Experimental groups
In this study, fifty male rats (weighing 200-250 g, 8-9 weeks) were enrolled in the study. These animals were kept in standard condition and permitted to access chewing food and water ad libitum. Fourteen days after accommodation, 40 rats were randomly selected and divided into the following groups; healthy rats received 50 µl of phosphate-buffered saline (PBS) intra-tracheally (C group); sensitized rats received 50 µl of PBS intra-tracheally (S group); sensitized rats received 50 µl of PBS intra-tracheally containing 3×105 C-kitˉ cells (S+C-kit- group); and sensitized rats received 50 µl of PBS intra-tracheally containing 3×105 C-kit⁺ cells (S+C-kit⁺ group). The remaining rats were used for the extraction of C-kit⁺ and C-kit- cells.
Induction of asthma
The asthma model of the rat was established according to the previously used method [31, 32]. Rats were sensitized by injection of 1 mg ovalbumin (OVA; Sigma-Aldrich, USA) mixed with 200 mg aluminum hydroxide (Sigma, Chemical Ltd, UK) dissolved in saline intraperitoneally on the first and 8th days. From the 14th day, rats were exposed to 4% OVA aerosol for 5 minutes daily for 6 weeks. The control group was challenged with normal saline instead of OVA. On the 33rd day, PBS, PBS containing C-kitˉ and C-kit⁺ cells were injected into the trachea through a cervical incision in S, S+C-kit- and S+C-kit+ groups respectively. All animals were humanely euthanized two weeks later by cervical dislocation after a high-dose injection of Xylazine and Ketamine.
Magnetic-activated cell sorting (MACS)
To isolate bone marrow-derived C-kit+ cells, rats were humanely euthanized by cervical dislocation after high-dose injection of Xylazine and Ketamine. Femurs were completely removed and medullary cells flushed out by PBS solution containing 2% fetal bovine serum (FBS; Gibco). The mononuclear cells were isolated by Ficoll (Sigma-Aldrich) gradient centrifugation at 400×g for 20 minutes. For the MACS procedure, harvested marrow mononuclear cells were incubated with 1% FBS for 30 minutes at 4°C. After that, cells were exposed to anti-human C-kit microbead (Catalog no. 130-091-224; Miltenyi Biotec) and cells passed through the LS column (Miltenyi Biotec) to isolate the c-kit+ and c-kit- cells [33].
Cell labeling
Both c-Kit+ and c-Kit- cells were labeled using 20 µM Cell TrackerTM CM-Dil at 37°C for 40 minutes followed by three-time PBS washes [34]. In this study, 50 µl PBS containing 3×105 of positive and negative C-kit cells were used for injection per rat.
Flow cytometric analysis of systemic CD4+ and CD8+ lymphocytes
The percentage of systemic CD4⁺ and CD8⁺ lymphocytes was measured before and after C-kit cell injection. For this purpose, blood cells were diluted with PBS solution (1:1) and mononuclear cells were isolated using Ficoll solution as above-mentioned. Then, cells were incubated with FITC-conjugated mouse anti-rat CD4 (eBioscience), and CD8 (eBioscience) for 30 minutes at 4˚C. Finally, cells were analyzed by the BD FACSCalibur flow cytometry system and FlowJo software (ver.7.6.1).
Western blotting
The right lungs of each group were homogenized using lysis buffer and then the samples were centrifuged at 4°C for 10 minutes at 10,000 rpm. The supernatant containing protein was determined by Bradford’s method. Equal amounts of protein were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were washed with TBST buffer and blocked with 5% skimmed milk for 1 hour. After that, membranes were incubated with primary antibodies anti-β-actin (Catalog No: sc-47778), ERK 1/2 (Catalog No: sc-292838), p-ERK 1/2 (Catalog No: sc-16981-R), NF-ƙB p65 (Catalog No: ab16636) at 4°C for 16-18 hours. Finally, the membrane was washed with TBST buffer and detected with an HRP-conjugated secondary antibody (Catalog No: sc-2357. The bands were visualized by enhanced chemiluminescence (ECL advanced reagents kit, Product Booklet, RPN2135).
Immunofluorescence imaging (IF)
Fourteen days after injection of Dil-labeled cells, 5-6 μm thick cryo-sections were prepared from lung tissues. The samples were incubated with anti-cytokeratin 19 antibody for 1 hour. After three-time PBS washes, the samples were exposed to an appropriate FITC-labeled secondary antibody for 1 hour at room temperature. The nuclei were stained using DAPI (4′, 6-diamidino-2-phenylindol). Here, we monitored the existence of Dil+/cytokeratin-19+ cells (dual red/green stained cells), indicating the differentiation of transplanted cells into the pneumocyte-like cells [35].
Data analysis
Results were displayed as mean±SEM and analyzed by one-way ANOVA with Tukey–Kramer post hoc test. Statistical p-values less than 0.05 were considered significant.