Journal: Neural Regeneration Research

Article Title: Fat mass and obesity–mediated N 6 -methyladenosine modification modulates neuroinflammatory responses after traumatic brain injury

doi: 10.4103/NRR.NRR-D-23-01854

Figure Lengend Snippet: ADAM17 is a downstream target of FTO-mediated m6A modification in microglia. (A) The m 6 A-modified genes were significantly different between control and LPS-treated BV2 cells. (B) The overlap between m6A-modified genes and inflammatory response–related genes was analyzed in control and LPS-treated BV2 cells using Venn diagrams. (C) Volcano plot of the gene enrichment pathway in control and LPS-treated BV2 cells. Compared with the control group, LPS-treated BV2 cells induced an up-regulation of the downstream effector molecules of the ADAM17/TNF-α/NF-κB pathway, including ADAM17, RELA and TNF-α. (D) m6A RNA-sequencing profile of the control and LPS-treated BV2 cells. By comparing m 6 A-sequencing with control BV2 cells, ADAM17, TNF-α and RELA mRNA in LPS-treated BV2 cells mainly concentrated in the coding sequences and 3′-UTR regions. (E, F) Compared with siFTO, siFTO + LPS BV2 cells, the levels of ADAM17, TNF-α, and RELA mRNA in oeFTO, oeFTO + LPS BV2 cells decreased (E) and protein levels also decreased (F). Values are expressed as mean ± SD from at least three independent experiments and the dots represent the value of each experiment. ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Student–Newman–Keuls tests). ADAM17: A disintegrin and metalloproteases 17; FTO: the fat mass and obesity-related protein; LPS: lipopolysaccharides; mRNA: messenger ribonucleic acid; m 6 A: N 6 -methyladenosine; N.S.: not significant; NF-κB: nuclear factor kappa B; RELA: v-rel avian reticuloendotheliosis viral oncogene homolog A; TNF-α: tumor necrosis factor-α; UTR: untranslated region.

Article Snippet: In some experiments, transfected BV2 cells were treated with the ADAM17 inhibitor TAPI-1 (1 μM; MedChemExpress, Monmouth Junction, NJ, USA) for another 48 hours at 37°C.

Techniques: Modification, Control, RNA Sequencing, Sequencing

Journal: Neural Regeneration Research

Article Title: Fat mass and obesity–mediated N 6 -methyladenosine modification modulates neuroinflammatory responses after traumatic brain injury

doi: 10.4103/NRR.NRR-D-23-01854

Figure Lengend Snippet: The epigenetic regulation of ADAM17 in an FTO-m 6 A-dependent mechanism. (A) Detection of ADAM17 precursor (pre-ADAM17) and mature transcripts (mat-ADAM17) by quantitative real-time polymerase chain reaction. The mat-ADAM17 mRNA was significantly enhanced in siFTO BV2 cells compared with oeFTO BV2 cells. (B) Detection of the half-life of ADAM17 in oeFTO and siFTO BV2 cells pretreated with actinomycin D and analyzed at 0, 2, 4, 6 and 8 hours. (C) The oeFTO and siFTO BV2 cells were pretreated with cycloheximide for 90 minutes; western blot analysis showed that ADAM17 protein had a longer half-life in the siFTO BV2 cells. (D) Schematic representation of positions of the m 6 A motifs within ADAM17 mRNA. (E) Schematic representation of the mutated (GGAC to GGTC) 3′-UTR pmirGLO vectors. (F) BV2 cells were transfected with pmirGLO-3′-UTR (wildtype, WT) or pmirGLO-3′-UTR-Mut1/2/3 (mutant, MUT1/2/3) reporter plasmids. The MUT3 construct had markedly decreased luciferase activity compared with WT BV2 cells. Values are expressed as mean ± SD from at least three independent experiments and the dots represent the value of each experiment. ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Student–Newman–Keuls tests). ADAM17: A disintegrin and metalloproteases 17; FTO: the fat mass and obesity-related protein; mRNA: messenger ribonucleic acid; N.S.: not significant; m6A: N6-methyladenosine; UTR: untranslated region.

Article Snippet: In some experiments, transfected BV2 cells were treated with the ADAM17 inhibitor TAPI-1 (1 μM; MedChemExpress, Monmouth Junction, NJ, USA) for another 48 hours at 37°C.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Transfection, Mutagenesis, Construct, Luciferase, Activity Assay

Journal: Neural Regeneration Research

Article Title: Fat mass and obesity–mediated N 6 -methyladenosine modification modulates neuroinflammatory responses after traumatic brain injury

doi: 10.4103/NRR.NRR-D-23-01854

Figure Lengend Snippet: Inhibition of ADAM17 in vitro blocks microglial activation after FTO-m6A modification. (A, B) In situ hybridization results indicated that ADAM17 co-localized with FTO in oeFTO and siFTO BV2 cells treated with or without LPS. (A) Double staining of FISH and IF demonstrated that FTO overexpression combined with LPS stimulus increased the co-localization of FTO protein and ADAM17 mRNA. Scale bars: 20 µm. (B) Line profile of colocalization. (C, D) M1-like (CD86 + /Iba-1 + ) microglia in the siFTO + TAPI group decreased compared with the siFTO group (C). Scale bars: 50 µm. The protein levels of CD86, ADAM17, and iNOS were down-regulated, while the expression of CD206 and Arg-1 increased in the siFTO + TAPI group (D). (E) ELISA results showed that the siFTO + TAPI group had significantly reduced expression of IL-1β, TNF-α, and IL-6 and increased expression of TGF-β1 compared with the siFTO group. Values are expressed as mean ± SD from at least three independent experiments and the dots represent the value of each experiment. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Student–Newman–Keuls tests). ADAM17: A disintegrin and metalloproteases 17; CD: cluster of differentiation; ELISA: enzyme-linked immunosorbent assay; FISH: fluorescence in situ hybridization; FTO: the fat mass and obesity-related protein; Iba-1: ionized calcium-binding adapter molecule 1; IF: immunofluorescence; iNOS: inducible nitric oxide synthase; IL-1β: interleukin-1β; LPS: lipopolysaccharides; N.S.: not significant; TGF-β1: transforming growth factor-β1; TNF-α: tumor necrosis factor-α.

Article Snippet: In some experiments, transfected BV2 cells were treated with the ADAM17 inhibitor TAPI-1 (1 μM; MedChemExpress, Monmouth Junction, NJ, USA) for another 48 hours at 37°C.

Techniques: Inhibition, In Vitro, Activation Assay, Modification, In Situ Hybridization, Double Staining, Over Expression, Expressing, Enzyme-linked Immunosorbent Assay, Fluorescence, Binding Assay, Immunofluorescence

Journal: Neural Regeneration Research

Article Title: Fat mass and obesity–mediated N 6 -methyladenosine modification modulates neuroinflammatory responses after traumatic brain injury

doi: 10.4103/NRR.NRR-D-23-01854

Figure Lengend Snippet: Enhancement of FTO reduces neuronal apoptosis and inhibits neuroinflammation after TBI in vivo. (A, B) The percentage of apoptotic cells determined by Nissl staining was higher in the TBI group than in the sham group. The neuronal apoptosis rate in the TBI + NADP group was significantly lower on day 3 after TBI compared with the TBI group. The arrows indicate apoptotic neurons. Scale bars: 50 µm. (C) Western blot analysis revealed the upregulation of apoptotic factors (caspase-3 and Bax) in the injured cortex on day 3 after TBI. The levels of cleaved caspase-3 and Bax in the TBI + NADP group were decreased compared with the TBI group, and the anti-apoptotic factor Bcl-2 was increased. (D) The M1-like microglial polarization was inhibited in the TBI + NADP group. Representative photomicrographs of M1-like (CD86 + , red, Alexa FluorTM 647/Iba-1 + , green, Alexa FluorTM 488) microglia are shown. Scale bars: 50 µm. (E) The TBI + NADP group showed significantly reduced expression of ADAM17, CD86, and iNOS and increased the expression of CD206 and Arg-1 compared with the TBI group. (F) ELISA results showed that the TBI + NADP group showed significantly reduced expression of TNF-α, IL-1β, IL-6 and IFN-γ compared with the TBI group. Values are expressed as mean ± SD from at least three independent experiments and the dots represent the value of each experiment. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Student–Newman–Keuls tests). ADAM17: A disintegrin and metalloproteases 17; Arg-1: arginase-1; Bcl-2: B-cell lymphoma-2; Bax: BCL2-Associated X; CD: cluster of differentiation; ELISA: enzyme-linked immunosorbent assay; FTO: the fat mass and obesity-related protein; Iba-1: ionized calcium-binding adapter molecule 1; IFN-γ: interferon-γ; IL-1β: interleukin-1β; iNOS: inducible nitric oxide synthase; N.S.: not significant; NADP: nicotinamide adenine dinucleotide phosphate; TBI: traumatic brain injury.

Article Snippet: In some experiments, transfected BV2 cells were treated with the ADAM17 inhibitor TAPI-1 (1 μM; MedChemExpress, Monmouth Junction, NJ, USA) for another 48 hours at 37°C.

Techniques: In Vivo, Staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Neural Regeneration Research

Article Title: Fat mass and obesity–mediated N 6 -methyladenosine modification modulates neuroinflammatory responses after traumatic brain injury

doi: 10.4103/NRR.NRR-D-23-01854

Figure Lengend Snippet: FTO regulates neuroinflammation in vivo by targeting ADAM17 in microglia after TBI. (A) Western blotting demonstrated that NADP intervention inhibited ADAM17, TNF-α, and NF-κB p65. (B) Schematic illustration of the isolation of exosomes in the supernatant of NC and oeADAM17 BV2 cells; exosomes were isolated and characterized through TEM, NTA, and exosomal biomarkers assay. Scale bars: 200 nm. (C) Compared with the TBI + NADP group, the TBI + NADP + exo-oeADAM17 group showed significantly increased expression of ADAM17, CD86, and iNOS and decreased the expression of CD206 and Arg-1. (D) ELISA results showed that the TBI + NADP + exo-oeADAM17 group showed significantly increased expression of TNF-α, IL-1β, IL-6 and IFN-γ. (E) The apoptosis rate of neurons in the TBI + NADP + exo-oeADAM17 group was significantly higher than that in the TBI + NADP group on day 3 after TBI. Representative photomicrographs of the Nissl-stained neurons are shown. The arrows indicate apoptotic neurons. Scale bar: 50 µm. (F) Representative photos of Evans blue dye extravasation. Values are expressed as mean ± SD from at least three independent experiments and the dots represent the value of each experiment. ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Student Newman–Keuls tests). ADAM17: A disintegrin and metalloproteases 17; Arg-1: arginase-1; Bcl-2: B-cell lymphoma-2; Bax: BCL2-Associated X; CD: cluster of differentiation; ELISA: enzyme-linked immunosorbent assay; FTO: the fat mass and obesity-related protein; Iba-1: ionized calcium-binding adapter molecule 1; IFN-γ: interferon-γ; IL-1β: interleukin-1β; iNOS: inducible nitric oxide synthase; NADP: nicotinamide adenine dinucleotide phosphate; NC: negative control; NF-κB: nuclear factor kappa-B; NTA: nanoparticle tracking analysis; N.S.: not significant; TEM: transmission electron microscope; TBI: traumatic brain injury; TNF-α: tumor necrosis factor-α.

Article Snippet: In some experiments, transfected BV2 cells were treated with the ADAM17 inhibitor TAPI-1 (1 μM; MedChemExpress, Monmouth Junction, NJ, USA) for another 48 hours at 37°C.

Techniques: In Vivo, Western Blot, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Binding Assay, Negative Control, Transmission Assay, Microscopy