Periodic red light irradiation treatment leads to increased H3K9ac and metabolism in skin tissues of a senescent mouse model.

a, CCK-8 analysis of keratinocyte activity after red light treatment, n=3. b, Fluorescence images of H3K9ac protein in keratinocytes after treatment with different doses of red light. c, Image of β-galactosidase staining in senescent keratinocyte model after red light irradiation treatment. d-h, ChIP-qPCR experiments were conducted to assess the enrichment of H3K9ac in the promoter regions of Ppargc1a (d), Sod2 (e), Lamin-b1 (f), Cdkn1a (g) and Cdkn2a (h). Following ChIP experiments using the H3K9ac antibody or control IgG, qPCR analysis was performed using primers targeting the promoters of these genes, RPL30 is used as an internal standard for the calibration of the target gene, n=3. i, Fluorescence images of H3K9ac (red) protein in skin tissues of mice of different ages (DAPI, blue), histogram shows the relative quantification of the red signal within the white dashed line in i, n=9. j, Fluorescence images of H3K9ac (red) proteins in skin tissues of one- and two-year old mice after red light irradiation treatment (DAPI, blue). histogram shows the relative quantification of the red signal within the white dashed line in j, n=9. k, The evels of H3K9ac and H3 proteins in the skin of mice in groups 2Y and 2Y+R, n=3. l and m, The levels of P16(l) and LAMB1(m) proteins in the skin of mice in groups 4M, 2Y and 2Y+R. n, The expression heatmap of SASP inflammatory factors in the skin of mice in groups 2Y, and 2Y+R, n=3. o, p, q and r, PET/CT images (o) and 18F-FDG Radiological signals in skin (p), liver (q) and brain (r) of mice in the 4M, 2Y and 2Y+R treatment groups, n=6. s, The conversion process of NAD+ and NADH in keratinocytes. t, Relative levels of NADH in serum of mice in the 4M, 2Y and 2Y+R groups, n=5. u, Relative content of Acetyl-CoA in the skin tissues of mice in groups 4M, 2Y and 2Y+R, n=5.

Red light irradiation promotes keratinocyte metabolic reprogramming.

a, Migration of keratinocytes after 24 h of red light treatment (n = 6). b, Proliferation of keratinocytes: 0–24 h after red light treatment (n = 3). c and d, Uptake of glucose (c, n=3) and fatty acid (d, n=6) in the culture medium by keratinocytes 24 h after red light treatment. e, Mitochondrial membrane potential of keratinocytes after red-light treatment. CCCP, Carbonyl cyanide m-chlorophenylhydrazone (n=6). f, Graded-dose red light treatment affects ATP production in keratinocytes (n = 4). g and h, Relative changes in cellular GSH (g) and NADPH (h) contents 12 h after treatment of keratinocytes with graded doses of red light (n=3). g and h, Fluorescence images of total reactive oxygen species (ROS) levels (i, n=6) and mitochondrial ROS levels (j, n=6) in keratinocytes under different red light doses. k-p, Relative changes in intracellular lactate (k, n=6), pyruvate (l, n=6), triglyceride (m, n=6), fatty acid (n, n=6), NADH (o, n=4), and acetyl-CoA (p, n=4) at 0h and 12h after treatment of keratinocytes with graded doses of red light.

Red light activated the Pi3k-akt signalling pathway and PPAR-α in keratinocytes simultaneously with H3k9ac.

a, Schematic representation of substance metabolism in mitochondria. b, Scatterplot of significantly enriched functions for transcript data from keratinocytes treated with red light (Top 20 KEGG for plotting). c, Differential gene module expression trend line graph. Coloured lines indicate the mean value of the change for this group of genes. d, Volcano plot of the differences in gene expression after red light treatment. e, Expression of keratinocyte metabolism-related genes after red light treatment. f, Enrichment analysis of metabolic pathways after red light treatment (P value > 0.5). g, Principal component analysis (PCA) of keratinocyte proteomics in the red light and dark treatment groups, n=3. h and i, GSEA analysis of gluconeogenesis (h) and PPAR signalling pathway (i) proteins in keratinocytes after red light irradiation. j, Expression levels of p-mTOR, Glut1, Pi3k, and p-AKT proteins in keratinocytes after red light treatment in the presence of AKT and PI3K inhibitors (n = 3). k and l, Immunohistochemical staining of PI3K (k) and Glut1(l) proteins in skin tissues after red light irradiation, n=9. m and n, Immunofluorescence staining of PPAR-α and PPAR-γ proteins in skin tissues after red light irradiation (n = 9). o, Changes in the levels of PI3K, p-mTOR, Raptor, Rictor, p-Akt, PPAR-α, PPAR-γ, p-S6, H3K9ac, and H3 proteins in keratinocytes at 0h, 12h, 18h, and 24h after red light treatment, n=3. p, Fluorescence images of H3K9ac protein at different time points after red light treatment in keratinocytes. q, Schematic diagram of increased glycolipid metabolism driving histone acetylation in cells after red light treatment of keratinocytes. Ctrl: Control; RL: Red light.

Red light drives metabolism through mitochondria and mediates histone-acetylation primarily through lipid metabolism.

a and b, Fluorescence images of H3K9ac (green, a, n=12) protein and levels of PI3K, p-mTOR, p-AKT, H3K9ac and H3 proteins (b, n=3) in keratinocytes under different concentrations of SC-79 treatment conditions (DAPI, blue). c and d, Fluorescence images of H3K9ac (green, c, n=12) protein and levels of PI3K, p-mTOR, p-AKT, H3K9ac and H3 proteins (d, n=3) in keratinocytes under different concentrations of MK2206 treatment conditions (DAPI, blue). e and f, Effects of different concentrations of SC-79 (e) and MK2206 (f) on Acetyl-CoA content in keratinocytes, n=4. g and h, Fluorescence images of H3K9ac (green, g, n=12) protein and levels of PI3K, p-mTOR, p-AKT, PPAR-α, PPAR-γ, H3K9ac and H3 proteins (h, n=3) in keratinocytes under different concentrations of WY14643 treatment conditions (DAPI, blue). i and j, Fluorescence images of H3K9ac (green, i, n=12) protein and levels of PI3K, p-mTOR, p-AKT, PPAR-α, PPAR-γ, H3K9ac and H3 proteins (j, n=3) in keratinocytes under different concentrations of GW6471 treatment conditions (DAPI, blue). k and l, Effects of different concentrations of WY14643 (k) and GW6471 (l) on Acetyl-CoA content in keratinocytes, n=4. m and n, Effect of red light treatment on the levels of PI3K, p-mTOR, Raptor, Rictor, p-AKT, PPAR-α, PPAR-γ, p-S6, H3K9ac and H3 proteins in keratinocytes in the presence of 5uM Nicur (m) and TSA (n), n=3. o and p, Effect of red light treatment on the levels of Pi3k, Raptor, Rictor, PPAR-α, PPAR-γ, p-S6, H3K9ac and H3 proteins in keratinocytes in the presence of 2uM Mk2206 (o) and 20uM GW6471 (p), n=3. q and r, Effect of red light treatment on the levels of PI3K, p-mTOR, p-AKT, PPAR-α, PPAR-γ, H3K9ac and H3 proteins in keratinocytes in the presence of 5uM Antimycin (q) and Elamipretide (r), n=3. s, t, u, and v, Relative contents of lactate (s, n=6), triglycerides (t, n=6), fatty acids (u, n=6) and Acetyl-CoA (v, n=4) in keratinocytes after irradiation of 5uM Elamipretide-treated keratinocytes with graded doses of red light.

Red light-induced downregulation of SIRT4 is primarily responsible for the activation of lipid metabolism, alleviation of inflammation, and resistance to aging in keratinocytes.

a, Heatmap showing the top 50 significantly differentially expressed proteins in red light-irradiated keratinocytes. b, Volcano plot of protein content changes in keratinocytes after red light treatment. c, Schematic representation of the role of SIRT4 in mitochondria and cell. d and e, Fluorescence images of Sirt4 (d) and SIRT1 (e) protein and nucleus in keratinocytes after red light treatment, n=9. f, Intracellular protein levels of SIRT1 and SIRT4 after dose-gradient red light treatment of keratinocytes, n=3. g, Protein levels of p-Nf-κB, PPAR-α and Sirt4 in keratinocytes after red light treatment, n=3. h, H&E staining images and fluorescence images of SIRT4 (green) and SIRT1 (red) proteins of mouse skin tissues in 4M, 2Y and 2Y+R treatment groups (DAPI, blue), n=3. i, The levels of SIRT4 and SIRT1 proteins in the skin of aged mice after cyclic red light treatment, n=3. j and k, Fluorescence images of p-Nf-κB (red, j) in mice skin tissues after red light treatment of 1Y and 2Y senescent mice (DAPI, blue) and the levels of p-Nf-κB protein (k) in skin tissues of 2Y senescent mice after red light treatment, n=3. l and m, Relative expression of SIRT4 mRNA (l, n=3) and fluorescence images of SIRT4 protein (green) (m, n=6) in keratinocytes after silencing of the SIRT4 gene in keratinocytes using siRNA. n and o, Changes in cellular activity (n, n=3), relative content of intracellular acetyl-CoA (o, n=6) in keratinocytes after silencing the SIRT4 gene in keratinocytes using siRNA. p and q, Fluorescence images of PPAR-α (p) and P16 (q) proteins in keratinocytes after silencing the Sirt4 gene in keratinocytes using siRNA, n=6. r, Protein levels of p-Nf-κB, LAMB1, PPAR-α, Sirt4, p21, p16, H3K9ac, and H3 in keratinocytes after silencing the Sirt4 gene in keratinocytes using siRNA, n=3. s, Fluorescence images of H3K9ac proteins in keratinocytes after silencing the SIRT4 gene in keratinocytes using siRNA, n=6. t, Relative expression of SASP inflammatory factors in keratinocytes following silencing of the SIRT4 gene in keratinocytes using siRNA, n=3. u, Images of β-galactosidase staining in keratinocytes after silencing the SIRT4 gene in keratinocytes cells using siRNA, n=6. N-Con: Negative Control; RL: Red light.

Red light activates lipid metabolism and increase H3K9ac levels in keratinocytes via SIRT4 downregulation-mediated MCD acetylation.

a, Heatmap showing the top 50 significantly differentially expressed acetylated proteins in red light-irradiated keratinocytes. b, Volcano plot of changes in the content of acetylation-modified proteins in keratinocytes after red light treatment. c, Acetylation modification proteomics top10 protein functional enrichment analysis (Protein functional annotation by InterPro). d, Proteomic top10 enriched pathway chordal map of red light irradiated keratinocytes (KEGG analysis). e, Top12 molecular process enriched by combined analysis of keratinocyte proteomics and acetylation modification proteomics after red light treatment (KEGG analysis). f, Top 10 pathways chordal map enriched by combined analysis of keratinocyte proteomics and acetylation modification proteomics after red light treatment. g, Schematic representation of the role of Sirt4 in mitochondria FAO and TCA cycle progress. h, i, j, k and l, Effects of red light on cell viability (CCK-8, h, n=12), fatty acid uptake (i, n=6), ATP production (j, n=4), NADH levels (k, n=4), and Acetyl-CoA content (l, n=4) in keratinocytes treated with 5 μM CBM-301940. m and n, Representative immunofluorescence staining of H3K9ac (m, n=9)and Western blot analysis of CPT1A, β-Actin, H3K9ac and H3 in keratinocytes treated with red light and 5 μM CBM-301940 (n, n=3).

Inflammation and senescence signaling in aging model mice alleviated after cyclic treatment with red light.

a, b, c and d, Fluorescence images of Sirt4 (red) proteins in the heart (a), liver (b), lung (c)and kidney (d) of mice in groups 4M, 2Y and 2Y+R (DAPI, blue), n=6. e, f, g and h, The levels of H3K9ac proteins in the heart (e), liver (f), lung (g)and kidney (h) of mice in groups 4M, 2Y and 2Y+R, n=3. i, j, k and l, Fluorescence images of H3K9ac (Red), p-Nf-κB (green) and p16 (yellow) proteins in the heart (i), liver (j), lung (k)and kidney (l) of mice in groups 4M, 2Y and 2Y+R (DAPI, blue), n=6. m, n, o and p, The levels of p-Nf-κB, LAMB1, p21 and p16 proteins in the heart (m), liver (n), lung (o)and kidney (p) of mice in groups 4M, 2Y and 2Y+R, n=3. q, r, s and t, The expression heatmap of SASP inflammatory factors in the heart (q), liver (r), lung (s)and kidney (t) of mice in groups 4M, 2Y, and 2Y+R, n=3.