Reagents and antibodies
Caerulein (CAE), lipopolysaccharide (LPS), Ang(1-7) (an agonists of Mas1), A779 (an antagonists of Mas1) and medium F-12 K were purchased from Sigma Chemical (Sigma-Aldrich, St. Louis, MO, USA). AR42J cell line was from the American Type Culture Collection (ATCC® CRL1492™; ATCC, Rockville, MD). The lentivirus containing Mas1 receptor shRNA and Mas1 receptor over-expression were purchased from Genechem (Shanghai, China); Antibodies against Mas1 receptor was from Alomone Lab (AAR-013, Jerusalem, Israel). Anti-LC3 was from MBL life science (M186-3, Japan). Anti-Trypsin (sc-137077) from Santa Cruz Biotechnology (Dallas, TX)；Anti-VAMP8 (ab76021), anti-Munc18c (ab175238), anti-SNAP23 (ab3340), anti-syntaxin4 (Syn4) (ab184545) and anti-SQSTM1 / p62 (ab56416) were from Abcam (Cambridge, UK). HRP-Conjugated GAPDH Antibody (HRP-60004) was from Protein tech (Rosemont, USA). Goat anti–rabbit and goat anti–mouse secondary antibodies from ZSJQ-BIO (Beijing, China); polyvinylidene fluoride membranes (0.45/0.22μm) and enhanced chemiluminescence from Millipore (Darmstadt, Germany); the Image Lab software was from Bio-Rad (Hercules, Calif); Trizol reagent was from Sigma-Aldrich; and SYBR Green PCR Master Mix, reverse transcription–polymerase chain reaction (RT-PCR) reagents, and cDNA Synthesis Kit from Applied Biosystems (Thermo Fisher, Waltham, Mass).
Human tissue specimens
A tissue microarray containing 6 clinically annotated pancreatic cancer specimens and corresponding non-cancerous tissues were obtained from Beijing Friendship Hospital from February to June in 2017. All the patients enrolled in this study provided written informed consent.
Animals and pancreatitis induction
Mice including wild-type (WT) C57BL6/J (6–8 weeks, 20–22g, male) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were housed under specific-pathogen-free conditions in individually-ventilated cages with wood shavings as bedding material (4–6 mice per cage), 12 h dark/light cycle at 22°C and free access to water and standard rodent diet. All mice (20–22g, male) were marked with an earmark and a randomized table was used; they were allocated into groups in a completely randomized manner (n = 6 per group). All the experiments involving animals were conducted under the principle for replacement, refinement and reduction (the 3Rs) and according to the legislation on the protection of animals and were approved by the Animal Ethics Committee of Capital medical university. Acute pancreatitis was induced in C57Bl/6 mice by hourly intraperitoneal injections of caerulein (100 mg/kg body weight) up to 8 hours, and one-dose LPS (10 mg/kg) at the last injection. Animals were sacrificed 0, 0.5, 2, 6, 12 and 24 hours after the last injection.
AR42J cell culture and transfection
AR42J cells were cultured in F-12 K medium supplemented with 20% fetal bovine serum at 37 °C with a humidified atmosphere containing 5% CO2. The lentivirus containing Mas1 receptor knockdown and over-express were purchased from Genechem (Shanghai, China); The cells were plated in six-well plates and transfection was conducted at 70–80% confluence. Transfection of AR42J cells with lentivirus according to the manufacturer's instructions. The sequences of the Mas1 receptor gene are GENE_ID (25153) from Genbank (XM_006227860). The Non-Targeting lentivirus Pool was used as controls. Since successfully transfected, the AR42J cells expressed green fluorescent protein, and the transfection efficiencies of Mas1 receptor lentivirus were observed using an Leica DMI3000B fluorescent microscope. Forty-eight hours after transfection, the cells were used for experiments. The effectiveness of lentivirus in changing Mas1 receptor expression was evaluated by RT-PCR and western blot.
Pancreas was snap frozen in liquid nitrogen and stored at -80 °C for measurement. For histology, tissue was fixed in 4% formalin for paraffin embedding or embedded in Tissue Tec (OCT, Sakura, Los Angeles, CA) for cryo sections. Collected blood samples were centrifuged and serum was stored at -80°C.
For light microscopy, fresh specimens of murine pancreas were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). The tissues were embedded in paraffin, and 5 mm sections were processed for hematoxylin and eosin (H&E) staining by standard procedures. Multiple randomly chosen microscopic fields from at least three mice in each group were examined and scored by two pathologists in a blind manner based on the presence of vacuolization, interstitial edema, interstitial inflammation, the number of acinar cell necroses, as previously described. The scoring assessment was performed on a scale of 0–3 (0 being normal and 3 being severe) on each parameter mentioned above, and the sum of the scores were used to evaluate the severity of acute pancreatitis.
Serum amylase, lipase, trypsinogen-activation peptide (TAP, Mouse, Rat) and Ang(1-7) was determined by means of acommercially available kit (Blue Gene, Shanghai, China), and expressed as units per liter (U/l). Assays were performed in duplicate using the Luminex 100 System (Austin, TX, USA).
Electron microscopy evaluation of cultured cells and mice pancreatic tissues
Acinar cell ultrastructure was examined by electron microscopy (EM). Cultured cells and pancreatic tissues from AP mice were fixed with 2.5% glutaraldehyde one hour and then in 0.1 M cacodylate buffer (pH 7.4) overnight post fixed in 1% OsO4 in the same buffer, enbloc stained with 2% aqueous uranyl acetate, dehydrated in ethanol and embedded in Poly/Bed 812 (Polysciences). Ultrathin sections were cut on Leica EM UC7 ultramicrotome, stained with lead citrate and examined in a HITACHIH7650 electron microscope at 60kV.
Pancreas tissues or AR42J cell were fixed in suspension with 4% paraformaldehyde (methanol free) for 30 minutes at room temperature and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline. Non-specific binding was blocked with 5% rabbit or 5% goat serum. Then, tissues or cells were stained with primary antibodies against Mas1 (1:500; Alomone Lab, AAR-013), LC3B (1:500; MBL, M186-3) and Trypsin (1:200, Santa Cruz, sc-137077). The cells were then labeled with Alexa Fluor 488 (green) and Alexa Fluor 633 (far red) econjugated secondary antibodies (Thermo Fisher Scientific), and Nuclei were stained with 4’6’-diamidino-2-phenylindol (DAPI, 1:10,000; Sigma-Aldrich, D8417) for 5 min. Images were acquired with a Zeiss LSM 710 confocal microscope (Carl Zeiss Microscopy, Thornwood, NY) .
Fresh intact mice pancreas were isolated and purified from mice killed by decapitation. All isolation procedures were performed on ice or at 4 °C. Pancreas was rinsed with ice-cold solation buffer A containing 320 mM sucrose, 3 mM MgCl2, and 20 mM Tris, pH 7.4. Pancreas pieces were homogenized in buffer A using a glass homogenizer. The homogenates were centrifuged for 15 min at 1000×g at 4 °C. The supernatants were removed, and the pellets were resuspended in 4 ml of buffer B (2.2 M sucrose, 1 mM MgCl2 , and 10 mM Tris, pH 7.4) and differentially centrifuged for 60 min at 60,000×g (Beckman XL-90 ultracentrifuge, Brea, CA, USA) using a swing out rotor at 4 °C. After centrifugation, the pellet containing the isolated nuclei was resuspended and washed by centrifugation in 2 ml of buffer A. Protein content in isolated nuclei was determined with the Pierce BCA Protein Assay Kit (Thermo Scientific, Fremont, CA, USA). For WB analysis, isolated nuclei were processed using the Nuclear Extract Kit (Active Motif, CA, USA) to eliminate the DNA and conserve only the nuclear proteins.
Reverse transcriptase PCR (RT-PCR)
Total RNA was extracted from acinar cells using TRIzol (Invitrogen), followed by reverse transcription with a DNA reverse transcription system (Invitrogen). And Quantitative RT-PCR was performed using the SYBR Green PCR Master Mix and RT-PCR reagents and the Prism 7500 Sequence Detection System. Polymerase chain reaction was carried out for 40 cycles (20 seconds at5 0 °C, 10 minutes at 95 °C, and 40 cycles of 15 seconds at 95 °C, and 1 minute at 60 °C). The primer sequences were as follows Table1. GAPDH (B661104-0001; B661304-0001; B661204-0001; Sangon Biotech (Shanghai) Co., Ltd.) was included in each reaction as an internal standard, and relative quantitative gene expression was calculated using the 2 −△△Ct method.
Western blot analysis
For western blot analyses, portions of frozen pancreas tissue and AR42J cells were rapidly homogenized in liquid nitrogen. Total protein and nuclear protein were extracted separately using the total Protein Extraction Kit (Sigma) according to the manufacturer's instructions. The concentrations of protein were determined using the BCA method (Pierce, Rockford, IL, USA). Each 30 μg aliquot of total protein or nuclear protein was loaded in a 10% or 12% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis gel, and then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After complete protein transfer, the membranes were blocked with 5% milk powder solution for 2 hat room temperature and incubated at 4 °C overnight with rabbit monoclonal anti-Mas1, anti-VAMP8, anti-SNAP23, anti-SYN4, anti-Trypsin, anti-HRP-Conjugated GAPDH and mice monoclonalanti-SQSTM1/p62, anti-LC3 antibody subunit diluted at a 1:1000 dilution in 5% milk powder solution. The following day, membranes were washed twice and incubated for 1 hour at 25 °C with anti-rabbit or anti-mice immunoglobulin G horseradishperoxidase-conjugated secondary antibody (1:5000). After 3 washes with Tris-HCl buffer salt solution + Tween later, enhanced chemiluminescence was used to detect immune reactive bands. Densitometric analysis of bands was performed using Image Lab software(170-8195) from Bio-Rad (Hercules, Calif), and data were expressed in arbitrary units.
All the experiments involving animals were conducted under the principle for replacement, refinement and reduction (the 3Rs) and according to the legislation on the protection of animals and were approved by the Animal Ethics Committee of Capital medical university (AEEI-2018-074). All the patients enrolled in this study provided written informed consent and have been approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
Data were presented as the means ± SD. Statistical analyses were done using SPSS V.16.0 for Windows (IBM) or GraphPad Prism (GraphPad Software, San Diego, California, USA). The student’s t-testor one-way ANOVA was used for comparison between groups. P values <0.05 were considered to be statistically significant.