Laboratory animal
A total of 40 8-week SPF C57BL/6J mice weighing 22.41 ± 2.34 g were purchased from the Beijing HFK Bioscience Co., Ltd., China. The weight and quality of laboratory mice met the national standards, and all mice were fed conventionally. The protocol and procedures employed were ethically reviewed and approved by the Animal Ethics Committee of The Second Hospital of Jilin University (No.D20190013), and experiments were performed in accordance with relevant institutional and national guidelines for the care and use of laboratory animals.
Establishment of colitis mice models
Twenty mice were randomly selected to establish colitis models, 10 mice were used as sham-operated mice, and 10 mice were fed conventionally. After the mice for establishing colitis models were allowed to acclimate for 1 week, they were induced using oxazolone. Oxazolone was dissolved in 50% ethanol to prepare 1% and 3% oxazolone solution. Mice were anaesthetized with intraperitoneal injection of 100 mg/kg 1% pentobarbital sodium. The abdomen of sham-operated mice and model mice was shaved, and the skin surface was smeared with 2 mL 3% oxazolone once a day for 5 d to generate sensitization. Then, 150 μL 1% oxazolone was infused by inserting a 2 mm silicone tube approximately 4 cm from the anus to conduct clysis. The skin surface of sham-operated mice was smeared with 2 mL 50% ethanol, and 150 μL 50% ethanol was used to conduct clysis; the remaining treatment was the same as that in model mice. The body weight loss and diarrhea occurred 24 h after clysis, indicating a successful modeling. Mice after clysis and healthy mice were bred conventionally under the same rearing condition.
Grouping
The mice were randomly and averagely divided into four groups: Normal group (healthy mice), control group (sham-operated mice), model group (model mice without any treatment), and K252a group (model mice with the treatment of 100 μmoL/kg TrkB-PLC/IP3 pathway inhibitor). Mice in K252a group before clysis were intraperitoneally injected with 100 μmoL/kg K252a once a day for 5 d. At 24 h after modeling, the disease activity index (DAI) of the mice was observed. The mice were anesthetized with administer intraperitoneal injection of 100 mg/kg, 1% sodium pentobarbital. Under anesthesia, eyeballs were picked out to collect peripheral blood. After mice died of excessive blood loss, the colon tissue was taken. The colonic mucosal injury index, histological injury index, and related molecular biological indicators were observed. The Ethics Committee of The Second Hospital of Jilin University had approved this method of euthanasia.
Disease activity index scoring
Detailed scoring rules on disease activity index (DAI) [8]: After 24 h of successful modeling the mice were weighed; fecal status was recorded; occult blood was detected; DAI was calculated. DAI was scored by overall consideration of mice weight loss, fecal viscosity and hemafecia status. Scoring was as follows: no weight loss of 0, weight loss less than 5% of zero, weight loss of 5%-10% of two points, weight loss of 10%-15% of three points, weight loss greater than 15% of four points; normal fecal viscosity of zero, loose stools of two points, diarrhea of four points; occult hemafecia of two points, revealed hemafecia of four points. The total score of weight loss, fecal viscosity and hemafecia was divided by three to obtain DAI score.
Colon mucosa damage index scoring
Detailed scoring rules on colon mucosa damage index (CMDI) [8]: After mice were sacrificed under anesthesia, the morphological changes of colon mucosa were observed with the naked eye to score CMDI. No mucosa damage was scored as zero; mild edema and hemorrhage on the surface of intestinal mucosa were scored as one point; moderate hyperemia, edema and erosion in the intestinal mucosa were scored as two points; severe hyperemia and edema, tissue necrosis, ulcer with a diameter of less than 1 cm in the intestinal mucosa, and thickening rectum wall with necrosis and inflammation were scored as three points; complete necrosis of the rectum wall and ulcer with a diameter of greater than 1 cm were scored as four points.
Tissue damage index scoring
Detailed scoring rules on tissue damage index (TDI) [8]: After 24 h of successful modeling, the lesions of mice were fixed with 100 mL/L formaldehyde and imbedded using paraffin to prepare paraffin sections. The sections were stained with hematoxylin and eosin (HE). The morphology of sections was observed under an optical microscope, and TDI was scored. The scoring standards of TDI mainly included inflammatory cell infiltration, the depth of infiltration, and the depth of an intestinal ulcer. No inflammatory cell infiltration of zero; mild inflammatory cell infiltration of one point; severe inflammatory cell infiltration of two points. The depth of infiltration was scored as follows: Infiltration to the mucosal layer of one point; infiltration to the mucosa and submucosa of two points; infiltration to the entire colon layer of three points. The depth of intestinal ulcer was scored as follows: No ulcer of zero; ulcer to the epithelium of one point; ulcer to the lamina propria mucosa of two points; ulcer to the muscularis mucosa of three points.
ELISA
The obtained peripheral blood was centrifuged at 3,000 rpm for 10 min at low temperature to collect the upper serum which was then centrifuged at 8,000 rpm for 10 min at low temperature to collect the upper serum. The serum of mice was detected in strict accordance with the instruction of the ELISA kit (ebioscience, Thermo Fisher, USA). The ELISA kit was placed at room temperature for 20 min to prepare the washing solution. The blank well and sample well to be tested were set. After the standard substance was dissolved, 100 μL washing solution were added to the sptting plate to make the standard curve. The sample of 50 μL was added to the sample well to be tested, including 10 μL samples to be tested and 40 μL diluent. After the coated plate was sealed, it was incubated at a constant temperature of 37°C for 30 min. The sample to be tested was washed after the plate was uncovered. The sample well, after patted dry, was added with 50 μL biotinylated antibodies working solution and incubated at a constant temperature of 37°C for 30 min. The sample to be tested was washed after the plate was uncovered. The sample well, after patted dry, was added with 50 μL color-developing agent A in a dark place, followed by the addition of 50 μL color-developing agent B. After the solution was mixed by gently shaking, the color was developed at 37°C for 15 min. After finishing color development, 50 μL stop buffers were immediately added. Optical density (OD) value at 450 nm in each well was measured using a microplate reader (BioTek Synergy 2, BioTek, USA). The contents (pg/mL) of tumor necrosis factor-α (TNF-α, ab6671, Abcam, USA), interleukin-4 (IL-4, ab11524, Abcam, USA), IL-8 (ab117318, Abcam, USA), IL-10 (ab108870, Abcam, USA), and TNF-γ (ab106099, Abcam, USA) in the serum of mice were analyzed through the standard curve drawn based on OD values.
Cell cycle and apoptosis detection by flow cytometry
The colon tissues of mice were put on a 60 μm-aperture nylon beaker, cut into pieces by an ophthalmological curved scissor, and washed with phosphate buffered saline (PBS). The single cells were collected, fixed with 95% ethanol, and washed with PBS once before centrifugation. Then centrifugation was performed at 1,500 rpm for 5 min. The supernatant was discarded. Cell cycle detection: Cells were treated in dark place, and incubated in a water bath with the addition of 100 μL RnaseA (SJ0709, Beijing Baiaolaibo Technology Co. Ltd., China) at 37°C for 30 min follow by the addition of 400 μL PI dye solution (Sigma, USA). After cells were treated in dark place at 4°C for 30 min, the red fluorescence intensity at 488 nm in flow cytometer (CytoFLEX, Beckman Coulter, CA 92821, USA) was recorded to determine the cell cycle distribution in each group. Cell apoptosis detection: Annexin-V-FITC, PI and HEPES buffer at the ratio of 1:2:50 were prepared into Annexin-V-FITC/PI dye liquor according to the instruction of Annexin-V-FITC apoptosis assays kit (Sigma, USA). A total of 1*106 cells were re-suspended per 100 μL dye liquor. The liquor was mixed by shaking and incubated at room temperature for 15 min. Then the liquor was added with 1 mL HEPES buffer and mixed by shaking. The 525 nm and 620 nm band-pass filters were excited with the wavelength of 488 nm to detect FITC and PI fluorescence expressions. The apoptosis was measured through FITC and PI fluorescence expressions.
qRT-PCR
The colon tissues of mice were used to prepare tissue homogenate. Total RNA in the tissue homogenate was extracted using Trizol (10296028, Thermo fisher, USA), and the purity and concentration of sample total RNA were determined. Sample RNA was reversed to cDNA using the reverse transcription kit (11939823001, Merck, USA). qRT-PCR was performed with cDNA as the template. Total reaction solution was 10 μL, including 0.5 μL PCR forward primer, 0.5 μL PCR reverse primer, 1 μL cDNA template, 3 μL ddH2O, and 5 μL (2×) SYBR® Premix Ex TaqTM II. qRT-PCR amplification procedure: pre-denaturation at 95°C for 4 min, denaturation at 94°C for 30 s and annealing at 60°C for 30 s for 35 circles, setting the solubility curve, and extension at 72°C for 5 min. The reference gene of qRT-PCR was GAPDH. The primers of Bax, Bcl-2, Caspase3, Fas, FasL and GAPDH were synthesized by the Sangon Biotech (Shanghai) Co., Ltd., China and shown in Table 1. Expressions of products were calculated using 2-ΔΔCt. ΔΔCt = ΔCtthe rest groups - ΔCtcontrol group. ΔCt = Cttarget gene - CtGAPDH. Ct represented the number of amplification cycles when the fluorescence intensity reached the threshold value, after qPCR was performed for samples to be tested.
Table 1 qRT-PCR primer sequence
Name
|
Sequence
|
TrkB
|
Forward: 5’- CTGGGGCTTATGCCTGCTG-3’
|
Reverse: 5’- AGGCTCAGTACACCAAATCCTA-3’
|
PLC
|
Forward: 5’- TTCCAGATGGTCTATTTCCGGG-3’
|
Reverse: 5’- CTTGGCACTTGCATCCTCC-3’
|
IP3
|
Forward: 5’- CGTTTTGAGTTTGAAGGCGTTT-3’
|
Reverse: 5’- CATCTTGCGCCAATTCCCG-3’
|
Bax
|
Forward: 5’- AGACAGGGGCCTTTTTGCTAC-3’
|
Reverse: 5’- AATTCGCCGGAGACACTCG-3’
|
Bcl-2
|
Forward: 5’- GCTACCGTCGTGACTTCGC-3’
|
Reverse: 5’- CCCCACCGAACTCAAAGAAGG-3’
|
Caspase3
|
Forward: 5’- CTCGCTCTGGTACGGATGTG-3’
|
Reverse: 5’- TCCCATAAATGACCCCTTCATCA-3’
|
Fas
|
Forward: 5’- GCGGGTTCGTGAAACTGATAA-3’
|
Reverse: 5’- GCAAAATGGGCCTCCTTGATA-3’
|
FasL
|
Forward: 5’- CAGCCCATGAATTACCCATGT-3’
Reverse: 5’- ATTTGTGTTGTGGTCCTTCTTCT-3’
|
GAPDH
|
Forward: 5’- AGGTCGGTGTGAACGGATTTG-3’
Reverse: 5’- GGGGTCGTTGATGGCAACA-3’
|
TrkB, protein-tyrosine kinase receptor B; PLC, phospholipase C; IP3, inositol triphosphate.
Western blot
The intestinal mucosa of mice was used to prepare tissue homogenate. The tissue was lysed on ice using RIPA to extract total protein. Centrifugation was performed at 13,000 rpm for 2 min to obtain the supernatant. After the protein concentration was adjusted using a BCA kit (P0012S, Beyotime Biotechnology Co., Ltd., Shanghai, China), 160 μL supernatant were mixed with 40 μL 5× SDS loading buffer. The mixture was put into a boiling water bath for 10 min. Then SDS-PAGE was performed. The protein was transferred to PVDF membranes by wet method; the PVDF membrane was pretreated by immersing in methanol for 30 s and washing with ddH2O. The membrane was sealed with 5% skimmed milk powder and incubated with primary antibodies at room temperature for 2 h. The primary antibodies included rabbit polyclonal antibodies p-TrkB (ab109684, 1/2,000, abcam, USA), TrkB (ab18987, 1/2,000, abcam, USA), PLC-γ1 (ab107455, 1/2,000, abcam, USA), PLC (ab154610, 1/3,000, abcam, USA), IP3 (ab5804, 1/1,000, abcam, USA), TNF-α (ab6671, 1/2,000, abcam, USA), TNF-γ (ab19139, 1/1,000, abcam, USA), IL-4 (ab9728, 1/2,000, abcam, USA), IL-8 (ab7747, 1/1,000, abcam, USA), IL-10 (ab6696, 1/3,000, abcam, USA), Bax (ab32503, 1/5,000, abcam, USA), Bcl-2 (ab182858, 1/2,000, abcam, USA), Caspase3 (ab13847, 1/500, abcam, USA), Fas (ab82419, 1/1,000, abcam, USA), FasL (ab15285, 1/2,000, abcam, USA), and GAPDH (ab181602, 1/10,000, abcam, USA). Then the membrane was rinsed with tris buffered saline tween (TBST) three times, 5-10 min every time. The membrane was incubated with second antibody goat anti-rabbit IgG (ab6721, 1/2,000, abcam, USA) at room temperature for 2 h. Then the membrane was rinsed with TBST three times, 5-10 min every time. The PVDF membrane was placed in an E-gel imager and covered using developing agent. The protein was photographed through Bio-Rad image analysis system (ChemiDoc MP, BIO-RAD, USA). The gray value of the protein band was analyzed by Quantity One software. The relative content of protein was calculated by the gray value of the protein band / the gray value of the internal reference band.
Statistical analysis
SPSS 21.0 software (SPSS, Inc, Chicago, IL, USA) was used to analyze the data. All measurement data were represented as mean ± standard deviation. Comparison among groups was performed using one-way ANOVA and post hoc test, Bonferroni pairwise comparison. There was a significant difference at P<0.05.