Cell culture
The human monocytic leukemia cell line THP-1 (purchased from American Type Culture Collection) was cultured in the RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 10,000 U/mL penicillin and 10,000 µg/mL streptomycin in a humidified incubator with 5% CO2 at 37 ℃. The experimental cells were treated with LPS to induce inflammation, followed by Thapsigargin (TG) or Pam3CSK4 treatment. The control cells were treated with dimethyl sulfoxide (DMSO) as the vehicle.
qRT-PCR
The Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract total RNA from THP-1 cells. The RevertAid 1st cDNA Synth kit (Thermo Fisher Scientific) was utilized to synthesize the cDNA for RT-PCR. The following sequences of PCR primers were synthesized by Sangon (Shanghai, China)
GAPDH Forward 5’-AATCCCATCACCATCTTCCA-3’and Reverse 5’-CCTGCTTCACCACCTTCTTG-3’;
GRP78 Forward 5’-CCCGAGAACACGGTCTTTGA-3’and Reverse 5’-TTCAACCACCTTGAACGGCA-3’;
CHOP Forward 5’-GCTCAGGAGGAAGAGGAGGA-3’and Reverse 5’-CTCCTTCATGCGCTGCTTTC-3’;
TLR1 Forward 5’-TAATGCATTTGATGCCCTGC-3’and Reverse 5’-CCTTCAAGGAAGTGCCAGAAT-3’;
TLR2 Forward 5’-TTCTTTTTCTTCCCTGGGCA-3’and Reverse 5’-TGAATTTGTTTCACCAGTGGA − 3’;
TLR3 Forward 5’- AAAACCTTTGCCTTCTGCAC-3’and Reverse 5’- GGAATCGTTACCAACCACATT-3’;
TLR4 Forward 5’-TGTGGCTCACAATCTTATCCA-3’and Reverse 5’- CTAAATGTTGCCATCCGAAA-3’;
TLR5 Forward 5’-GCTTTTCAGGGACTGTTCCAT-3’and Reverse 5’- CAAACCCATGTGAAAGATCCA-3’;
TLR6 Forward 5’-TCTTGGGATTGAGTGCTATGA-3’and Reverse 5’-CGGTGTACAAAGCTGTCTGTG-3’;
TLR7 Forward 5’-GCCAACTTCACTTGAATCTCC-3’and Reverse 5’-GCTGACAAATTTGGAGTTGCT-3’;
TLR8 Forward 5’-GCTGACAAATTTGGAGTTGCT-3’and Reverse 5’-AAATGCAATGCCCGTAGAGA-3’;
TLR9 Forward 5’-TGAGGACCTGGCCAATCTGA-3’and Reverse 5’-AAGGCCCTGAAGATGCCGA-3’;
TLR10 Forward 5’-CAAATGCACAAATGCCACAC-3’and Reverse 5’-AAAATCCAGATGGGCTGA-3’;
TNF-αForward 5’-AAGCCTGTAGCCCATGTTGT-3’and Reverse 5’-AGTCGGTCACCCTTCTCCA-3’;
IL-1βForward 5’-GACGGACCCCAAAAGATGAA-3’and Reverse 5’-CAGCCACGAGGCTTTTTGTT-3’;
IFN-βForward 5’- TCTCCTCCAAATTGCTCTCCT-3’and Reverse 5’-TACTCCTTGGCCTTCAGGTAA-3’;
IL-8 Forward 5’-TTGGCAGCCTTCCTGATTT-3’and Reverse 5’-TCAAAAACTTCTCCACAACCC-3’.
The amplification protocol was: 95°C for 10 min, 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The GAPDH was used to normalize gene expressions. The relative mRNA levels were calculated using the 2 − ΔΔCt method.
Enzyme-linked immunosorbent assay (ELISA):
The supernatants were collected from THP-1 cells, and the TNF-α, IL-1β, IFN-β and IL-8 levels in the supernatants were measured using ELISA kits (4A Biotech Co. Ltd., Beijing, China) following the manufacturer's instructions.
Western blot analysis:
Following the protocol provided by the manufacturer, THP-1 cells were collected and lysed with RIPA (Beyotime Institute of Biotechnology, China). The protein concentrations were quantified using BCA Protein Assay kits (Beyotime Institute of Biotechnology, China). Equal amounts (50 µg) of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membrane was blocked with 3% skim milk solution in phosphate buffered saline (PBS) containing 0.1% (v/v) Tween-20 (PBST) for 1 h at room temperature and then incubated with primary antibodies for Grp78 (Cell Signaling Technology, USA) (1:1,000), TLR2 (Abcam, USA) (1:300) and GAPDH (Beyotime Biotechnology, Suzhou, China) (1:1,000) in PBST overnight at 4°C. Subsequently, the membrane was further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Beyotime Biotechnology) (1:1,000) at room temperature for 1 h, followed by the incubation with enhanced chemiluminescence (ECL) solution, and exposure to the X-ray films. The protein bands were quantified and analyzed using Image Pro Plus 6.0 software.
Animals
Six-week-old female BALB/c mice were purchased from Hunan Slake Jingda Laboratory Animal Co. Ltd (Changsha, China). Prior to the experimentation, the mice were acclimatized for about one week. Each mouse was individually placed in a room maintained at 22°C under a 12 h day/night cycle. The Changsha Central Hospital Ethics Committee reviewed and approved all the animal experiments (Approval No. 20200512). The mice were randomized into three groups: the normal control group (n = 15), the TNBS modeling group (n = 15), and the TNBS + TUDCA treatment group (n = 15). Moreover, mice weight among various groups was not significantly different. First, the mice were kept on a 24 h fast with only drinking allowed and weighed before modeling. the animals were anesthetized through an intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine For the treatment and modeling groups, 150 µL TNBS (75 µL TNBS (5% w/v) mixed with 75 µL alcohol) was infused into the intestinal cavity through the mice's anus. For the control group, the same volume of saline was infused using a similar method. The next day following the infusion, TUDCA (50 mg/kg) was intraperitoneally injected daily into the mice of the TNBS + TUDCA treatment group. For the TNBS modeling and control groups, the same volume of saline was injected using a consistent methodology for seven days. Disease Activity Index (DAI) score was recorded by assessing the body weight, stool and hematochezia of the mice on a daily basis. On day 7, all the mice were sacrificed under anesthesia. Then, the mice were dissected to separate the colon and samples from the area with the most obvious inflammation were taken. For fixation and preservation, samples were fixed in 10% neutral buffer tissue fixative solution and made into histopathological sections. Histological disease scores were evaluated based on the presence of crypt loss and inflammatory cell infiltration, as described previously[12].
Clinical samples
The surgical pathological paraffin sections from 10 Crohn's disease (CD) patients (six males and four females; aged 20–60; three patients had lesions in the terminal ileum, and seven patients had lesions in the right colon), who visited Changsha Central Hospital from 2012–2018, were taken. Moreover, 10 pathological paraffin sections of normal human intestinal tissues were collected from the colon of five males and five females aged 20–60. All participants provided written informed consent. All experiments followed the Declaration of Helsinki. The experimental protocol was approved by the Ethics Committee of the Changsha Central Hospital (Approval No. 20200512).
Immunohistochemical staining
Furthermore, 5 µm of paraffin-embedded colonic or ileal sections from patients and mice were cut. For antigen retrieval, the sections were boiled in a 10 mM sodium citrate buffer. After blocking endogenous peroxidase with 3% hydrogen peroxidase, sections were blocked for 30 min with 3% BSA, followed by the incubation with primary antibodies for Grp78 (Abcam, Cambridge, UK) (1:500) and TLR2 (Abcam, USA) (1:300). PBS was utilized as a negative control instead of primary antibodies. After 1 h at room temperature incubation with horseradish peroxidase-labeled secondary antibodies (Abcam) (1:200), sections were incubated with 3,3′-diaminobenzidine for 2 min at 20°C. Under a light microscope, tissues were viewed, and photos were acquired.
Statistical Analyses
GraphPad Prism 8.0 was used to conduct statistical analyses, and all values were given as means ± SD. Using the Student's t-test, the differences between the groups were compared. Furthermore, p < 0.05 was considered statistically significant.