Cell culture
Ishikawa cell line was cultured in our lab of Shanghai General Hospital affiliated to Shanghai Jiao Tong University since Oct, 2017, catalogue number: GCC-UT0004RT/CS. HEC1A cell line was purchased from Cell Bank of the Chinese Academy of Sciences in January, 2016, catalogue number: TCHu149. AN3CA cell line was purchased from ATCC in Mar, 2016, catalogue number: ATCC®HTB-111. All the above cell lines have recently been tested for no mycoplasma contamination, and were authenticated using Short Tandem Repeat (STR) analysis as described in 2012 in ANSI Standard (ASN-0002) by the ATCC Standards Development Organization (SDO) and in Capes-Davis’s reference [
31], according to the GENECHEM performs STR Profiling following ISO 9001:2008 and ISO/IEC 17025:2005 quality standards by GeneMapper Software 5. All the cell lines have ethics approval for their use by the Ethical Committee on Human Research of Shanghai General Hospital affiliated to Shanghai Jiao Tong University. Cells were cultured in DMEM for 24 h or 48 h: F12, containing 10% FBS, 100 U/mL of penicillin G, and 100 μg/mL of streptomycin at 37 °C in a humidified atmosphere of 5% CO
2.
Secretory phase endometrium specimens were taken from 3 cases of patients who underwent curettage due to abnormal uterine bleeding in Shanghai General hospital affiliated to Shanghai Jiao Tong University in October 2018. For primary human endometrial glandular cells culture, 3 to 5 g endometrial tissue was scraped and placed in DMEM: F12, double antibiotics (penicillin 100 U/ml + streptomycin 100 U/m1) and metronidazole in a sterile collection tube. Then tissue was washed 3 times with D-Hank’s solution, double antibiotics, 100 U/ml metronidazole, and soaked for 5 min before cell culture solution was added to remove red blood cells under super clean operation; Next, the tissue was cut into 2~3 mm3 pieces, digested with 0.1% trypsin-EDTA, for 30 min at 37 °C, shaken every 10 min; Filter solution and 400 mesh (38 pupils) was added to remove mucus, undigested tissue, epithelial and interstitial cells. Centrifuged at 1000 rpm for 10 min, discarded the supernatant. Finally, the endometrial gland cells were resuspended and cultured into DMEM: F12 containing 10% FBS, placed at 37 °C incubator with a relative volume fraction of 5% CO2 for use.
Both endometrial cancer and primary human endometrial glandular cell lines were cultured with fresh H-CM for 24 h or 48 h to ensure that the cells are in good condition. About 20 min before each the follow-up functional experiment, we changed fresh H-CM again to ensure the duration of H2 in the medium. Hydrogen concentration detector was used for H2 measurement in the medium at the beginning of each functional experiment.
In vivo tumorigenesis
The 4 weeks age female SPF grade BALB/c-nude mice were purchased from Shanghai Ling Chang BioTech Co., Ltd. affiliated to Shanghai Slake Laboratory Animal Co., Ltd. from Mar, 2019, License number: SCXK (shanghai) 2018–0003 and used as a human tumor xenograft model; The nude mice are homozygous mutations, hairless and thymic defects which belong to T-lymphocyte dysfunction animals. The protocols were approved by the Ethical Committee on Human Research of Shanghai General Hospital affiliated to Shanghai Jiao Tong University, China, reference number: 2019SQ054. Our research was in compliance with the Helsinki Declaration and the institutional guidelines for care and use of animals. BALB/c mice weighing 18–25 g were implanted with 1 × 107 AN3CA-LUC cells at right shoulder, housing under barrier environment, with each mouse in an independent ventilation cage, at 22–26 °C and humidity 45–65%, on a 12/12-h light/dark cycle (lights on at 08:00 h). Mat and feed were changed every 2–3 days. The mice were divided into two groups: 6 mice in hydrogen-rich water group with the concentration of 1.0 ppm (HRW:H1, H2, H3, H4, H5, H6), and 3 mice in purified normal control group (NC:P1, P2, P3) randomly when the tumors grew 3 to 4 mm in diameter and were visible. The mice were separately treated by gavage with either hydrogen-rich water or pure water control (20 mL/kg/d). We changed the hydrogen-rich water every 2 h each day, and the treatments were maintained for 24 days. Mouse weight and tumor volume were measured and recorded every 3–4 days. Photographs were taken on days 12, 13, and 14 of the experiment with a living imaging system.
We anesthetized and sacrificed the animals with an overdose of 2% sodium pentobarbital (0.5 mL), and then used cervical dislocation to confirm death. We took the endometrial cancer tissues from mice from different groups and performed immunohistochemistry to determine NLRP3, caspase-1, GSDMD, and IL-1β protein expression as described previously. All samples were histologically diagnosed as endometrial cancer.
Materials
DMEM: F12 (1:1, Gibco, USA),
FBS: fetal bovine serum (Gibco, Gaithersburg, MD, USA),
streptomycin (Life Technologies, Inc., Rockville, MD),
Triton-X100(Weiao, WF0193, Shanghai, China),
anti-rabbit NLRP3 (ab210491, Abcam, USA,1:200),
anti-human caspase-1 (2225, CST, USA,1:200),
anti-human GSDMD (PA5–60727, ThermoFisher, USA, 1:100),
ECL chemiluminescence (Millipore).
ImageJ software (National Institutes of Health, Bethesda, MD, USA).
anti-human IL-1β (12,242, CST, USA,1:200).
PVDF membrane (Millipore, Billerica, MA, USA).
anti-mouse ASC (sc-514,414, SANTA CRUZ, USA, 1:1000),
BSA (Roche, Mannheim, Germany),
Tris-buffered saline (TBST),
secondary antibody (DAKO, K5007, Proteintech, Chicago, USA, 1:5000 dilution),
dichlorofluorescin diacetate (DCFH-DA, Beyotime, CA1410, Jiangsu, China),
MitoSOX™ reagent stock solution (iYEASEN, 40778ES50, Shanghai, China),
MTT (3–4,5-dimethylthiazol2-yl-2,5- diphenyltetrazolium bromide, Genview, USA).
fluorescence microscope (IX71, Olympus, Japan).
TUNEL Apoptosis Assay Kit (Beyotime, C1091, Jiangsu, China),
LDH Cytotoxicity Assay Kit (Beyotime, C0016, Jiangsu, China),
IL-1β ELISA kit (CSB-E08053h, Cusabio, USA),
GV493 vectors (Shanghai GeneChem Co., Ltd.),
GSDMD-specific shRNA (Shanghai GeneChem Co., Ltd.),
polybrene (Genomeditech Co., Ltd., Shanghai, China),
puromycin (Clontech Laboratories, Inc., Mountainview, CA, USA),
TRIzol (Thermo Fisher Scientific, Inc.) method,
A reverse transcription kit (Promega Corporation, Madision, WI, USA),
SYBR-Green (DRR041B; Takara Biotechnology Co., Ltd., Dalian, China),
fluorescence microscopy (model no. IX71; Olympus Corporation, Tokyo, Japan),
a living imaging system (Perkin Elmer, Lumina LT).