Reagents
Imatinib mesylate (IMT) was procured from Sigma Aldrich, USA. Cerulein was obtained from Ana Spec, France. TGF-β1 was procured from Bio-legend, USA. Antibodies used in this study were β-actin (Catalogue no. sc-47778), collagen1a (Catalogue no. sc-393573), collagen3a (Catalogue no. sc-271249), α-SMA (Catalogue no. sc-53142), CTGF (Catalogue no. sc-373936), TGF-β1 (Catalogue no. sc-146), DDR2 (Catalogue no. sc-81707), pSmad2/3 (Catalogue no. #8828), Smad2/3 (Catalogue no. #8685), pDDR1 (Catalogue no. #14531), DDR1 (Catalogue no. #5583), pNF-κB (Catalogue no. #3033), NF-κB (Catalogue no. #8242) were procured from Santa Cruz Biotechnology Ltd and Cell Signaling Technology, USA. The ELISA kits of TGF-β, IL-1β, IL-6 and TNF-α (catalogue no. TGF-β: 88-8350, IL-1β: 88-7013, IL-6: 88- 7064, and TNF-α: 88-7324) were obtained from eBioscience, USA.
PSCs isolation, culture and treatment
PSCs were isolated from the mice according to the methods as described with slight modifications [14]. Briefly, pancreas was isolated from adult male Swiss albino mice. Isolated pancreas was immediately kept in sterile PBS. Further, small portion of pancreas was cut, minced with scissors and single cell suspension was prepared by using 1 ml syringes in Dulbecco's Modified Eagle (DME) medium. Then, cells suspension was centrifuged at 3000 rpm for 3 min followed by two times washing with PBS. Cells pellet was mixed and cultured in DMEM medium containing 1% antibiotic solution and supplemented with 20% fetal bovine serum (FBS). PSCs were separated by using the Histodenz density gradient centrifugation [15].
Oil-red O staining for PSCs identification
After 70% confluency, PSCs were fixed with 4% paraformaldehyde and 0.1% Triton X-100 reagents and stained with oil-red O working solution (1% in isopropanol) at 60℃ for 30 min. Next, PSCs were counterstained using hematoxylin, mounted with DPX and observed under the microscope [16].
Confocal microscopy
Freshly isolated and cultured PSCs were treated with TGF-β1 (10 ng/ml) and IMT (1 μM). After 24 hr of treatment, cells were fixed with 4% paraformaldehyde and 0.1% Triton X-100 followed by blocking with 3% bovine serum albumin (BSA) for 1 hr. Then cells were labeled with primary antibodies against α-SMA, collagen 1a, pSmad2/3, pDDR1, and DDR2 at 4°C for overnight. Next day, cells were probed with secondary antibody conjugated with rhodamine (1:200 dilution) or fluorescein isothiocyanate (FITC) (1:100 dilution) for 1 hr. After washing, cells were mounted with fluoroshield DAPI (Sigma-Aldrich, USA) medium and fluorescent signals were captured by confocal laser-scanning microscopy (Leica, Germany) [17].
Animals and experimental design
Animals were kept in well-controlled housing facilities at temperature (25 ± 2°C) and 12/12 hr light/dark cycle with free access to water as ad libitum and pellet food. The Animal experiments were designed, conducted and reported as per the ARRIVE guidelines [18]. Specifically, all the animal experiments were performed according to the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), which is the approval body for animal experimentation in India. The CPCSEA certified Institutional Animal Ethical Committee (IAEC) of National Institute of Pharmaceutical Education and Research (NIPER)-Hyderabad, has reviewed the animal protocol and approved it (IAEC Protocol Approval No.: NIP/01/2019/RT/365).
Male Swiss albino mice (age: 6–8 weeks, body weight 25-30 g) were purchased from Palamur Biosciences Pvt. Ltd, Mahabubnagar, India. All mice were randomly divided into 6 groups (7 mice per group, n = 42): Normal control, IMT alone, Cerulein, Cerulein + IMT low dose (LD - 1 mg/kg), Cerulein + IMT mid dose (MD - 3 mg/kg) and Cerulein + IMT high dose (HD - 10 mg/kg). CP was induced by 6-hourly exposures of cerulein (50 μg/kg/intraperitoneal (i.p.), 3 alternative days per week, for the period of 3 weeks as per our previously published protocol [19]. Cerulein + IMT group mice were administered IMT (1, 3 and 10 mg/kg) orally, every other day after cerulein exposures for 3 weeks. Normal control animals received i.p. injection of sterile normal saline same as cerulein group. IMT alone group mice were administrated with IMT (10 mg/kg) orally 4 alterative days in the week for 3 weeks (Fig. 1). Mice were sacrificed after the completion of 21 days. Doses of IMT were selected on the basis of previous literature [20]. However, cerulein needed minimum 12 hr to produce maximum inflammatory response, IMT was given every other day after cerulein exposures for the period of 3 weeks [19].
Blood collection and plasma analysis
Blood samples were collected by cardiac punctured on the day of sacrifice in heparin containing tubes and plasma was separated after centrifugation. Amylase and lipase levels were assessed in plasma and measured by using Accurex kinetic enzymatic kits and values are expressed as IU/L [19].
Collagen estimation by Sircol assay
Pancreatic tissues were homogenized in PBS and then supernatants were incubated with collagen binding dye picrosirius red for 1 hr at 37°C. Next, samples were centrifuged and obtained pellet was resuspend in 100% ethanol followed by centrifugation at 10,000 rpm for 10 min. Then pellet was mixed with 0.5 M NaOH solution and incubated for 30 min at 37°C. The final absorbance was taken at 540 nm and values were expressed as relative collagen content per milligram of protein [21].
Enzyme-linked immunosorbent assay
Cytokines concentrations in pancreas were determined by eBioscience ELISA kits of IL-6, IL-1β, TNF-α and TGF-β1 as per the previously described protocol [22]. Concentration of cytokines were expressed as pg per milligram of protein. Total protein was estimated by Bicinchoninic acid assay kit (Sigma Aldrich, USA).
Histopathology and immunohistochemistry analysis
Pancreas were fixed with 10% formaldehyde solution and paraffin-embedded pancreatic sections were taken at 5 µm. Further, pancreatic sections were stained with H & E, picrosirius red (PSR) and Masson’s trichrome (MT) as per previously described protocol [23]. For immunostaining, antigen retrieval was carried out by using citrate buffer. After washing, sections were incubated with 3% hydrogen peroxide for 15 min followed by blocking with 3% BSA. The primary antibodies against α-SMA, collagen1a, pDDR1 and DDR2 (1:100) were added and kept at 4℃ overnight. The HRP-conjugated secondary antibodies were added and sections were stained with DAB reagent. Further, sections were counter stained with hematoxylin and images were observed using a light microscope [24]. Quantitative analysis of fibrotic and immunopositive area were analyzed by using ImageJ software (NIH, USA).
Immunoblotting
To analyze the pancreas protein expression, immunoblotting technique was used as described earlier [25, 26]. Small portion of pancreatic tissues were homogenized in RIPA lysis buffer. Then equal amounts of proteins were separated using SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane. The protein blots were then detected using ECL (Bio-Rad Laboratories, USA) and densitometry analysis of respective protein band was carried out by ImageJ software (NIH, USA).
Statistical analysis
All results are given as mean ± SEM. Student's t-test was used to determine the difference between two groups. However, differences among more than two groups were analyzed using nonparametric test one-way analysis of variance by GraphPad Prism scientific software version 6.01. Value of P less than 0.05 was considered statistically significant.