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 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 with 1 mmol/L NAC for 4 hours, then stimulated with acid, bile hydrochloric acid, and LPS 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 [NLRP-3/caspase-1/intereukin (IL)-1β/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 Hoechst33342 staining. Results MCC950 intervention (0.023) and NAC intervention (0.031) effectively inhibited HEECs apoptosis induced by acid (0.042), bile salt (0.047), and LPS (0.054). The results of RT-PCR experiments showed that MCC950 intervention and NAC intervention significantly inhibited the high expression of Nox-4 mRNA (MCC950: 1.68; NAC: 1.62) in HEECs cells induced by acid (2.40), bile salt (3.07), and LPS (3.52), and significantly upregulated the mRNA expression levels of antioxidant proteins Nrf-2 (MCC950: 0.72; NAC: 0.57) and HO-1 (MCC950: 0.74; NAC: 0.57). MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA expression levels of NLRP-3 (MCC950: 1.58; NAC: 1.47), ASC (MCC950: 1.56; NAC: 1.93), caspase-1 (MCC950: 1.64; NAC: 1.96), IL-1β (MCC950: 1.66; NAC: 1.82), IL-18 (MCC950: 1.58; NAC: 1.84) in HEECs cells induced by acid stimulated, bile salt stimulated, and LPS. MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA expression levels of apoptosis pathway markers such as caspase-4 (MCC950: 1.51; NAC: 1.61), caspase-5 (MCC950: 1.38; NAC: 1.64), and GSDMD (MCC950: 1.41; NAC: 1.54) induced by acid stimulation, bile salt stimulation, and LPS in HEECs cells. The electrophoresis results were similar with RT-PCR. Conclusion Acid, bile salt, and LPS can all induce the overexpression of oxidative stress markers in HEECs, reduce the expression of antioxidant proteins, and activate the NLRP-3 inflammasome signaling pathway and cell pyroptosis pathway, promoting cellular inflammatory damage, but MCC950 has a protective effect.

Citation： ABULIKEMU Wulayin, MAIMAITI Yisireyili, WANG Yongkang, WU Zhaoyang, KELIMU Abudureyimu. Study on the inhibiting mechanism of MCC950 on activation of NLRP-3 inflammasome and pyroptosis in HEECs cells. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2025, 32(1): 93-99. doi: 10.7507/1007-9424.202408101 Copy

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