ABULIKEMU Wulayin 1,2 , MAIMAITI Yisireyili 1,3 , WANG Yongkang 1 , YU Wei 1 , WU Zhaoyang 2 , KELIMU ABUDUREYIMU 1,3
  • 1. Graduate School of Xinjiang Medical University, Urumqi 830054, P. R. China;
  • 2. Department of Emergency Trauma Surgery, People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi 830001, P. R. China;
  • 3. Department of Minimally Invasive, Hernia and Abdominal Wall Surgery, People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi 830001, P. R. China;
KELIMU ABUDUREYIMU, Email: klm6075@163.com
gastroesophageal reflux disease; MCC950; human esophageal epithelial cell; NOD like receptor protein 3; acid stimulation; pyroptosis
10.7507/1007-9424.202408101
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Objective  To investigate the inhibitory effects and related mechanisms of NOD like receptor protein 3 (NLRP-3) inflammasome inhibitor MCC950 on oxidative stress, inflammation, and pyroptosis in human esophageal epithelial cells (HEECs). Methods HEECs cells were passaged and divided into blank control group, acid stimulation group (stimulated 3 times a day with pH 4 acidic medium for 15 minutes each time, cultured for 48 hours), bile salt stimulation group (stimulated 3 times a day with 400 μmol/L bile salt mixture for 15 minutes each time, cultured for 48 hours), lipopolysaccharide (LPS) group (stimulated with 10 μL of 100 ng/mL LPS for 48 hours), MCC950 group (stimulated HEECs cells with 10 μL of 7.5 ng/mL MCC950 for 4 hours, then stimulated with acid, bile hydrochloric acid, and LPS for 48 hours), and N-acetyl-L-cysteine (NAC) group (stimulated HEECs cells with 1 mmol/L NAC for 4 hours, then stimulated with acid, bile hydrochloric acid, and LPS and incubated for 48 hours). Three culture dishes were used in each group to detect the mRNA and protein expression levels of oxidative protein/antioxidant protein [Nox-4 (NADPH oxidase 4), nuclearfactor erythroidderived 2-like 2 (Nrf-2), heme oxygenase-1 (HO-1)], NLRP-3 signaling pathway [NLRP3/caspase-1/intereukin-1β (IL-1β)/intereukin-18 (IL-18)], and cell apoptosis pathway [caspase-4/caspase-5/GSDMD] using real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting experiments. Cell apoptosis were observed through Hoechst 33342 staining. Results MCC950 intervention (average optical density: 0.023) and NAC intervention (average optical density: 0.031) effectively inhibited HEECs apoptosis induced by acid (average optical density: 0.042), bile salt (average optical density: 0.047), and LPS (average optical density: 0.054). The results of RT-PCR and Western blotting experiments showed that MCC950 intervention and NAC intervention significantly inhibited the high expression of Nox-4 mRNA (MCC950:1.68±0.18, NAC: 1.62±0.17) and protein in HEECs cells induced by acid (1.00±0.05), bile salt (3.07±0.25), and LPS (3.52±0.37); And significantly upregulated the mRNA and protein expression levels of antioxidant proteins Nrf-2 (MCC950: 0.72±0.12, NAC: 0.57±0.12) and HO-1 (MCC950: 0.74±0.12, NAC: 0.57±0.12). MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA and protein expression levels of acid stimulated, bile salt stimulated, and LPS induced HEECs cell NLRP-3 (MCC950 intervention: 1.58±0.06, NAC intervention: 1.47±0.09), ASC (MCC950 intervention: 1.56±0.09, NAC intervention: 1.93±0.17), caspase-1 (MCC950 intervention: 1.64± 0.13, NAC intervention: 1.96±0.20), IL-1β (MCC950 intervention: 1.66±0.18, NAC intervention: 1.82±0.20), IL-18 (MCC950 intervention: 1.58±0.13, NAC intervention: 1.84±0.16) and other indicators. MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA and protein expression levels of apoptosis pathway markers such as caspase-4 (MCC950 intervention:1.51±0.03, NAC intervention: 1.61±0.12), caspase-5(MCC950 intervention: 1.38±0.13, NAC intervention: 1.64±0.21), and GSDMD (MCC950 intervention: 1.41±0.04, NAC intervention: 1.54±0.10) induced by acid stimulation, bile salt stimulation, and LPS in HEECs cells. Conclusion Acid, bile salts, and LPS can all induce the overexpression of oxidative stress markers in HEECs, reduce the expression of antioxidant proteins, and activate the NLRP3 inflammasome signaling pathway and cell pyroptosis pathway, promoting cellular inflammatory damage, but MCC950 has a protective effect.