At least 80 adult C57Bl/6 mice weighing 22 ± 2 g, male or female, purchased from Shanghai SLAC Laboratory Animal Co. Ltd. were used in this experiment. The animals were housed under standard conditions at a 12:12 h light-dark cycle, with free access to water ad libitum and lab chow at 23 ±2 °C and 55 ± 5% humidity. Same sex litter mates were housed together in individually ventilated cages with two or four mice per cage, and the animals’ welfare and the experimental procedures were approved by the Animal Ethics Committee of Tongji University, Shanghai, China.
Establishment of AP mouse model and experimental protocol
Experimental mouse AP model was induced by caerulein (Sigma Chemical, St. Louis, USA) in the same way as a previous study . In brief, mice fasted for 12 hours before receiving intraperitoneal injections (i.p.) of 50 μg/kg caerulein hourly, totaling six injections to induce mouse AP model, and mice received normal saline (NS) i.p. instead of caerulein as control.
C57Bl/6 Mice were randomly assigned into the following 4 groups: NS group, HNS group, AP group and HAP group, and each group were set 3 observation time point as 1h, 3h and 5h, and at each time point n=6 – 8 for each group (“n” indicate the total number of mouse used and mice died during model replication were excluded). Mice in AP group and HAP group both received the caerulein injections and mice in NS group and HNS group both received the NS injections as above mentioned. But mice in HAP group and HNS group were treated with hydrogen-rich gases (42% H2 + 21% O2 + 37% N2) inhalation for 3 days before the first injection of caerulein or saline, while mice in AP group and NS group in normal air condition. 1h, 3h or 5h after the final injection of caerulein or NS, mice were euthanized under isoflurane anesthesia. Blood samples were taken, placed in the heparin-treated tubes, centrifuged immediately at 3000 rpm for 10 minutes, and the plasma was collected and stored in liquid nitrogen. Meanwhile, the pancreas tissue were dissociated, rinsed and divided to 3 parts. The head portions of the pancreas were fixed in 4% paraformaldehyde for pathological evaluation, and the rest of the pancreatic tissues placed in empty tubes or in tubes with 0.5 ml RNAiso Plus (Takara Bio, Beijing, China). Both were stored in liquid nitrogen for later usage.
The pancreatic tissues fixed in the 4% paraformaldehyde above were paraffin embedded, sliced (5 μm), and stained by hematoxylin and eosin (HE). The slides were observed and studied under a high power microscope. At least 15 randomly chosen microscopic fields from three slides of three mice in every group were examined and evaluated blindly. Images were captured by digital image analysis system Motic Med 6.0 (Motic Software Engineering Co. Ltd., Xiamen, China). Pancreatic histopathological changes were given scores as described previously , Scoring for (ⅰ) The interstitial and acinar edema: 0 absent, 1 focal expansion of interlobular septa, 2 expanded expansion of interlobular septa, 3 focal expansion of intralobular septa, 4 expanded expansion of intralobular septa; (ⅱ) the presence of vacuolization: 0 absent, 1 less than 20%, 2 20% to 35%, 3 35% to 50%, 4 more than 50%; (ⅲ) the interstitial infiltration of neutrophils or lymphocytes: 0 absent, 1 1 to 10 /high power field (HP), 2 11 to 20 /HP, 3 20 to 30 /HP, 4 >30 /HP; (ⅳ) the necrosis of acinar cell: 0 absent, 1 1 to 5 /HP, 2 5 to 10 /HP, 3 11 to 15 /HP, and 4 >15 /HP. In each group, means of the scores in edema, vacuolization, infiltration and necrosis were presented as results of the final pathological scores.
Measurement of plasma amylase and lipase activity
The activity of plasma amylase and lipase were determined using the respective enzymatic reaction commercial kits (Jiancheng Technology, Nanjing, China). Methods were according to the manufacturer`s instructions (Cat No. C016 and Cat No. A054-1-1), samples were mixed, water bath, optical density (OD660nm and OD420 nm) detection, and plasma amylase and lipase activity were calculated according to formula provided on the instruction. Result of the amylase activity was presented as unit per deciliter (U/dL), and the lipase activity was presented as unit per liter (U/L).
Assay for plasma IL-1 and IL-6
Plasma interlukin-1 (IL-1) and IL-6 levels were quantified by mouse IL-1β or IL-6 commercial available enzyme-linked immune-sorbent assay (ELISA) kits (R&D Systems, Abingdon, UK). Protocols were according to the manufacturer`s instruction of the corresponding kits, and each specimen was measured twice. Levels of IL-1and IL-6 in plasma were calculated as pg/ml, and the results presented as a percentage of the NS group of 1h (the NS1h group).
Measurement of pancreatic GSH and MDA
The pancreatic tissues in empty tube stored in liquid nitrogen were thawed on ice and homogenized in NS. Part of the homogenate was used for protein quantification by the use of Pierce BCA Protein Assay Kit (Thermo, Rockford, USA). 0.1 ml of the homogenate was centrifuged at 3500 rpm, 4 °C for 10 minutes, and the supernatant used for the reduced glutathione (GSH) detection. The content of GSH in pancreas was determined by commercial GSH Assay Kits (Jiancheng Technology, Nanjing, China) which contain total glutathione (T-GSH) and oxidative glutathione (GSSG) standard and based on dithio-bis-nitrobenzoic acid (DTNB) cycle reaction. Method was according to the instruction of the manufacturer (Cat No. A061-1) and content of GSH calculated by T-GSH minus double GSSG according to the instruction. Data was calculated and presented as μmol per gram of total protein (μmol /g).
0.1 ml of the homogenates was used for the malondialdehyde (MDA) detection. The content of MDA in pancreas was determined by commercial MDA Assay Kits (Jiancheng Technology, Nanjing, China) based on thiobarbituric acid (TBA), and method was according to the instruction of the manufacturer (Cat No. A003-1). Data was calculated and presented as nano-mole per milligram of total protein (nmol /mg).
The expression of in situ Hsp60 protein in pancreas was detected by immunohistochemistry, with the same protocol as the former . Slides of the paraffin section of mouse pancreatic tissue dewaxed, hydrated, and heat-mediated antigen retrieval, and then blocked in 5% bovine serum albumin (BSA) (Sangon Biotech, Shanghai, China) for 1 h at room temperature. The slides were subsequently incubated with rabbit anti-Hsp60 antibody (1:600 diluted) (Enzo, Pennsylvania, USA) overnight at 4 ℃, and horse radish peroxidase (HRP)-conjugated secondary antibody (Beyotime, Shanghai, China) for 2 h at room temperature. Finally, the slides were stained with diaminobenzidine (DAB) (Sangon Biotech, Shanghai, China) and hematoxylin.
Positive signals with brownish staining were observed under microscopy. Images were captured by Motic Med 6.0 ((Motic Software Engineering Co. Ltd., Xiamen, China), and the optical density (OD) of the positive signals was measured semi-quantitatively by the software of Image J. Results were presented as a percentage of the NS1h group.
The expression of Hsp60 protein in pancreatic tissue was detected by Western blotting as well, and the protocol was similar to that of the former . Mouse pancreatic tissues preserved in liquid nitrogen were thawed and homogenized, and the total protein was extracted. Protein quantification was carried out with the Pierce BCA Protein Assay Kit (Thermo, Rockford, USA). Protein samples loaded and separated on 10% SDS-PAGE gels was transferred to PVDF membrane. The PVDF membrane was blocked in 5% BSA (Sangon Biotech, Shanghai, China) for 2 h at room temperature, and subsequently incubated with rabbit anti-Hsp60 antibody (1:1000 diluted) (Enzo, Pennsylvania, USA) or β-actin antibody (1:2000 diluted) (Cell Signaling Technology, Boston, USA) overnight at 4 ℃, and a HRP-conjugated secondary antibody (1:2500 diluted) (Beyotime, Shanghai, China) for 1 h at room temperature. Positive signals in membrane were detected by chemiluminescence with the use of ECL reagent (Beyotime, Shanghai, China) and the integrated density (IntDen) of the positive signals was analyzed by the software of Image J. Results was calculated as a relative expression of Hsp60 protein adjusted to the corresponding β-actin expression, and data was shown as a percentage of the NS1h group.
Quantitative real time PCR
The expression of Hsp60 mRNA in pancreas was detected by real-time quantitative PCR (RT-PCR), and protocols were similar to that of the former . Mouse pancreatic tissues in tubes with RNAiso Plus (Takara Bio, Beijing, China) stored in liquid nitrogen were thawed and homogenized, and total RNA was extracted. 1mg of RNA was reverse transcribed to single-stranded complementary DNA (cDNA) according to the manufacturer’s recommended procedure of the HiScript III RT SuperMix Kits (Vazyme, Nanjing, China). Next, quantitative RT-PCR was performed on QuantStudio 6 Flex System (Therm Lifetech, Walsham, USA) by using Power SYBRTM Green PCR Master Mix (Thermo Fisher, Walsham, USA), with GAPDH as the house-keeping gene, and the annealing temperature was 58 °C and the number of PCR cycles was chosen to stop the reaction in the linear phase of amplification (40 cycles). Primer sequences were used as follow: hsp60, forward (5`-CAGTGAAGGATGGAAAAACCCT-3`) and reverse (5`-TCTTTGGTGACAATGACCTCC C-3`); GAPDH, forward (5ʹ-GCATCTTCTTGTGCAGTGCC-3ʹ) and reverse (5ʹ-TACGGCCAAATCCGTTCACA-3ʹ). Relative quantification was calculated by the double ΔCt method, and results were present as a percentage of the NS1h group.
Data analysis was performed using statistical software SPSS version 22.0. All results were expressed as mean ± standard error of the mean (SEM), Comparisons were made by one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. Values of P < 0.05 were considered statistically significant.
At least 6 mouse models were constructed at each time point in each group. The number of observed animals per indicator varies according to source of specimen and the method used and for each indicator the number of samples in each group of the test indexes is equal. For pathological evaluation, at least 15 randomly chosen microscopic fields from three slides of three mice in every group were examined and evaluated blindly. Plasma amylase and lipase activity, pancreatic GSH and MDA content which detected by biochemical methods as well as IL-1 and IL-6 which detected by ELISA each with animal number 6, the included animal number n is 6. Molecular biology indicators such as Hsp60 protein and mRNA expression which detected by immunohistochemistry, Western blotting or RT-PCR, each contain at least 3 animals.