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AIM: To investigate whether Panax notoginseng saponins (PNS) attenuate arecoline (ANE)-induced human oral submucous fibrosis (OSF) by regulating ferroptosis, and to study its mechanism. METHODS: The OSF model was established by treating human immortalized keratinocyte cell line HaCaT with ANE. There were 6 groups created: blank group, model group(50 mg/L ANE), low-, medium- and high-dose (12. 5, 25 and 50 mg/L)PNS groups, and liproxstatin-1 (ferroptosis inhibitor) group. The morphological changes of the cells were observed under a light microscope. Cell viabilty was detected by CCK-8 assay. Collagen type I (Col-l), solute carrier family 7 member 11(SLC7A11) and glutathione peroxidase 4(GPX4)protein expression levels were detected by Western blot. The morphological changes of mitochondria were observed by transmission electron microscopy. The Fe2+ level was detected by FerroOrange fluorescent probe. The intracellular reactive oxygen species (ROS) content was detected by DCFH-DA fluorescent probe. The content of glutathione (GSH)was determined by colorimetry. RESULTS: Compared with blank group, the majority of the cells in model group displayed long spindle shape, fibrotic appearance, unstable adhesion, and obvious morphological changes. Under electron microscope, mitochondrial double membrane density increased, and cristae decreased significantly, presenting typical ferroptosis. The expression of Col-I increased, GPX4 and SLC7A11 protein expression decreased, Fe2+ and ROS levels increased, and GSH content decreased (all/><0. 05). Compared with model group, most of the cells in low-, medium- and high-dose PNS groups were round and epit he lo id, with decreased mitochondrial membrane density, increased ridge number and normal morphology, decreased Col-I expression and ROS level, and increased GSH content (all P<0. 05). Compared with model group, the protein expression levels of GPX4 and SLC7A11 in medium- and high-dose PNS groups were significantly increased, while the content of Fe2+ was significantly decreased (all P<0. 05). There were no significant differences in cell and mitochondrial morphology and indicator levels between liproxstatin-1 group and high-dose PNS group (all 05). CONCLUSION: Panax notoginseng saponins can alleviate OSF by preventing the ferroptosis of HaCaT cells induced by ANE. [ABSTRACT FROM AUTHOR]