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AIM: To investigate the anti-fibrotic effect of Panax notoginseng saponins (PNS) in arecoline (ANE)-induced oral submucous fibrosis, and to analyze the effect of PNS on nuclear factor E2-related factor 2(Nrf2)/glutamate-cysteine ligase catalytic subunit (GCLC) signaling pathway. METHODS: CCK-8 assay was used to evaluate the effects of different concentrations of PNS and arecoline on the survival rate of human immortalized keratinocyte cell line HaCaT. The results of CCK-8 were used to select 75 mg/L arecoline, and 25，50 and 100 mg/L PNS as subsequent experimental concentrations. The cells were set as blank control group, model group, and low, medium and high doses (25，50 and 100 mg/L) of PNS groups. The protein and mRNA expressions of collagen type I (COL-I), E-cadherin, Nrf2, GCLC and glutathione reductase (GR) in each group were detected by Western blot and RT-qPCR. Immunofluorescence method was used to detect the entry of Nrf2 into the nucleus. Biochemical kits were used to detect the content of glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH) and malondialdehyde (MDA), and superoxide dismutase (SOD) activity in each group of cells. DCFH-DA fluorescent probe was used to detect the content of intracellular reactive oxygen species (ROS). RESULTS: Compared with the blank control group, the protein and mRNA expression of COL-I in the model group was up-regulated, and the protein and mRNA levels of E-cadherin, Nrf2, GCLC, nuclear Nrf2 and GR were down-regulated. The content of NADPH, MDA and ROS in the cells increased, and the content of GSH and the activity of SOD was significantly reduced. Compared with the model group, the protein and mRNA expression of COL-I was down-regulated, and the protein and mRNA expression of E-cadherin, Nrf2, GCLC, nuclear Nrf2 and GR were up-regulated in PNS 50 and 100 mg/L groups. Compared with the model group，the content of NADPH, MDA and ROS in cells decreased, and the content of GSH and the activity of SOD was significantly enhanced (P<0. 05 or P<0. 01). CONCLUSION:Panax notoginseng saponins have anti-fibrosis effects in HaCaT cells, and their mechanism may be related to the activation of Nrf2/GCLC signaling pathway, thereby resisting oxidative stress and improving oral submucosal fibrosis. [ABSTRACT FROM AUTHOR]

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]

AIM: To investigate the expression of syntaxin binding protein 4 (STXBP4) in oral squamous cell carcinoma (OSCC) and its effects on the malignant biological behavior of OSCC cells. METHODS: (1) The TIMER, GEPIA2, UALCAN, ENCORI, GEO and HPA databases were used to comprehensively study the expression of STXBP4 in head and neck squamous cell carcinoma (HNSCC) tissues at mRNA and protein levels. The mRNA and protein expres⁃ sion levels of STXBP4 in OSCC cell lines were detected by qPCR and Western blot, respectively. (2) The expression of STXBP4 in SCC-9 cells was down-regulated by small interfering RNA (siRNA), and knockdown (si-STXBP4-1, si-STXBP4-2 and si-STXBP4-3) groups and negative control (si-NC) group were established. Overexpression of SCAL-27 cells was achieved by lentivirus transfection, and overexpression (LV-STXBP4) group and negative control (LV-NC) group were established. The viability, colony formation and migration of OSCC cells were examined by CCK-8, plate colony formation, and Transwell assays, respectively. Western blot was used to detect the expression of epithelial-mesen⁃ chymal transition (EMT)-related proteins. (3) By establishing a subcutaneous xenograft tumor model in nude mice, we in⁃ vestigated the effect of STXBP4 on the proliferation of OSCC cells in vivo. RESULTS: (1) Analysis of the TIMER, GE⁃ PIA2, UALCAN, ENCORI and GEO databases revealed that HNSCC tissues had higher STXBP4 mRNA levels than nor⁃ mal tissues. Immunohistochemical staining from HPA database showed that STXBP4 protein in HNSCC tissues was higher than that in normal oral mucosa tissues. In contrast to normal oral mucosa cells, STXBP4 was highly expressed in 5 OSCC cell lines (P<0. 05). (2) The relative expression of STXBP4 in SCC-9 cells transfected with si-STXBP4-1, si-STXBP4-2 and si-STXBP4-3 was significantly lower than that in si-NC group (P<0. 01). Compared with si-NC group, knockdown of STXBP4 resulted in decreased proliferation and migration of SCC-9 cells (P<0. 05). The CAL-27 cells transfected with LV-STXBP4 had considerably higher STXBP4 level than the cells in LV-NC group (P<0. 01), and had markedly better proliferation and migration abilities than those in LV-NC group (P<0. 01). The protein expression of N-cadherin decreased and that of E-cadherin increased after STXBP4 expression was knocked down, while STXBP4 overexpression exerted the opposite effects (P<0. 05). (3)In vivo, overexpression of STXBP4 increased the volume and weight of xenograft tumors in nude mice (P<0. 05). CONCLUSION: High expression of STXBP4 promotes the proliferation and migration of OSCC cells, and its effect on the migration behavior of OSCC cells may be related to EMT. [ABSTRACT FROM AUTHOR]