 1:200 dilution, rabbit polyclonal anti-RAMP1 (sc-11379; Santa Cruz Biotechnology, Inc.) at a 1:300 dilution and mouse polyclonal anti-β-actin (Boster) at a 1:1,000 dilution. Following washing, the membranes were incubated with horseradish peroxidase-conjugated enhanced chemiluminescence (ECL) rabbit anti-goat, goat anti-rabbit or rabbit anti-mouse secondary antibodies. The ECL western blotting system (Beyotime Institute of Biotechnology, Haimen, China) was used for detection. Proteins were quantified with a digitized image and normalized against β-actin.

Values are expressed as the mean ± standard error of the mean. MAP, PaO₂/FiO₂ and α-CGRP analyses between groups were assessed by repeated measures tests followed by Tukey’s test. Differences in other data were examined using one-way analysis of variance followed by least significant difference test or unpaired Student’s t-test. All statistical assessments were two-sided and P<0.05 was considered to indicate a statistically significant difference between values. Statistical analyses were carried out using either SAS 9.2 (SAS Institute Inc., Cary, NC, USA) or SPSS 21.0 (IBM, Armonk, NY, USA).

The MAP was decreased in the four groups during the course of the experiment. There were no significant differences in MAP between the four groups at any time-point (P>0.05; Fig. 1A).

Instillation of LPS resulted in a significant decrease in PaO₂/FiO₂ in the L-S and L-C groups compared to that of the S-S and S-C groups following 1 h of injury (P<0.05 at each time-point). Animals in the L-C group had significantly higher PaO₂/FiO₂ levels at 3 and 4 h after LPS instillation compared with those in the L-S group (312.50±17.27 vs. 270.90±27.80 mmHg, P<0.05 at 3 h; 307.4±25.05 vs. 252.6±23.88 mmHg, P<0.05 at 4 h). There were no significant differences between the two S-S and S-C groups (Fig. 1B).

The lung W/D weight ratio was significantly increased 4 h following LPS instillation (P<0.05 vs. S-S group) (Fig. 2A). α-CGRP infusion significantly attenuated the lung W/D weight ratio increase (4.674±0.22 vs. 5.16±0.21, P<0.05).

LPS induced an increase in EB dye accumulation in lung parenchyma (P<0.05 vs. S-S group; Fig. 2B). α-CGRP treatments resulted in a significant decrease in EB dye accumulation (20.35±4.51 vs. 27.8±5.29 μg/g lung, P<0.05).

Total BALF cell numbers were significantly increased 4 h following LPS instillation (P<0.05 vs. S-S group; Fig. 2C) and L-C group cell numbers were significantly lower than those of the L-S group (4.94±0.78×10⁶ vs. 6.34±1.06×10⁶, P<0.05).

The TNF-α, IL-1β, IL-6, MIP-2, and ICAM-1 protein levels in BALF were significantly increased in the L-S and L-C groups compared with those of the S-S and S-C groups. The L-C group had significantly lower TNF-α, IL-1β, MIP-2, and ICAM-1 levels in BALF compared with those of the L-S group (P<0.05; Fig. 3A–D).

Histological analysis demonstrated that lungs in the S-S and S-C groups exhibited no morphological injuries (Fig. 4). A representative lung tissue section from the L-S group demonstrated increased wall thickness and marked inflammatory cell infiltration compared with that of the S-S group. Furthermore, α-CGRP pre-treatment decreased wall thickness and inflammatory cell infiltration.

The lung homogenate α-CGRP levels in the L-S group were significantly higher than those in the S-S group from 1 h following LPS administration at each time-point (P<0.05; Fig 5).

Immunohistochemistry detected CRLR expression in pulmonary microvascular endothelial cells and alveolar macrophages in LPS-induced lung injury (Fig. 6Aa and Ab). RT-qPCR and western blot analysis revealed lower mRNA and protein expression levels of CRLR and RAMP1 in the L-S group compared with those in the S-S group (P<0.05; Fig. 6B and C; Fig. 7).

RT-qPCR assays demonstrated that ICER mRNA expression levels were significantly higher in the L-C group at two and 4 h than those in the L-S group (P<0.05; Fig. 8A). TNF-α mRNA expression was significantly lower in the L-C group compared with those in the L-S group at two and 4 h (P<0.05; Fig. 8B).