Animals and water avoidance stress (WAS)
Animals were raised in the Animal Experimental Center of Tongji Medical College. As in previous research [14, 15], 9-week-old male C57BL/6J mice were placed on a small platform surrounded by water at a temperature of 25 °C for 2 h per day for 10 days. Mice were randomly divided into three groups (n = 10): the control group, WAS group and WAS + Yoda1 group. In the WAS + Yoda1 group, 1 h before being subjected to WAS, the mice were injected intraperitoneally with Yoda1 (50 μmol/L, 4 ml kg−1 day−1; Tocris); the mice were treated in this manner consecutively for 10 days.
Mice carrying a floxed Piezo1 locus (Piezo1LoxP/LoxP) on a C57BL/6 J background were crossed with mice expressing a tamoxifen-inducible Cre-recombinase transgene under the control of a Mucin2 promoter (Mucin2-CreERT2) to generate Piezo1LoxP/WT; Mucin2-CreERT2 mice (with the capacity to induce a heterozygous deletion of intestinal Piezo1 after tamoxifen treatment). Subsequent cross-breeding of Piezo1LoxP/WT; Mucin2-CreERT2 mice resulted in the generation of Piezo1 flox-mucin2 Cre mice (Piezo1 flox-mucin2 Cre mice, with the capacity to induce a homozygous deletion of intestinal Piezo1 after tamoxifen treatment). Cre was induced in 8–12-week-old male mice by intraperitoneal injection of tamoxifen (1 mg/day in sunflower oil for 5 days; Sigma, Millipore, St. Louis), while tamoxifen-treated Piezo1LoxP/LoxP mice were used as controls.
Abdominal withdrawal reflex (AWR)
Visceral sensitivity to colorectal distention (CRD) was measured by the AWR test as described previously . CRD was conducted at constant pressures of 20-, 40-, 60- and 80-mm Hg by a custom-made distension control device. The responses were considered stable if there was less than 20% variability between 2 consecutive trials of CRD at 60 mm Hg. The AWR score was recorded as described in previous research .
Horizontal Ussing chamber and mucus thickness measurement
According to previous research , we made a horizontal perfusion chamber. For the ex vivo experiments, mice were anesthetized with isoflurane (R510-22, RWD Life Science Co., Ltd) and killed by cervical dislocation. The distal colon was dissected and flushed, and the muscle layer was removed. The mucosal tissue explant was mounted in a horizontal perfusion chamber. The upper surface of the colonic mucus was visualized by the addition of charcoal particles. The mucus thickness was determined by measuring the distance between the epithelial surface and the mucus surface by a micropipette attached to a patch clamp viewed through a stereomicroscope. Specifically, the height of the micropipette attached to the patch clamp was noted on the stereoscopic microscope using the homemade spatial positioning device. When the front end of the micropipette is located on the mucus surface, the corresponding height is recorded as H1. While keeping the horizontal position unchanged, when the front end of the descending micropipette falls into contact with the epithelial surface, the corresponding height is recorded as H2. H1 minus H2 to get the mucus thickness of the corresponding position. The mean value was calculated after repeated measurement for 3 times, then the position of the micropipette was changed and the mucus thickness at 6 different positions was measured.
Colonic smooth muscle contraction
Colonic smooth muscle contraction was recorded as previously described . The mean contractile amplitude of each strip was recorded with an RM6240 multichannel physiological signal system. A 1-cm segment of the distal colon longitudinal muscle strip was removed and placed in an organ bath system containing oxygenated (95% O2 + 5% CO2) Krebs solution (119 mmol/L NaCl, 4.7 mmol/L KCl, 25 mmol/L NaHCO3, 1.2 mmol/L NaH2PO4, 2.5 mmol/L CaCl2, 1.2 mmol/L MgSO4, and 11.1 mmol/L glucose; pH 7.30–7.40) at 37 °C. The mean contractile amplitude of each strip was recorded with a LabChartReader_8.1.13 multichannel physiological signal system.
Spontaneous smooth muscle activity was recorded, and the motility index (MI) was calculated. The MI is defined as the area under the contractile curve in unit time, which could be considered a comprehensive evaluation of muscle activity containing muscle tension, amplitude, and frequency. Additionally, 10 mM acetylcholine (Ach; A6500, Sigma, Millipore, St. Louis) was added into the bath chamber to observe the excitatory nerve–mediated contractions.
Ink propulsion test
For measurement of the intestinal transit rate (ITR), mice received an oral administration of 200 μL of Indian ink. The specific process was conducted as described in previous literature . The percentage of blackened intestinal tracts was calculated: ITR (%) = pushing length/total length × 100%.
Hematoxylin and eosin (H&E) and Alcian blue staining
After the mice were sacrificed, the mouse colon samples were fixed in 10% neutral-buffered formalin and Carnoy’s solution (60% methanol, 30% chloroform, 10% glacial acetic acid, G1120, Servicebio) overnight and then processed by standard methods and embedded in paraffin. H&E and Alcian blue were used to stain paraffin section (3–5 μm) for mucus characterization, which was performed according to a standard protocol . The investigators photographed the sections by using a BX51 Olympus microscope. Another two investigators who were not clear about the grouping measured the Mucus layer thickness (5 measurements per section/2 sections per animal/5 animals per group, 200X) and goblet cells/epithelial cells (10 measurements per section/2 sections per animal/5 animals per group, 200X) with ImageJ software respectively .
Fluorescence in situ hybridization (FISH)
As in previous research , paraffin-embedded Carnoy’s-fixed sections were hybridized with 10 ng/ml of a general bacterial 16S rRNA probe (EUB338, red, 5′-GCT GCC TCC CGT AGG AGT-3′, Boster) and immunostained for Mucin2 using the UEA-1 (Bauhinia bean lectin, FL-1061-2, Vector labs). Images were observed with a Hitachi U8010 transmission electron microscope (Hitachi, Ltd., Tokyo, Japan).
Western blot (WB) and immunofluorescence
Immunoblotting was conducted as described previously . Colon tissues from mice were dissected the next day after completing the WAS procedure. The epithelial layers were gently scraped off, and proteins were extracted. In separate studies, human LS174T cells were collected and processed with the same method. Proteins were separated and blotted with the following primary antibodies: Piezo1 (1:500, 15939-1-AP, Proteintech), H3K9me3 (1:500, 13969, Cell Signaling Technology), T-H3 (1:500, GTX122148, GeneTex), SUV39h1 (1:500, PA5-29470, Thermo-Fisher), Histone Deacetylase 3 (Hdac3) (1:500, GTX1096679, GeneTex), and GAPDH (1:1000; Proteintech).
For immunofluorescence staining, paraffin sections were stained as described in previous research . Primary antibodies used for incubation were as follows: Piezo1 (1:200, 15939-1-AP, Proteintech), H3K9me3 (1:500, 13969, Cell Signaling Technology), AGR2 (1:200, AF6068, R&D Systems), and Mucin2 (1:200, GTX100664, GeneTex). DAPI (D9542, Sigma, Millipore, St. Louis) was used for nucleic acid staining.
Cell culture and treatment
Briefly, the LS174T cell line was purchased from Shanghai Zhong Qiao Xin Zhou.
Biotechnology. Cells were cultured in DMEM containing 10% FBS and 1% nonessential amino acids. Culture medium was changed every other day. Piezo1 knockdown (Piezo1-KD) LS174T cells were infected with lentivirus packaging Piezo1 siRNA as described in previous research .
For shear force intervention, the cells were inoculated on 24-well and 6-well culture plates (Corning, NY, USA). After growing to fusion, the cells were placed on a rocking board in an incubator. The parameters were 0.3 Hz and shear stress = η × φ × 125 ≈ 0.1 N (η: viscosity of the medium; φ: flow rate) as previous research . The Piezo1 agonist Yoda1, antagonist GsMTx4 (HY-P1410, MCE) and the methylation inhibitor UNC0638 (HY-15273, MCE) were added during mechanical stimulation.
Quantitative real-time RT-PCR (RT-PCR) was carried out in a LightCycler 480 system using SYBR Green Transcription Master Mix (Roche Diagnostics, IN, USA) as previously described .
Mouse primers were as follows.
GAPDH: forward “5′-TGAAGCAGGCATCTGAGG-3′”; reverse “5′-CGAAGGTGGAAGAGTGGGAG-3′”.
Mucin2: forward “5′-GCTGACGAGTGGTTGGTGAATG-3′”; reverse “5′-GATGAGGTGGCAGACAGGAGAC-3′”.
EDEM1: forward “5′-TGAAAGCATGTGAGGGTAGTG-3′”; reverse “5′-GAGAGAAGGGAAGACAGGATAGA-3′”.
GRP78: forward “5′-AAGAATGAAGGAAAAACAGGACAAAA-3′”; reverse “5′-CAAATGGAGAAGATTCCGCC-3′”.
ATF4: forward “5′-CCACTCCAGAGCATTCCTTTAG-3′”; reverse “5′-CTCCTTTACACATGGAGGGATTAG-3′”.
Samples were obtained according to instructions (Human Mucin 2 ELISA Kit Catalog Number: HM10493, Bioswamp). The concentration of mucin2 in LS174T cells was measured as previously described .
Chromatin immunoprecipitation (ChIP) assay
Chromatin immunoprecipitation was performed with a ChIP kit (26157, Pierce). Specific procedures were conducted as described in previous research . LS174T cells after 24 h of mechanical stimulation and control treatment were evaluated. Chromatin was subjected to immunoprecipitation using the following antibodies: H3K9me3 (1:500, 13,969, Cell Signaling Technology). DNA was finally eluted in elution buffer and used for real-time PCR amplification using the same primer sets as MeDIP-qPCR. Primer: TTGGCATTCAGGCTACAGGG; GGCTGGCAGGGGCG-GTG.
The data were analyzed with SPSS 21.0 and GraphPad Prism 8.0 software. All data in the figures are expressed as the mean ± SEM. Differences between groups were evaluated with either one-way analysis of variance (ANOVA) with the Holm-Sidak post hoc test or with a t test using SigmaPlot. The significance level was set at P value < 0.05 (compared to control *P < 0.05, **P < 0.01 and ***P < 0.001).