〔Abstract〕 To investigate the mechanism of Anemoside B4 (AB4) on glutamine metabolism in oral li- chen planus (OLP) epithelial cells via the nitric oxide synthase 3 (NOS3)-dihydrofolate reductase (DHFR) axis . Methods Bioinformatics analysis was performed to identify the intersection of molecular targets of OLP , AB4 , and genes related to glutamine metabolism . A lipopolysaccharide ( LPS)-induced HOK-16B model of OLP was estab- lished . HOK-16B were divided into Ctrl group , OLP group , AB4 group , OLP + oe-NOS3 group , OLP + sh-NOS3 group , OLP + sh-NOS3 + oe-DHFR group , and OLP + sh-NOS3 + AB4 group . Cell proliferation was detected by cell counting kit-8 (CCK-8) ; cell apoptosis was detected by TdT-mediated dUTP Nick-End Labeling ( TUNEL) ; inflammatory factors iInterleukin (IL)-1β, tumor necrosis factors-α ( TNF-α) concentrations in cell supernatants were measured using enzyme-linked immunosorbent assay (ELISA) kits; glutamine uptake and glutamate produc- tion were determined using kits; and the protein expression of alanine-serine-cysteine transporter2 ( ASCT2) and glutamine synthase (GLS) was assessed by Western blotting. Results Bioinformatics analysis of molecular targets of OLP , AB4 , and genes related to glutamine metabolism revealed three intersection targets : NFE2L2 , NOS1 , and NOS3 . Compared with the Ctrl group , the OLP group exhibited decreased HOK-16B cell viability (P < 0. 001) , increased apoptosis rate (P < 0. 01) , upregulated concentrations of IL-1βand TNF-α(P < 0. 001) , elevated glu- tamine uptake and glutamate production (P < 0. 01) , and enhanced expression of ASCPT2 and GLS proteins (P < 0. 001) . Compared with the OLP group , the AB4 group showed improved cell viability (P < 0. 05) , reduced apop- tosis rate and release of IL-1βand TNF-α(P < 0. 05) , decreased glutamine uptake and glutamate production (P < 0. 05) , and downregulated expression of ASCPT2 and GLS proteins ( P < 0. 001) . Compared with the OLP group , the OLP + oe-NOS3 group had increased HOK-16B cell viability (P < 0. 01) , reduced apoptosis rate (P < 0. 05) , decreased concentrations of IL-1βand TNF-α(P < 0. 05) , lowered glutamine uptake and glutamate pro- duction (P < 0. 05) , and weakened expression of ASCPT2 and GLS proteins (P < 0. 01) ; whereas the OLP + sh- NOS3 group had decreased HOK-16B cell viability ( P < 0. 05) , increased apoptosis rate ( P < 0. 05) , elevated concentrations of IL-1βand TNF-α ( P < 0. 01 ) , increased glutamine uptake and glutamate production ( P < 0. 05) , and enhanced expression of ASCPT2 and GLS proteins (P < 0. 001) . Compared with the OLP + sh-NOS3 group , both the OLP + sh-NOS3 + oe-DHFR group and the OLP + sh-NOS3 + AB4 group showed increased HOK- 16B cell viability (P < 0. 001) , reduced apoptosis rate (P < 0. 05) , decreased concentrations of IL-1βand TNF-α (P < 0. 01) , lowered glutamine uptake and glutamate production ( P < 0. 05) , and weakened expression of AS- CPT2 and GLS proteins ( P < 0. 05) . Conclusion AB4 inhibits the progression of OLP by mediating glutamine metabolism via the regulation of the NOS3-DHFR axis .