Animals
Specific pathogen-free (SPF) female C57BL/6 mice, 6-week old with body weight of 18.64 ± 0.22 g, were purchased from Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. The animals were bred in a SPF room (ambient temperature 22–26℃, humidity 60–70%) and scrutinized for fitness upon arrival for three days, then randomized into different study groups and used within 2 weeks. The laboratory animal management rules of Tongji Medical College of Huazhong University of Science and Technology were strictly adhered to.
Polyvalent bacterial lysate and other laboratory materials
A mixture of polyvalent bacterial lysates (PVBLs) was used as the immunostimulant (Broncho-Vaxom™, batch No. S20150042, OM Pharma, Switzerland), it contains extracts derived from 8 bacterial species (Staphylococcus aureus, Streptococcus viridans, Streptococcus pyogenes, Diplococcus pneumoniae, Klebsiella pneumoniae, Klebsiella ozaenae, Neisseria catarrhalis, and Haemophilus influenza) [16]. Injection formulation of Bleomycin hydrochloride (BLM, batch No. H20055883) was purchased from HiSun Pharmaceutical CO., LTD, Zhejiang, China. A self-made transparent polyethylene exposure chamber (35cm×25cm×20 cm) with a connection to the outlet of a nebulizer (type NE-C900, OMRON) was set for the aerosolized PVBLs exposure.
Animal models
Firstly, we investigated the impact of aerosolized PVBLs on the animals’ general condition and lung morphology. The mice were randomized into 2 groups and put in the exposure chamber alternatively during an episode of the aerosol exposure, when the animals were either exposed to the aerosol of 10.5 mg PVBLs dissolved in 6 ml sterile PBS solution or to the aerosol of 6 ml sterile PBS solution (shamed exposure as control) at the 1st, 4th and 7th day of the experimental course. At the 8th day, we anesthetized the mouse by intraperitoneal injection of 50mg/kg·body weight (bw) sodium pentobarbital solution 1 ml and fixed the animal on a small animal surgery board when the anesthesia took into effect (lack of withdrawal reflex upon pinching a toe). We exposed the trachea through a standard aseptic surgery and infused 0.5 ml of sterile normal saline (NS) solution into the trachea during inspiration. To ensure a homogenous distribution, we blew 0.5 ml air into the trachea twice and then rotated the animal body for 2 minutes. After closing and sterilizing the wound, the animal was cared for on a warm pad (36℃) until fully recovery from anesthesia.
In the second step, we explored the effect of the multiple pre-exposures of the PVBLs on BLM-induced pulmonary fibrosis. Similarly, at each episode of the PVBLs exposure, the mice were put into the chamber and exposed to the aerosol of 10.5 mg PVBLs dissolved in 6 ml sterile PBS solution at the 1st, 4th and 7th day of the experimental course. At the 8th day, the animal was intra-tracheally infused 4mg/kg·bw BLM dissolved in 0.5 ml sterile NS, followed by blowing 0.5 ml air into the lung twice and 2-minute body rotation. The care for the animal after that was the same as described above.
Pulmonary morphology and morphometry
On the 22nd day of the experiment, i.e., 14 days later after intra-tracheal BLM infusion, the mice were euthanized by intraperitoneal injection of sodium pentobarbital 100mg/kg·bw and exsanguinated by severing the abdominal aorta immediately after death. We intratracheally intubated the animal, removed its right lung en bloc upon ligation of the right main bronchus, infused the left lung via the tracheal catheter with 4% paraformaldehyde (PFA) solution at a constant hydrostatic pressure of 20 cm for at least 20 minutes (until the pleural surface became smooth), then we fully immersed the left lung in PFA solution for at least 4 hours before further histological processing. The right lung was stored in -80℃ for further biochemistry measurement. The PFA fixed lungs were paraffin embedded and sagittal sections of mid-part of the lung sample with 4 µm thickness were deparaffinized and stained with hematoxylin and eosin.
Morphometric evaluation was referred to a modified Ashcroft’s scale [19]. Briefly, ten non-overlapping pulmonary microscopic fields under a 10-fold magnification were randomly selected for each slice of a specimen. The assignment of grades from 0 to 8 was based on the extent of fibrosis in alveolar septa and lung structure. To ensure the objectivity, three experts of pulmonary fibrosis pathology who blinded to the study design did the scoring. The average of the three scores was taken as the final score.
Determination of Hydroxyproline in lung tissue
The hydroxyproline content in lung tissue was measured using a Hydroxyproline Assay Kit (batch No. A030-2, JianCheng Bioengineering Institute, Nanjing, China).
Determination of IL-4 and IFN-γ in bronchoalveolar lavage fluid
For determination of IL-4 and IFN-γ in bronchoalveolar lavage fluid (BALF), the mice were also euthanized by intraperitoneal injection of sodium pentobarbital 100mg/kg·bw at the 22th day of the experiment. The procedure for collecting BALF was as that reported preciously [20]. IL-4 and IFN-γ in the BALF was measured using an ELISA kit for IL-4 and IFN-γ (batch No. E-EL-M0727, Elabscience Biotechnology Co.,Ltd., Wuhan, China) according to the manufacturer’s instruction.
Statistics
Statistical analyses were performed using SPSS 19.0 for Windows (IBM, USA) by a statistician who blinded to the study design. Test of normal distribution was by Kolmogorov-Smirnov method, the data approximated as normal distribution was expressed as mean ± Sd, the parametric statistics such as ANOVA and post hoc Dunnett’s comparisons were applied where appropriate. Results of Ashcroft’s scale were presented as median and 95% confidence interval (CI), the non-parametric statistics such as Kruskal-Wallis ANOVA & Median test and Mann-Whitney U test were used for inter-group comparisons of the Ashcroft’s scores. P < 0.05 was defined as statistical significant.