Experimental animals and group assignment
The present study was done by enrolling 20male Wistar rats (8–10 weeks old, weighing 200–250 g). Animals were kept at 22°C ± 2°C temperature with 12 h light/dark cycle. All rats were allowed to access to water and rodent pellets. After a two-week inhabitation period, 10 rats were blindly selected for isolation of bone marrow content c-Kit+ cells. Animals were randomly allocated into four groups (each in 6 rats) as follows: Healthy rats only received 50 μl normal saline intratracheally (group C); sensitized rats received 50 μl normal saline intratracheally (group A); Sensitized rats received 50 μl PBS intratracheally containing 3×105 c-kit- cells (group A+ c-kit-); and Sensitized rats received 50 μl normal saline intratracheally containing 3×105 c-kit+ cells (group A+ c-kit+) (Fig. 1a, b).
Animals sensitization protocol
We used a protocol a period of 32 ± 1 days for induction of asthmatic changes according to our previous experiment . For this propose, rats were received 1 ml sterile normal saline containing 1 mg ovalbumin (Sigma -Aldrich, USA) and 200mg aluminum hydroxide intraperitoneally on days 1and 8. Six days post intraperitoneal injection; the animals were exposed to aerosolized ovalbumin (4% w/v) from day 14 to 32 ± 1 for 5 min daily using an ultrasonic nebulizer (CX3, Omron Co., Netherland) connected to a Plexiglas chamber (30 cm × 20cm × 20 cm). The control rats were treated with saline instead of OVA. After completion of OVA treatment, all rats were anesthetized on day 33, dissected via the ventral neck and received PBS, c-kit+ and c-kit- cells according to group allocation [8, 17]. Animals were kept for the next 14 days .
Isolation of c-Kit+ cells by Magnetic Activated Cell Sorting (MACS)
After cervical dislocation, upper and lower extremities of femurs were cut by sterile scissors. Medullary contents were washed by pushing PBS containing 2% fetal bovine serum (FBS; Gibco, USA) using a syringe connected to 18-gauge needle. Mononuclear cells (MNCs) were collected by gradient centrifugation using Ficoll-Hypaque® solution (Sigma-Aldrich, USA). To this end, cells were centrifuged at 400gfor 20 min and monolayer cell located interphase gently collected. Following twice wash with PBS, MNCs were incubated in PBS containing 1% FBS and incubated at 4˚C for 30 min. Then, cells were incubated with mouse-anti human c-Kit microbead (Miltenyi Biotech, Germany) according to manufacturer's instructions. By passing cells through the LS columns (Miltenyi Biotec, Germany) and c-Kit+ and c-Kit- cells were isolated and used for different analyses.
Immunophenotyping of c-kit+ cells by flow cytometry
The multipotentiality of isolated cells was studied after MACS by flow cytometry analysis .In short, cells both groups were incubated with FITC-conjugated mouse-anti human CD117 (c-kit+) at 4˚C for 30 min. The samples were analyzed by BD FACS Calibur and raw data processed using FlowJo software (Ver. 7.6.1).
Cells were labeled by using Cell TrackerTM CM-Dil as previously described . Cells were re-suspended in 20 µM Cell TrackerTM CM-Dil solution and kept at 37˚C for 30-40 min. Thereafter, cells were washed with PBS three times (each for 5 min). 50 µl PBS aliquots containing 3×105 c-Kit+ and/or c-Kit- cells were prepared.
Serum levels of IL-4 and IFN-γ
Fourteen days after completion of asthma induction, animals were euthanized by the overdose of Ketamine and Xylazine. Blood samples were collected via the inferior vena cava, allowed to clot at RT condition and serum harvested by centrifugation at 3000 rpm at 4°C for 10 min. The serum levels of cytokines IL-4 and IFN-γ were measured using rat ELISA kits (Sigma-Aldrich, USA) according to the manufacturer’s instructions .
Real-time PCR analysis
Transcription of miRNA-126 and miRNA-133 was quantitatively measured by conventional real-time PCR assay.the Total RNA content was extracted from left lungs using a total RNA extraction mini kit (Yekta Tajhiz, Iran) and quantified using a Nano Drop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, 19810 USA). Total RNA was transcribed into cDNA using cDNA synthesis kit (Yekta Tajhiz, Iran). Real-time PCR reaction was done on a Corbett Rotor-Gene 3000 instrument (Corbett Life Science, Australia) using SYBR Green master mix (Yekta Tajhiz, Iran). In the current experiment, miRNA-191 was used to normalize miRNA-126 and miRNA-133 values. The data were analyzed in accordance with 2−ΔΔCtmethod.Primers used in this study are listed in Table 1.
Right pulmonary lobes were fixed in 10% neutral buffered formalin solution. Paraffin-embedded samples were cut into the 4-μm thick longitudinal sections by using Leica microtome. Hematoxylin-Eosin (H&E) and Periodic acid–Schiff (PAS) staining was used to show the pathological changes [2, 20]. The existence of chronic pathological features such as hyperemia, emphysema, interstitial pneumonitis and epithelial cells injury was scored by an independent pathologist. A four-point semi-quantitative score system ranging from 0 to 3 [0: absence, 1: mild injury, 2: moderate injury, and 3 severe injuries was used to report the extent of pathological changes.
All quantitative results are presented as mean ± SEM and analyzed using a One-Way ANOVA and Tukey–Kramer post hoc test. Pathological scores were evaluated using the Kruskal-Wallis test followed by post-hoc Mann-Whitney analysis. Statistical significance was set at p<0.05.