Fig 2: FMN supplementation suppresses LPS-induced inflammation in BV2 cells and primary microglia. A) Schematic model of the riboflavin biosynthesis metabolic pathway. RFK, riboflavin kinase; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide. B-D) The supernatant levels of IL-1ß, IL-6, and TNF-a in LPS-treated BV2 cell cultures with different doses of FMN or FAD n = 3 per group. E–G) Representative blots and quantification of RFK, TNFR1, p-NF-?B, NF-?B, p-I?Ba, and I?Ba expression levels in LPS-treated BV2 cells with 200 µM FMN. n = 3 per group. H,I) Immunofluorescence staining and quantification of TNFR1 with the membrane dye DIL in LPS-treated BV2 cells with FMN n = 6–8. Scale bars, 20 µm. Magnified images are shown in the right columns. Scale bars, 5 µm. (J) The mRNA expression levels of Il-1b, Il-6, and Tnfa in LPS-treated primary microglia with 200 µM FMN n = 3 per group. K-M) Representative blots and quantification of RFK, TNFR1, p-NF-?B, NF-?B, p-I?Ba and I?Ba expression levels in LPS-treated primary microglia with 200 µM FMN n = 3 per group. N,O) Immunofluorescence staining and quantification of RFK with Iba1 in LPS-treated primary microglia with 200 µM FMN n = 7–9. Scale bars, 40 µm. Magnified images are shown in the right columns. Scale bars, 13 µm. Results are expressed as mean ± SEM. ** p < 0.01, *p < 0.05 versus Ctrl; ## p < 0.01, # p < 0.05 versus LPS. Statistical significance was determined using one-way ANOVA and Tukey's tests for post hoc comparisons.

Fig 3: RFK is mainly located in microglia and its expression is increased in the inflammation-based cognitive impairment model. A) Immunofluorescence staining of RFK with Iba1 in the CA1 of the hippocampus and cortex. Scale bars, 50 µm (top), 10 µm (middle), and 5 µm (bottom). B) Immunofluorescence staining of RFK with GFP in the hippocampi of Cx3cr1-GFP mice. Scale bars, 8 µm. C and D) Representative blots and quantification showing RFK expression levels in Control and LPS-treated BV2 cells n = 3 per group. E,F) Representative blots and quantification showing RFK expression levels in the cortex and hippocampus of Control mice and the LPS mouse model n = 4 per group. G,H) Representative blots and quantification showing RFK expression levels in the cortex and hippocampus of WT mice and the 5xFAD mouse model n = 4 per group. I,J) Representative blots and quantification showing RFK expression levels in BV2 cells treated with Control siRNA and three targeting RFK siRNAs n = 3 per group. (K) Effects of Rfk siRNA on the mRNA expression levels of Il-1b, Il-6, Tnfa, Ifng, and Tgfb in BV2 cells n = 3 per group. Results are expressed as mean ± SEM. ** p < 0.01, *p < 0.05 versus Ctrl or Ctrl siRNA or WT. Statistical significance was determined using Student's t-test.

Fig 4: RNA-seq analysis of MNPs@FMN treatment in the LPS-induced mouse model. A,B) Representative blots and quantification showing KMT2B and RFK expression levels in the hippocampi of LPS mice treated with NPs@FMN, MNPs, or MNPs@FMN, n = 4 per group. C) Schematic illustration of RNA-seq. D) Volcano plot showing the DEGs between the LPS and WT groups. E) KEGG pathways enriched by downregulated DEGs in the LPS compared with WT group. F) Downregulated DEGs in the LPS groups that are enriched in the “Alzheimer's disease” KEGG pathway. G) Venn diagram showing 38 DEGs that were downregulated by LPS and upregulated by MNPs@FMN. H) KEGG pathways are enriched by these 38 DEGs. I,J) KEGG and GO pathways enriched by upregulated DEGs in LPS + MNPs@FMN compared with LPS. K) Hierarchical clustering of the 38 LPS + MNPs@FMN reversed DEGs. L) The mRNA expression levels of Uqcrb, Cox8b, and Calml4 in the hippocampi of the WT, LPS, and LPS + MNPs@FMN groups. n = 3 per group. M–O) The mRNA expression levels of Uqcrb, Cox8b, and Calml4 upon treatment with different concentrations of FMN in LPS-treated BV2 cells n = 3 per group. P) The mRNA expression levels of Uqcrb, Cox8b, and Calml4 upon FMN and RFK plasmid treatment in LPS-treated BV2 cells. n = 3 per group. Results are expressed as mean ± SEM. ** p < 0.01, *p < 0.05 versus WT or Control; ## p < 0.01, # p < 0.05 versus LPS-MNPs@FMN or LPS. Statistical significance was determined using one-way ANOVA and Tukey's tests for post hoc comparisons.

Fig 5: FMN regulates RFK expression via KMT2B. Wild-type mice were intraperitoneally injected with 40 mg/kg FMN for 5 weeks, and the hippocampal samples were subjected for RNA-seq. A) Volcano plot showing the DEGs between FMN and Ctrl mice. B,C) Representative KEGG and GO pathways enriched by downregulated DEGs between FMN and Ctrl mice. D) DEGs enriched in the “Cytokine-mediated signaling pathway”, “Cell activation involved in immune response”, and “Positive regulation of cytokine production”. E,F) Representative blots and quantification showing KMT2B expression levels in Control and LPS-treated primary microglia n = 3 per group. G,H) Representative blots and quantification showing KMT2B expression levels in the hippocampus of Control mice and the LPS mouse model n = 4 per group. (I) Kmt2b mRNA expression levels in the hippocampus of WT mice and the 5xFAD mouse model. n = 3 per group. J,K) Representative blots and quantification showing KMT2B expression levels in the hippocampus of WT mice and the 5xFAD mouse model n = 4 per group. L,M) Representative blots and quantification showing KMT2B and RFK expression levels in LPS-treated primary microglia with Control siRNA or Kmt2b siRNA n = 3 per group. N–P) Representative blots and quantification of KMT2B, RFK, TNFR1, p-NF-?B, and NF-?B expression levels in LPS-treated primary microglia with FMN and KMT2B recombinant protein n = 3 per group. Q) The mRNA expression levels of Il-1b, Il-6, Tnfa, and Ifng in LPS-treated primary microglia with FMN and KMT2B recombinant protein n = 3 per group. Results are expressed as mean ± SEM. ** p < 0.01, *p < 0.05 versus Ctrl or Ctrl siRNA; ## p < 0.01, # p < 0.05 versus LPS or LPS + Ctrl siRNA; && p < 0.01, & p < 0.05 versus LPS + FMN. Statistical significance was determined using Student's t-test (for E-K) and one-way ANOVA and Tukey's tests for post hoc comparisons (for L-Q).