Plasmids of cDNA and shRNA of p65 (cDNA: 55304-1; shRNA: 5405-1, 5406-1, 5407-1) and Cas3 (cDNA: 55749-1; shRNA: 5402-1), and negative controls were purchased from Shanghai Jikai Gene Medical Technology Co., Ltd. (Shanghai, China). Primary antibodies against caspase-3 (19677-1-ap), p65 (10745-1-ap), GAPDH (10494-1-ap), and α-actinin (11313-2-ap) were purchased from Proteintech Group Inc. (Wuhan, China), while p-p65 antibody was purchased from Abcam (ab86299, Cambridge, UK). Secondary antibodies included goat anti-rabbit IgG labeled with Cy3 (A0516, Bi-Yun-Tian Biotechnology Co., Ltd, Shanghai, China) and horseradish peroxidase (HRP) (CST, 7074S, MA, USA). The following reagents were used to quantify gene expression: RNAiso Plus reagent (Takara Bio Inc.), ReverTra Ace qPCR RT Master Mix kit (Toyobo Co., Ltd. Life Science Department, Osaka, Japan), and SYBR1 Green Real-time PCR Master Mix kit (Toyobo Co., Ltd.). A protease inhibitor cocktail was obtained from Roche Diagnostics GmbH (11836145001). All drug concentrations are expressed as final working concentrations in buffers. A bicinchoninic acid (BCA) assay kit was supplied by Bi-Yun-Tian Biotechnology Co., Ltd (P0010, Shanghai, China). Enhanced chemiluminescent (ECL) reagents were purchased from Bi-Yun-Tian Biotechnology Co., Ltd. (P0018AS, Shanghai, China).
TC cells(thymocytes) were primary cell cultures prepared by our laboratory. V79 cells were obtained from the Chinese Academy of Sciences Cell Bank (CS0199) while MTEC-1 cells were purchased from Shanghai Hongshun Biological Technology Co., Ltd (HSC9942). All cell types were cultured in suspension in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO2.
Cells were irradiated in vitro at room temperature at a dose rate of 100 cGy/min for a total dose of 6 Gy . Cells in the treatment group were supplemented at concentrations of 20 µg/mL for 2 hours prior to irradiation.
For the in vivo studies, animals were placed in bespoke boxes  prior to exposure to 6 Gy of total body radiation at a dose rate of 100 cGy/min. OB was administered orally for 4 days at a dose of 2.5 mg/kg prior to irradiation. At various time points, the mice were sacrificed, tissue blocks were harvested and different physiological indicators were measured.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from 30-50 mg tissue using 1 mL RNAiso Plus reagent. The corresponding cDNA was created from 1 µg of the RNA from each sample using a ReverTra Ace qPCR RT Master Mix kit, in accordance with the manufacturer’s instructions. The reaction system was heated to 37°C for 15 min, 50°C for 5 min, then 98°C for 5 min using BIO-RAD T100 Thermal Cycler. PCR product was diluted 10 times for qPCR. The reaction system was then heated to 95°C for 1 min, then taken through 40 cycles of heating to 95°C for 15 s, 60°C for 30 s and 72°C for 45s. The following primer sequences were used (mus/ham):
Caspase3-F: GGACTGATGAGGAGATGG, ATCGTGACACACACTGGACC;
Caspase3-R: AAAGGGACTGGATGAACC, CCATGAGACTGCAGCACAGA.
p65-F: GGACCTATGAGACCTTCAAGAG, GATGCGATTAGTTTTGGCTTCC;
p65-R: ACAGAAGTTGAGTTTCGGGTAGG, CCCGTGTAGCCATTGATCTGT
GAPDH-F: AGGTCGGTGTGAACGGATTTG and GAPDH-R: TGTAGACCATGTAGTTGAGGTCA. mRNA expression levels of the target genes were calculated relative to the endogenous control gene, GAPDH, using the 2-ΔΔCT method.
Cells were lysed using cell-lysis buffer (20 mM Tris-HCl, 2 mM EDTA, 137 mM NaCl, 1% NP-40, and 10% glycerol, with an aliquot of 1 mM PMSF and proteinase inhibitor cocktail added just prior to use). Lysis was conducted on ice for 30 min, with vortex mixing at 5 min intervals, after which the suspension was centrifuged at 12000 rpm for 15 min at 4°C. The tissue samples were homogenized on ice in cell-lysis buffer, as described above, except that lysis was conducted on ice for 45 min. The supernatant after centrifugation represented total tissue or cellular protein. The concentration was assayed using a BCA protein assay. A total of 40-80 µg protein was separated by SDS-PAGE after which the protein bands were transferred to a membrane. Each membrane was incubated with an appropriate dilution of a primary antibody (p65: 1;2000; p-p65: 1:1000; Cas3: 1:1000; GAPDH: 1:10000; or α-actinin: 1:2000), followed by incubation with horseradish peroxidase-conjugated secondary antibody diluted 1:2000. Protein bands were visualized using enhanced chemiluminescence (ECL). The intensity of bands on the Western blots was measured using ImageJ software. Background intensity was subtracted from each calculated band density.
Preparation and identification of p65+/- mice
C57BL/6J-Relaem1Smoc mice (NM-KO-190139), referred to as p65+/- mice, were prepared by Shanghai Southern Model Biotechnology Co., Ltd, with transcript Ensembl number: Rela-201 ENSMUST00000025867.5. CRISPR/Cas9 technology was used to knock out exon 4 of the p65 gene and then non-homologous recombination was used to repair the introduced mutations, resulting in a frameshift of the Rela gene protein reading frame and loss of function (sgRNA1: GCCCCAGCAGACTTGCCTCCTGG; sgRNA2: GGCTGGCCTGTCCAGCCATAGGG)
A 0.1-0.2 cm section of mouse tail was placed into an EP tube and 60 μL of solution A(an alkaline lysis reagent: 25mM NAOH and 0.2mM disodium EDTA, pH 12 not adjusted) were added. The mouse tail was lysed for 30 minutes in a closed metal bath at 100°C. The lid was opened and excess pressure released every 5 minutes. The lysed mouse tail was flicked with a finger, then cooled to 4°C . An 80 μL aliquot of solution B (a neutralizing reagent: 40mM Tris-HCl, pH 5, not adjusted) was added to the EP tube which was then centrifuged to evenly mix the liquids at 10000 rpm for 5 min at 4°C, after which the supernatant was removed. PCR was then performed to confirm mouse tail gene transformation (P1: AGGGTGGGCACTGGAGTTTATTGA Common,
P2: GAGGCCCAGGGAAGGTGACAGAGA Mutant,
P3: GATGAGGCCGGTGAGGTGGAT Wildtype（WT）;
Mutant=846/997bp, WT =546bp).
Flow cytometry assays (FCM)
An Annexin V-FITC flow cytometry assay was used to detect cellular apoptosis. Cells were incubated with treatment drugs for 2 hours then irradiated with 6 Gy radiation. After different durations (TC cells: 6 h, V79 cells: 48 h, MTEC-1 cells: 24 h), the cells in each group were pelleted by centrifugation at 1800 rpm for 5 min, washed three times with cold PBS then gently resuspended in 195 µL Annexin V-FITC binding solution. An aliquot comprising 10 µL Annexin V-FITC and 5 µL propidium iodide (PI) dye was carefully added to the cells and incubated at room temperature for 15 min in the dark. Apoptosis was identified by flow cytometry (FCM) as green fluorescence, cell death was represented by red and green fluorescence, while living cells emitted no fluorescence.
Harvested tissue was fixed in 4% paraformaldehyde overnight, processed, then embedded in paraffin. The tissue blocks were then sliced into 4µm-thick sections and placed on polylysine-treated slides, and dewaxed by placing in xylene prior to incubation through a gradient of alcohol concentrations. The sections were then placed in a 0.01M sodium citrate solution at pH 6.0 for antigen retrieval and cells were permeabilized by treatment with 0.3% Triton X-100 for 20 min. Non-specific staining was blocked by placing the slides in a humid container and incubating with 10% sheep serum for 60 min at room temperature. The slides were then incubated with primary antibody (1:200) overnight at 4°C, then with Cy3-labelled secondary antibody (1:500) in a humid chamber at room temperature for 1 h, washing with PBS after each incubation. The slides were incubated with DAPI for 2 minutes at room temperature then rinsed 3 times in PBS for 3 min each time. An anti-fluorescent quencher was added to the tissue sections in the dark. The fluorescence of tissue sections was imaged using Pannoramic a MIDI digital slide scanner (3DHISTECH).
Histological analysis of tissue
Paraffin sections were deparaffinized and stained with hematoxylin and eosin, dehydrated, then mounted with neutral gum. Finally, the sections were examined using a Pannoramic MIDI digital slide scanner (3DHISTECH).
Staining of tissue with Hoechst
Paraffin sections were placed in an oven at 60°C and warmed for 30 minutes, then a small volume of Hoechst 33342 staining solution was added to cover the samples and incubated for 5 min. The Hoechst 33342 staining solution was then aspirated off the slides which were washed in PBS 3 times, 5 min each time. The stained slides were then directly observed using an inverted fluorescence microscope. The nuclei of cells undergoing apoptosis were densely stained.
Data were expressed as means ± standard deviation (SD). The data were analyzed by analysis of variance (ANOVA) using SPSS/PC* (statistical package for social sciences, personal computer) and ImageJ software. The group means were compared using a Duncan’s Multiple Range Test (DMRT). The means of the treated groups were compared with those of the radiation-alone or non-irradiated groups. A value of P < 0.05 was considered statistically significant.