The current study was carried out with the approval of the Ethics Committee of Harbin Medical University Cancer Hospital, and all animal experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Extensive efforts were made to minimize the suffering of the included animals.
Microarray-based gene expression profiling
The AP-related microarray GSE121038, with better data availability and quality obtained from 16 microarrays, was retrieved from the GEO database with “acute pancreatitis” serving as the key word, “Mus musculus” as the species and “Expression profiling by array” as the type. The samples were obtained from mouse pancreatic tissues, and the sequencing platform file was GPL10787. Sample grouping information is illustrated in Additional file 1: Table S1. Differential analysis of the GSE121038 microarray was performed using the R language “limma” package, with |logFC|> 1, p value < 0.05 serving as the threshold to screen DEGs in pancreatitis samples. Additionally, genes related to AP were downloaded from the GeneCards and CDT databases. Intersection analysis was subsequently conducted on the top 300 DEGs in the GSE121038 dataset and the AP-related genes using the jvenn tool. In addition, the binding sequence of transcription factor Rbpjl and the promoter region of Arid5a gene was predicted using the JASPAR website. Lastly, the interaction network of Arid5a and IL-6 was obtained from the STRING website.
Cell culture and transfection
Mouse pancreatic acinar cell line (MPC-83, CL-0518), procured from Procell (Wuhan, Hubei, China), was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (HyClone Company, Logan, UT), supplemented with 100 U/mL penicillin and 100 U/mL streptomycin (Gibco Company, Grand Island, NY) in a 5% CO2 incubator at 37 °C. Upon reaching 80% confluence, the cells were passaged and collected for follow-up experiments.
BLOCK-iT™ RNAi Designer was adopted to design Rbpjl and Arid5a short hairpin RNA (shRNA) sequences. The obtained shRNAs were subsequently cloned into pLKO.1-puro lentivirus plasmids. Mouse Rbpjl and Arid5a full-length sequences were cloned into pCDH-EF1-MCS-copGFP lentivirus plasmids. MPC-83 cells were stimulated with LPS (2 µg/mL, L8880, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) to mimic the microenvironment of pancreatitis. The specific action time was detailed in the legend of relevant results. In the LPS treatment groups, the cells were transfected with plasmids of oe-NC, oe-Rbpjl, sh-NC, sh-Rbpjl (5′-CCATCCAAACCAGAGTCTGAT-3′), sh-Arid5a (5′-GCCTGAGTATTCAGATGACAA-3′), oe-Rbpjl + oe-NC, oe-Rbpjl + oe-Arid5a, oe-Arid5a + DMSO, and oe-Arid5a + JSI-124 (JSI-124: 1 µM for 24 h, Bio-Techne), alone or in combination. Simultaneously, in control groups, the cells were transfected with plasmids of oe-NC, oe-Rbpjl, sh-NC, sh-Rbpjl, oe-Rbpjl + oe-NC, sh-Arid5a, oe-Rbpjl + oe-Arid5a, oe-Arid5a + DMSO and oe-Arid5a + JSI-124, alone or in combination. Thereafter, the HEK-293 T cells (CL-0005, Procell) were seeded in a 6-well plate, and 4 µg of over-expression or silencing plasmids were added with 8 µL Lipofectamine 3000 (L3000015, Thermo Fisher Scientific Inc., Waltham, MA) for transient transfection. Lentivirus (virus titer of 2 × 108 TU/mL) was collected, isolated, and purified. Afterwards, the purified lentivirus was utilized to transduce MPC-83 cells, which were collected after 72 h.
Tissue or cell samples were lysed with the TRIzol reagent (16,096,020, Thermo Fisher Scientific) for 10–30 min at room temperature. Total RNA content was extracted with the addition of phenol/chloroform, and RNase-Free DNase I was used to digest and extract RNA. The concentration and purity of the extracted RNA (260/280 = 1.8—2.0) were detected with a nucleic acid quantitative analyzer (ND-1000, NanoDrop Technologies Inc.). Next, 400 ng total RNA was reverse-transcribed into cDNA with PrimeScript RT Reagent Kit (D7168l, Beyotime, Shanghai, China). RT-qPCR was subsequently performed using SYBR® Premix Ex Taq™ II kits (RR820A, Takara). PCR primers were synthesized by Riobio Biotechnology Co., Ltd (Guangzhou, China). The specific sequences are illustrated in Additional file 4: Table S4. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was adopted as internal reference control of mRNAs. The 2−ΔΔCT method was utilized to express the relative expression of the gene.
Western blot assay
Total protein was extracted from tissues or cells using radioimmunoprecipitation assay (RIPA) buffer (R0010, Solarbio) with 1 mM PMSF protease inhibitor. BestBio Beibo Nuclear Protein Extraction Kit (BB-3102–1, Invent Biotechnologies Inc., Eden Prairie, MN) was used to extract nuclear protein. Briefly, appropriate amounts of cytoplasmic extraction buffer were added to the cells or tissues, and the homogenate was shaken and centrifuged at 16,000 rpm and 4 °C for 5 min. Next, the supernatant (cytoplasmic fraction) was transferred to a new pre-cooled 1.5 ml centrifuge tube. The pellet was resuspended in 0.5 mL of pre-cooled PBS, and then centrifuged at 10,000 rpm for 3–5 min. The collected pellet was supplemented with an appropriate amount of nucleus extraction buffer, and later incubated on ice for 1 min. Afterwards, the nuclear extract was centrifuged at 14,000–16,000 rpm for 30 s, and the nuclear protein was stored at − 80 °C.
The protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit (G3522, Jiebeisi Biotechnology, Guangzhou, China). Next, 40 µg protein was extracted from each sample and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The obtained proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Billeria, MA), which was sealed with TBST containing 5% bovine serum albumin (BSA) at room temperature. The membrane was subsequently incubated with diluted primary antibodies against PAP-I (0.1 µg/mL, PA5-47,700, Thermo Fisher Scientific), Rbpjl (dilution ratio of 1:1000, ab25949, Abcam Inc., Cambridge, UK), Arid5a (dilution ratio of 1:2000, ab81149, Abcam), phosphorylated (p)-STAT3 (dilution ratio of 1:1000, ab76315, Abcam), STAT3 (dilution ratio of 1:1000, _#9139, Cell Signaling Technologies [CST], Beverly, MA), tumor necrosis factor-α (TNF-α) (dilution ratio of 1:1000, #3707, CST), IL-6 (dilution ratio of 1:1000, # 12,153, CST), IL-1β (dilution ratio of 1:1000, #12,153, CST), histone H3 (dilution ratio of 1:1000, # 4499, CST) and GAPDH (dilution ratio of 1:2000, TA-08, Zhongshan Gold-bridge, Beijing, China) overnight at 4 °C. The following day, secondary goat anti-rabbit against IgG antibody (dilution ratio of 1:2000, Zhongshan Gold-bridge) or goat anti-mouse against IgG (dilution ratio of 1:2000, Zhongshan Gold-bridge) was added to the membrane for 1 h incubation at room temperature. Enhanced chemiluminescence reagent (Beyotime) was employed to visualize images using the Bio-Rad Gel Doc XR gel imager (Bio-Rad Laboratories, Hercules, CA). Image J software was adopted to quantify the gray value of the protein bands. The semi-quantitative expression of protein was expressed by the ratio of gray value of the target protein to that of internal reference protein. GAPDH was adopted as the internal reference of total protein, and histone 3 as the internal reference of nuclear protein.
Initially, 1 µg of purified Rbpj1 protein was incubated with 1 μL DNA probe (0.08 μM) in the Arid5a promoter binding region (WT sequence: forward: 5′-CCACCAAGCTCACTGGTGTCTGCTCCAGGCTGAGC-3′; reverse: 5′-GCTCAGCCTGGAGCAGACACCAGTGAGCTTGGTGG-3′; MUT sequence: forward: 5′-CCACCAAGTTTTTTTTTTTTTTCTCCAGGCTGAGC-3′; reverse: 5′-GCTCAGCCTGGAGAAAAAAAAAAAAAACTTGGTGG-3′) in a binding buffer supplemented with 25 mM HEPES buffer (pH 7.6), 50 mM KCl, 0.1 mM EDTA (pH 8.0), 12.5 mM MgCl2, 1 mM DTT, 0.5% BSA, and 5% glycerol at room temperature for 30 min. Subsequently, the binding reaction mixture was loaded onto a 7% non-denaturing polyacrylamide gel and run at 80 V for 2 h. Afterwards, the bound DNA was visualized by ethidium bromide staining .
Pancreatic acinar cells were trypsinized and dispersed into a single cell suspension. Next, the cells were seeded into 96-well plates, at a density of 6 × 103 cells per well. Subsequently, 200 μL cell suspension was added to each well, with 6 parallel wells set for each group. At the 24 h, 48 h, 72 h and 96 h time intervals, 10 μL CCK-8 solution (96,992, Sigma-Aldrich Chemical Company, St Louis, MO) was added to each well. After a 2 h period of culture, the optical density (OD) values at 450 nm were detected with a microplate reader (NYW-96 M, Beijing N.Y.W. Instrument Co., Ltd., Beijing, China).
Early and late apoptotic MPC-83 cells were detected by flow cytometry. Briefly, MPC-83 cells (at a density of 1 × 104/well) were trypsinized, rinsed with PBS and resuspended in a binding buffer. Subsequently, the cells were stained with 5 µL Annexin V FITC and PI (BD Biosciences) for 30 min in conditions void of light at 37 °C. The cells were subsequently detected on a flow cytometer (FACScan™; BD Biosciences), and the results were analyzed with the FlowJo software (version 10.6.2; BD Biosciences) to count the proportion of apoptotic cells.
ROS production assay by dihydroethidium (DHE) fluorescence staining
DHE-ROS reactive oxygen detection kits (BB-47051, Shanghai Best Biotechnology Co., Ltd., Shanghai, China) were adopted to detect ROS accumulation in pancreatic acinar cells. Briefly, DHE solution was diluted with PBS buffer at a ratio of 1:200, added to pancreatic acinar cells, incubated at 37 °C in conditions void of light for 30 min, and then rinsed thrice with PBS. Finally, the cells were visualized under an inverted fluorescence microscope (BX63, Olympus Optical Co., Ltd., Tokyo, Japan). Following excitation by blue light, ROS-positive cells presented with red staining in the whole nuclear area; meanwhile, following excitation with ultraviolet light, the unoxidized dihydroethidine in the cytoplasm presented with blue fluorescence.
Cultured pancreatic acinar cells were cross-linked with 1% paraformaldehyde at room temperature for 5 min, and then added with glycine at a final concentration of 127 mM to terminate the cross-linking. Next, the cells were lysed with cell lysis buffer (50 mM Tris HCl, pH = 8.1, 1% sodium dodecyl sulfate, 10 mM EDTA) containing protease inhibitor on ice for 30 min, and then subjected to ultrasonification to produce 200—1000 bp chromatin fragments.
Afterwards, ChIP Assay Kits (#9002, CST) were utilized. Protein A/G agarose (SC-2003, Santa Cruz Biotechnology, Inc, Santa Cruz, CA) and ChIP antibody to Rbpjl (ab25949, Abcam) or IgG (#14,705, CST) were added to 50 μg DNA and incubated overnight at 4 °C. Normal IgG antibody was adopted as NC. The DNA complex was washed successively with low-salt buffer and high-salt buffer. DNA was subsequently extracted and purified with phenol/chloroform. Specific ChIP-RT-qPCR primers (forward: 5′-TTGGAAAGGATGGAACCGGC-3′; reverse: 5′-CTTTGCAGCTAGGGGCTGAT-3′) were designed for the Arid5a promoter.
Dual luciferase reporter assay
Arid5a promoter region with WT and MUT Rbpjl binding site was constructed into the pGL3-Basic vector (Promega Corp., Madison, Wisconsin) to obtain the recombinant vector of WT-Arid5a promoter (5′-CAGACACCAGTGAG-3′) and MUT-Arid5a promoter (5′-CAGGACTTGACGAG-3′). Subsequently, HEK-293 T cells (CL-0005, Procell) were seeded in 24-well plates, at a density of 3 × 104 cells/well. WT-Arid5a promoter and MUT-Arid5a promoter were then co-transfected with oe-NC and oe-Rbpjl into HEK-293 T cells. At a 48-h period of transfection, the cells were collected and lysed, after which the luciferase activity was detected using the luciferase detection kit (K801-200, BioVision, Milpitas, CA) on a Glomax20/20 Luminometer (Promega). The relative luciferase activity was expressed as the ratio of the firefly luciferase activity to the Renilla luciferase activity. The relative luciferase activity of different treatment groups was normalized to the ratio of 1 of the control group.
TUNEL cell apoptosis detection kits (C1098, Beyotime) was adopted for this experiment. Following pre-processing, the cells were treated with 20 µg/mL of proteinase K without DNase at 37 °C, incubated with 50 µL of TUNEL detection solution at 37 °C for 60 min, and then added with DAB (DA1010, Solarbio) as a substrate. Apoptotic cells were subsequently observed under a microscope. Under a light microscope, the nuclei of the apoptotic cells appeared as brown spots, which were manually counted and quantitatively analyzed in a blind manner.
Detection of MDA and GSH
GSH and MDA production was determined with GSH (r-glutamyl cysteingl + glycine, #S0056, Beyotime) and MDA kits (#S0131, Beyotime), in accordance with the provided instructions. Briefly, pancreatic tissues (100 mg) were lysed with cell lysis buffer (# p0013, Beyotime) on ice for 30 min, followed by 10-min centrifugation at 12,000g and 4 °C, with the supernatant harvested. Next, BCA protein concentration determination kit (#2020LES76, Yeasen Biotechnology, Shanghai, China) was utilized to detect the total protein concentration. Afterwards, GSH activity was detected at 340 nm and MDA activity at 520 nm.
Mouse serum or supernatant of pancreatic acinar cells was collected. ELISA kits for proinflammatory factors TNF-α (ab181421), IL-1β (ab214025) and IL-6 (ab46029) were purchased from Abcam. Briefly, 96-well plates were placed in the microplate reader (Thermo Fisher Scientific) to analyze the OD values at 562 nm. A standard curve was subsequently drawn with standard protein concentration as the X axis and OD value as the Y axis.
Establishment of AP mouse models
Six-week-old C57/B6 male mice were purchased from Beijing Vital River Biological (Beijing, China) and raised in a specific-pathogen-free animal laboratory. The mice were allowed to acclimatize for two weeks, and then randomly assigned into the following 6 groups (with 8 mice in each group): the sham group, the AP group, the oe-NC group (AP mice injected with lentivirus carrying oe-NC), the oe-Rbpjl group (AP mice injected with lentivirus carrying oe-Rbpjl), the oe-Rbpjl + oe-NC group (AP mice injected with lentivirus carrying oe-Rbpjl + oe-NC) and the oe-Rbpjl + oe-Arid5a (AP mice injected with lentivirus carrying oe-Rbpjl + oe-Arid5a). AP was induced with an intraperitoneal injection of supramaximal cerulein (Sigma-Aldrich) at a dose of 100 mg/kg, for a total of ten times with a 1-h interval between injections. Sham-operated mice were subjected to treatment with PBS. To achieve effective interference, 10 μL of lentivirus (final titer of approximately 1 × 108 TU/mL) was injected via tail vein of mice 1 week prior to AP establishment . Lentiviruses carrying oe-Rbpjl and oe-Arid5a were procured from Genechem (Shanghai, China). The titer of lentivirus was above 1 × 109 TU/mL. Each mouse was injected with 300 µL of corresponding lentivirus for a duration of 14 days. Pancreatic tissues were collected and weighed. The organ coefficient was calculated using the following formula: organ coefficient = organ weight (mg)/body weight (g).
Paraffin-embedded tissue sections were stained with hematoxylin (C0007, Baoman Biotechnology Co., Ltd., Shanghai, China) at room temperature for 10 min, hydrolyzed with 1% hydrochloric acid alcohol, and then reverted to blue in 0.6% ammonia. Next, the tissue sections were counterstained with eosin at room temperature. The morphological and structural changes of pancreatic tissues were subsequently observed under an optical microscope (XSP-36, Boshida Optical Instruments Co., Ltd., Shenzhen, China). Pathological scoring was evaluated, and the parameters included edema, fat necrosis, hemorrhage, inflammatory cell infiltration, and acinar necrosis. The severity of pancreatic injury and the corresponding score were recorded as follows: 0 point indicates no injury; 1 point indicates mild injury; 2 points indicate moderate injury; 3 points indicates severe injury; 4 points indicates very severe injury .
Pancreatic tissues were sectioned at the thickness of 5 μm, dehydrated, treated with 3% hydrogen peroxide, and then subjected to peroxidase blocking with normal goat serum. Immunohistochemical staining was subsequently performed by incubating the tissue sections with primary antibody to Rbpjl (dilution ratio of 1:100, 14,613–1-AP, Proteintech) overnight at 4 °C. The following day, the tissues sections were incubated with biotin-labeled goat anti-rabbit secondary antibody (dilution ratio of 1:200, BA1003, Boster Biological Technology Co. Ltd., Wuhan, Hubei, China) for 20 min at 37 °C. Afterwards, the sections were exposed to DAB substrate, followed by hematoxylin counterstaining and standard dehydration treatment. Staining images were obtained using a microscope. Finally, the ImageJ software was adopted for statistical analysis of positive staining. Five different fields of view were randomly selected to count the number of positive cells, and the total number of cells counted was more than 100. The proportion of positive cells = (number of positive cells/total cells) × 100% .
Statistical analyses were performed using the SPSS 21.0 statistical software (IBM Corp. Armonk, NY). Measurement data, obtained from three independent experiments, were expressed as mean ± standard deviation. Data between two groups were compared using unpaired t test, and those among multiple groups were compared with one-way analysis of variance (ANOVA), combined with Tukey’s post-hoc tests. Data at different time points among multiple groups were compared by repeated measures ANOVA, followed by Bonferroni post-hoc tests. A value of p < 0.05 was regarded statistically significant.