This study was approved by the ethics committee of Shengjing Hospital of China Medical University (2020PS388K(X1)). The ARM rat model was referred to previous literatures13. Sixty pregnant Wistar rats were randomly divided into two groups: one group was given 125mg/ mL ETU and the other group was given equal dose of normal saline without ETU.Pregnancy was continued under the original conditions. The fetuses were obtained by cesarean section on GD14–GD16, and the anorectal part was sampled under a microscope.
Cell transfection and processing
We purchased rat intestinal epithelial cells (IEC-6) from iCell Bioscience Inc., Shanghai, China. The cells were cultured in DMEM (Gibco, NY, USA) supplemented with 10% fetal bovine serum (HyClone Laboratories Inc., Utah, USA), 1% double antibody, and 89% high glucose, as a complete medium. Lip3000 (Invitrogen,Grand Island, NY, USA) was used to transfect an mir-NC/mimic, si-NC/TXNIP (RiboBio, Guangzhou, China), in order to construct an inflammatory state in vitro, IEC-6 was incubated with 20μg/mL LPS (Sigma, USA) for 4 hours. After supplementing the medium, then added 5 mM ATP (Sigma, USA) to the cells and incubate for 30 min.
After deparaffinization of 4-µm fetus tissue sections, the antigen was thermally repaired in citrate buffer (pH 6.0) at 98 ℃ for 2 minutes, and then naturally cooled to 25℃. PV-9000 (ZSJQB Co., Wuhan, China) two-step method was used for immunohistochemical staining, according to the manufacturer’s instructions. The sections were incubated with primary antibody TXNIP (Affinity, Shanghai, China; 1:1000), NLRP3 (Abcam, USA; 1:1000), caspase-1 (Novus, USA; 1:1000), IL-1β (Affinity; 1:1000), and IL-18 (Affinity; 1:1000) at 4°C for 16 h. After adding the amplification enhancer, the enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer was added dropwise on the tissue. DAB chromogenic solution was added dropwise on the tissue, and then. hematoxylin staining was performed for 1 min. Finally, dehydration was performed, and the tissue was mounted with neutral resin and photographed under a microscope (Nikon Corp., Tokyo, Japan).
The protein lysate was prepared using RIPA buffer:PMSF (100:1). The protein sample (20/40 µg) was mixed with 6× buffer, and then placed at 95℃ in a metal bath for 5 min. The proteins were resolved by SDS-PAGE gel electrophoresis and transferred on to PVDF membranes (Milipore,MA, USA). The membranes were washed with TBST (TBS+Tween) and blocked with 5% skim milk (TBST configuration) at room temperature for 2 h. Thereafter, the membranes were incubated with the following antibodies for 14 h at 4℃ after washing: TXNIP (Affinity; 1:1000), NLRP3 (Abcam; 1:1000), caspase-1 (Novus; 1:1000), IL-1β (Affinity; 1:1000), IL-18 (Affinity; 1:1000), β-actin (Affinity; 1:5000), and Tublin (Affinity; 1:5000). After washing, the membrane was incubated with the secondary antibody (goat anti-rabbit, Affinity; 1:8000) for 2 h. Thereafter, the membrane was washed again, and incubated with ECL luminescent solution (Thermo, USA). The PVDF membrane was placed in the developing instrument for exposure and development (Azure Biosystems, Inc., CA, USA).
We used 4.0-μm tissue sections and 5'-CY3-AGTAGTGCA ACCTAGTCAGAGC-3' probe (ServiceBio, Wuhan, China) for FSH. The paraffin sections were deparaffinized, digested with proteinase K (ServiceBio, Wuhan, China) for 20 min, and incubated at 37°C for 1 h for pre-hybridization. Thereafter, the hybridization solution containing the probe was added dropwise, and hybridization was performed overnight at 42°C in a thermostat. After washing, rabbit serum was added to block the membrane for 30 min at 25℃, and then mouse anti-DIG-HRP was added, and the sections were incubated for 50 min at 37℃; CY3-TSA (Servicebio, Wuhan, China) was added after PBS washing the sections. Finally, DAPI was added for counterstaining and allowed to react for 5 min in the dark. After mounting, the sections were photographed (Nikon DS-U3,Tokyo,Japan).
We inoculated 8 × 103 IEC-6 cells per well in a 96-well plate. According to the CCK-8 instructions, 10 µl of CCK-8 reagent was added to each well. After incubating the cells at 37°C for 2 h, a microplate reader was used to measure the optical density of the samples at 450 nm.
Dual luciferase assay
TXNIP wild-type (WT) and mutant (WUT) plasmids were constructed by Hanbio Biotechnology Co., Shanghai, China. According to the manufacturer’s instructions (Promega, Wisconsin, USA), lip3000 was used to transfect mir-NC+WT TXNIP, mir-NC+MUT TXNIP, mimic+WT TXNIP, and mimic+MUT TXNIP into 293T cells. The Du Al-Luciferase® reporter system was used to detect the luciferase activity 48 h after transfection.
Real-time quantitative PCR
The total RNA was extracted using TRIzol (Invitrogen, Carlsbad, USA), according to the manufacturer’s instructions. Follow the instructions of PrimeScriptTMRT regent Kit (Takara Biotechnology co., Ltd., Dalian, China) for reverse transcription and using a SYBR
Premix Ex Taq Kit(Takara Biotechnology co., China) for gene amplification.Applied Biosystems Fast 7500 Real Time PCR System was used to perform pre-denaturation and PCR. The 2-ΔΔCt was used to calculate the relative expression of the target gene. The primer sequences are as follows:
β-actin: F: TGCTATGTTGCCCTAGACTTCG, R: GTTGGCATAGAGGTCTTTACGG; TXNIP: F: AGTCAGAGGCAATCACAT, R: ATCAGCAAGGAGTATTCAAC; u6: F: CTCGCTTCGGCAGCACA, R: AACGCTTCACGAATTTGCGT; mir-301b-5p: F: ACACTCCAGCTGGGGCTCTGACTAGGTTGC, R: TGGTGTCGTGGAGTCG.
The level of IL-1β (Elabscience, Wuhan, China) and IL-18 (Boster, Wuhan, China) in the cell supernatant was determined according to the manufacturers’ instructions.
All data were statistically analyzed using SPSS19.0 and prism 8.0. The results are expressed as mean ± SD of three experiments. One-way ANOVA and Student's t-test were used to analyze the differences between groups. Results with P < 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05, ##p < 0.01, ###p < 0.001).