Tissue Sample
10 cases of colitis and 5 normal colon tissues were purchased from Xi'an Best Biotechnology Co., Ltd. (Xi'an, China).
Animals And Treatments
C57BL/6 male p38αΔMφ mice and wild type (WT) littermates (6–8 weeks of age) were provided by Medical College of Xiamen University and housed in the SPF standard laboratory conditions.
As previously described 13, p38αΔMφ mice (n = 5) and WT mice (n = 6) were administrated by the addition of 3% DSS (36–50 kDa, MP Biomedicals, USA) in the drinking water for 7 consecutive days. In another cohort, male C57BL/6 mice (6–8 weeks old) were intraperitoneally injected with p38 MAPK inhibitors or vehicle (DMSO) for 7 consecutive days. According to previous literature 12, the injection doses of p38 MAPK inhibitors SB203580 and BIRB796 were 50 mg/kg/day and 15 mg/kg/day, respectively. Mouse body weight, colon length, clinical phenotype and pathological scoring were assessed.
Evaluation Of Dss-induced Colitis
The mice were observed every morning. Weight loss, fecal consistency and intestinal bleeding were recorded in accordance with the previous report 13. The DAI value was evaluated based on the above records.
Histopathological Assessment
The colon from the anus to the cecum was obtained and measured. The lower colon tissues in each group were fixed in paraformaldehyde solution ( 4% ) for 48 hours, and then embedded in paraffin. Histopathological assessment of the inflammatory responses in colon tissues in each group was determined by hematoxylin and eosin (H&E) staining in accordance with the previous report 14.
Immunohistochemistry And Immunofluorescence
Immunohistochemistry (IHC) and immunofluorescence (IF) analyses were performed to evaluate the protein levels of claudin-2 (AF0128, Affinity Biosciences, USA), MUC-2 (DF8390, Affinity Biosciences, USA), OAS2 (DF12680, Affinity Biosciences, USA), OAS3 (ER1914-27, HuaBio, China), P53 (AF0879, Affinity Biosciences, USA), ki-67 (ab16667, Abcam, United Kingdom), c-myc (AF0358, Affinity Biosciences, USA), TNF-α (17590-1-ap, Proteintech, China), IL-1β (ab33591, Lianke Biotechnology, China), IL-6 (21865-1-AP, Proteintech, China), and p-p38 (AF4001, Affinity Biosciences, USA), in paraffin-embedded colonic sections in each group. The detailed description of IHC and IF procedure is performed according to the previous reports 15, 16. The sum of the area% was analyzed by ImageJ software.
Rna Sequencing And Bioinformatic Analysis
Briefly, total RNA was extracted from the colon tissues of the WT and p38αΔMφ groups. The quantitative and quality control of total RNA were detected by NanoDrop ND-1000. 1–2 µg RNA was extracted from each sample to generate the RNA library, which were sequenced on an Illumina Hiseq 4000 platform after cluster generation. Data were analyzed as follows: sequencing quality control, sequencing data comparison, quantitative analysis of differentially-expressed genes, and GO and KEGG enrichment analysis.
Quantitative Real-time Pcr (Qrt-pcr)
Trizol reagent (Invitrogen, USA) was used to isolate total RNA from colon tissues Complementary DNA (cDNA) is obtained by RNA reverse transcription. The reaction procedure is based on our previous reports 17. The 2−ΔΔCt method was used to calculate the gene expression level. GAPDH was used as the internal control. All reactions were performed more than twice. All primers were listed in Table S1.
Western Blot Analysis
Western blot analysis
The protein levels of Claudin-2, OAS2 and OAS3 in colon tissues in each group were detected by Western blotting. In briefly, total proteins from colon tissues were obtained with RIPA lysis buffer (Beyotime, Shanghai, China) and protease inhibitor (Servicebio, China). After the determination of protein concentration, the protein was denatured by boiling water for 15 minutes. The primary antibodies claudin-2 (AF0128, Affinity Biosciences, USA), OAS2 (DF12680, Affinity Biosciences, USA), OAS3 (ER1914-27, HuaBio, China), and anti-β-actin (AF7018, Affinity Biosciences, USA) were thinned and incubated overnight at 4°C. We added HRP-labeled goat anti-rabbit secondary antibodies in a 1:3000 dilution, and incubated them at room temperature for 30 minutes.
Wash the membranes three times with PBST. The immunoreactive bands were detected with ECL Luminescent Solution (Servicebio, China).
Tdt-mediated Dutp Nick-end Labeling (Tunel) Assay
Apoptosis of IECs was detected by TUNEL kit (Servicebio, China). In short, colon tissue sections were cultured in TUNEL reaction mixture. Then, it was stained with 4’, 6-diamino-2-phenylindole (DAPI). After that, all samples were observed under the fluorescence inverted microscope. Each sample was observed in three random fields.
Fluorescence In Situ Hybridization (Fish) Analysis
Oligonucleotide modified probe sequences for P38α (probe1: 5'-Dig-CGGGCACCTCCCAGATTGTCTTGTT 3', probe2: 5'-Dig-GCCGAGCCAGTCCAAAATCCAGAAT 3', probe3: 5'-Dig-TCCAATACAAGCATCTTCTCCAGC 3') and CD68 (probe1: 5'-Bio-GGGGAATGAGAGAAGCAGGTGGGG 3', probe2: 5'-Bio-GCAATAAGCACCAGGGCGAGGAGGC 3', Probe3: 5'-Bio-GCCGAGAATGTCCACTGTGCTGCGT 3') were applied for FISH. The detailed operation steps were performed as described above 18. The images were analyzed by pathological image quantitative analysis–HALO software (Indica Labs, USA).
Statistical analysis
Statistical analyses were performed with SPSS 20.0 and GraphPad Prism 8.0.1. All data were displayed as the mean ± SEM. The differences between groups were analyzed by One-way ANOVA. P < 0.05 means statistical difference.