5.1 Establishment of FD model
All animal experiments were performed at the Model Animal Research Institute at Wuhan Myhalic Biotechnology Co., Ltd. (Wuhan, China). The procedures conformed to the Guidelines for Animal Care and Use and have been approved by the institutional review board (approval number HLK-20180420-01). To establish the in vivo model of FD, male Sprague-Dawley rats (specific-pathogen-free, 6-week-old, purchased from China Three Gorges University) were subjected to multi-factor stress intervention to mimic the symptoms of FD using a method described by Guo et al. . This method applies a tail-clamping approach to elicit stress in the experimental animals, resulting in behaviors of FD such as anxiety, mood swing, and depression . Five rats were kept in each cage, and the rats were enraged using a Foerster clamp to clip the distal end of their tail. This action resulted in pain and discomfort, provoking violent fights among the rats in the same cage. This procedure was performed twice a day for 14 days, each fight lasting 30 min. During the 14 days, the rats were given normal access to drinking water but made to fast every other day. The rats were also subjected to intraperitoneal administration of iced physiological saline (2 mL, 4°C, 0.9% NaCl) twice a day. During the 14-day intervention period, rat weight, water intake, and food intake were monitored every other day. Successful induction of FD was indicated by weight loss, dry and yellow hair, loose stool, reduced food and water intake, inactivity, slow response, and irritability and depression due to mood changes.
5.2 EA and/or miR-221-3p intervention
During the 14-day period, FD-modeled rats were subjected to EA and/or injection of miR-221-3p agomirs or antagomirs. For EA, acupuncture needles were inserted at the Zusanli (ST36) and Taichong (LV3) acupoints on both sides of each rat. The needles were connected to an acupoint stimulator (SDZ-II, Hwato, Suzhou, China) and a continuous wave was applied at a frequency of 10 Hz and an intensity of 1 mA to stimulate both acupoints simultaneously for 30 min. Sham operation was performed without electric stimulation The entire EA procedure was performed once a day. After acupoint stimulation, 5 nmol of miR-221-3p agomirs (miR40000890-4-5, Ribobio, Guangzhou, China) or antagomirs (miR20000890-1-5, Ribobio) in physiological saline were administered via tail vein injection, once every five days. Injection of vehicle only (physiological saline) was treated as a negative control (NC). At the end of day 14, the rats were sacrificed by an overdose of pentobarbital sodium via intraperitoneal injection. The gastric antrum and serum were preserved at -80°C for further analysis.
The morphology of ICCs in the gastric antrum was observed using TEM. Gastric antrum tissues were cut into 1-mm3 pieces and fixed in 2.5% glutaraldehyde at 4°C for 30 min. The fixed tissues were washed three times with 0.1 M phosphate-buffered saline (PBS) for 10 min each and fixed again in 1% osmium acid for 1 h. After three washed in 0.1 M PBS for 10 min each, the tissue specimens were dehydrated using a graded concentration series of ethanol (5 min at 50%, 70%, 80%, 90%, 90%; then 4 min at 100% twice). The specimens were then half-immersed in a 1:1 mixture of acetone:epoxy resin 812 at 40°C for 6 h and fully immersed in pure epoxy resin at 40°C for 4 h. After fixing, the specimens were embedded in a polymerization box for 4 h at 40°C, 2 h at 50°C, and 12 h at 90°C. The embedded specimens were cut into ultrathin sections (60 nm in thickness) using an ultramicrotome and subjected to double-staining. The sections were first dyed in uranium acetate (Xi’an Ding Tian Chemical Co., Ltd., Xi’An, China) in the dark for 20 min, washed three times with double-steamed water, and dried. The sections were then dyed with lead citrate (Boliante, Xi’An, China) for 15 min, and excess lead solution was washed with double-distilled water. Thereafter, the specimens were subjected to TEM analysis using a Hitachi HT770 apparatus.
5.4 Identification and analysis of differentially expressed miRNAs
After FD induction, EA treatment, and/or miR-221-3p agomir/antagomir intervention, RNA from was extracted from the gastric antrum of the experimental rats. Small RNA libraries construction and deep sequencing were carried out using the TruSeq Small RNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions on an Illumina Hiseq system. The raw reads were filtered by removing poor-quality reads, 5' adapter reads, reads without 3' adapters, reads containing poly (A) tails, rRNA, or tRNA, and reads < 18-nt in length to obtain clean reads. Known miRNAs were determined from the clean reads using miRbase and their expression levels were determined by the number of reads per million clean tags. Differentially expressed miRNAs were analyzed using DEGseq using the following criteria: p ≤ 0.05 and fold change ≥ 2. Target genes of the differentially expressed miRNAs were predicted using TargetScan.
RNA was isolated from gastric antrum tissue samples using TRIzol (15596026, Ambion, Inc., Foster City, CA) and reverse-transcribed into complementary DNA using the Advantage RT-for-PCR Kit (639505, TaKaRa, Dalian, China). qRT-PCR was performed using the SYBR Green PCR kit (KM4101, KAPA Biosystems, Wilmington, MA) with the primers listed in Supplementary Table S1. The experiment proceeded as follows: initial denaturation at 95°C for 3 min; 39 cycles of denaturation at 95°C for 5 s, annealing at 56°C for 10 s, and extension at 72°C for 25 s; and final extension at 65°C for 5 s and 95°C for 50 s. Data were acquired using qbase plus software and analyzed with the 2−ΔΔCt method.
5.6 Dual luciferase activity assay
Based on preliminary screening, miR-221-3p was identified to bind to the 3'UTR of c-kit at a seven-base binding site. pmirGLO reporter plasmids (Addgene, Watertown, MA) were constructed and wild-type (WT) and mutant (MUT) 3'UTR segments were prepared as follows: c-kit WT forward, 5'-CTAGCGTAAATATTGAAATGTAGCAAUAATGTCTTTTGAATATTCCTGAGCCCCATGAGTCCCTGAAAAT-3', WT reverse, 5'-CTAGATTTTCAGGGACTCATGGGGCTCAGGAATATTCAAAAGACATTTTGCTACATTTCAATATTTAC-3'; MUT forward, 5'-CTAGCGTAAATATTGAATAGGCATAAUAATGTCTTTTGAATATTCCTGAGCCCCATGAGTCCCTGAAAAT-3', reverse, 5'-CTAGATTTTCAGGGACTCATGGGGCTCAGGAATATTCAAAAGACATTTTATGCCTATTCAATATTTAC-3'. For the dual luciferase assay, miR-221-3p mimics (or negative control/NC) were co-transfected with c-kit 3'UTR-WT or c-kit 3'UTR-MUT in HEK293T cells using Lipofectamine 2000 according to the manufacturer’s instructions. Firefly luciferase and renilla luciferase signals were detected using a LucPair™ Duo-Luciferase Assay Kit (LF001, GeneCopoeia, Rockville, MD) with a Glo-MAX 20/20 analyzer.
5.7 Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed using kits from Bioswamp (CX43: RA20769; Raf: RA21040; Erk: RA21301; SCF: RA20600; SCF receptor: RA21199) with all required reagents provided. Prior to the assay, the wells of a 96-well plate were coated with respective antibodies. Rat serum (~ 40 µL) collected after treatment were added to each well and incubated, and 10 µL of biotinylated antibodies against the respective protein was added to each well. Then, 50 µL of horseradish peroxidase-conjugated reagent was added to each well and incubated for 30 min at 37°C. For color detection, chromogen was added to each well and incubated for 10 min at 37°C. After stop solution was added, the absorbance of the wells was evaluated using a plate reader (Labsystems Multiskan MS) at 450 nm.
5.8 Western blot
Tissue samples were lysed using radioimmunoprecipitation assay buffer supplemented containing protease and phosphate inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using 20 µg of protein in denaturing conditions, and the separated proteins were transferred onto polyvinylidene fluoride membranes (IPVH00010, Millipore, Burlington, MA). After 2 h of blocking in 5% skim milk at room temperature, the membranes were incubated at 4°C overnight with rabbit primary antibodies against CX43 (1:8000; ab11370, abcam, Cambridge, UK), SCF (1:1000; ab64677, abcam), c-kit (1:500; ab5505, abcam), p-Raf (1:5000; ab173539, abcam), Raf (1:5000; ab137435, abcam), p-Erk (1:1000; ab201015, abcam), Erk (1:1000; ab17942, abcam), and GAPDH (1:2000; PAB36264, Bioswamp). Thereafter, the membranes were incubated at 4°C for 1 h with goat anti-rabbit IgG (1:10000, PAB160011, Bioswamp) secondary antibodies in the absence of light. The membranes were immersed in an enhanced chemiluminescence reagent (WBKLS0010, Millipore) for color detection. Protein bands were visualized using an automatic analyzer (Tanon-5200, Tanon, Shanghai, China) and gray values were generated using Tanon GIS software.
5.9 Hematoxylin & eosin (HE) staining
Gastric antrum tissues embedded in paraffin, sliced into 4-µm sections using a microtome, and fixed on microscopic slides. For HE staining, tissue sections were dehydrated and washed with water for 1 min. Hematoxylin staining (PAB180015, Bioswamp) was performed for 3 min, following by 5 min of washing under running water. The sections were differentiated in 1% hydrochloric alcohol for 1 min, washed with water for 3 min, and immersed in bluing solution for 1 min. After washing in water for 3 min, the sections were stained in 0.5% eosin solution (PAB180016, Bioswamp) for 3 min and rinsed with distilled water for 10 s. The sections were then washed with 80% ethanol for 15 s, 95% ethanol for 15 s, and anhydrous ethanol for 3 min. Thereafter, the sections were immersed twice in xylene for 5 min each and sealed with neutral balsam.
5.10 Statistical analysis
The data are presented as the mean ± standard deviation (SD). The means among more than two groups was compared using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. p < 0.05 indicates a statistically significant difference