Fig 1: Resolvin D1 neither enhances nor perturbs basal in vitro myogenesis but suppresses myokine production and protects muscle cells against the deleterious effects of chronic inflammation.(A) Murine C2C12 myoblasts were treated with RvD1 (100 nM) at onset of myogenic differentiation. At 3 days postdifferentiation, myotubes were fixed in 4% paraformaldehyde (PFA) and stained for sarcomeric myosin. Cell nuclei were counterstained with DAPI. Quantitative analysis was performed on 6 nonconsecutive fields of view per well to determine overall cell density (DAPI+ nuclei/mm2), the extent of myogenic differentiation (% DAPI+ nuclei within myosin+ cells), and mean myotube (multinucleated cell) diameter. (B) Confluent C2C12 myoblasts were induced to differentiate in the presence of RvD1 (0.1–1 µM), or NSAIDs, including NS-398 (50 µM), ibuprofen (500 µM), and indomethacin (200 µM). (C) C2C12 myotubes at day 3 postdifferentiation were pretreated with RvD1 (100 nM) for 30 minutes before stimulation with lipopolysaccharide (LPS, 100 ng/mL) for 3 hours in the continued presence of RvD1. mRNA expression of cytokines, including IL-6, MCP-1, and TNF-a, was determined by RT-PCR. (D) C2C12 myotubes at day 3 postdifferentiation were pretreated with RvD1 (100 nM) for 30 minutes and then stimulated with LPS (100 ng/mL) for 24 hours in the continued presence of RvD1. Conditioned culture medium was collected from the cells and analyzed for concentrations of the cytokines IL-6, MCP-1, and TNF-a by ELISA. (E) Confluent C2C12 myoblasts were induced to differentiate in the presence of exogenous TNF-a (20 ng/mL), with or without RvD1 (100 nM) cotreatment. At day 3 postdifferentiation, myotubes were fixed, stained, and quantified as described in A. Scale bars: 400 µm. Bars show the mean ± SEM of 3–8 replicates per group with dot representing data for a single independent culture well. P values were determined by 2-tailed unpaired t tests (A) or 1-way ANOVA followed by pairwise Holm-Šidák post hoc tests (B–E).
Fig 2: Adipose tissue inflammation in Ts65Dn male mice fed a high-fat diet. A-B) Representative H&E stained histological sections of visceral (gonadal) white adipose tissue (gWAT) of male (A) and female (B) Ts65Dn and euploid mice. The quantifications of adipocyte cell size (CSA, cross sectional area) in male (Euploid, n = 14; Ts65Dn, n = 7) and female (Euploid, n = 10; Ts65Dn, n = 9) mice are indicated by bar graphs. C-D) Representative H&E stained histological sections of subcutaneous (inguinal) white adipose tissue (gWAT) of male (C) and female (D) Ts65Dn and euploid mice. The quantification of adipocyte CSA in male (Euploid, n = 14; Ts65Dn, n = 8) and female (Euploid, n = 9; Ts65Dn, n = 9) mice are indicated by bar graphs. E) Serum concentrations of IL-1ß, IL-6, TNF-a, and macrophage chemotactic protein 1 (MCP-1) in male (Euploid, n = 22; Ts65Dn, n = 9) and female (Euploid, n = 13; Ts65Dn, n = 9) mice. F) Volcano plot of the iWAT RNA-seq data. The dotted y-axis line shows the adjusted p-value cut-off for significance (adj. p-value = 0.05). The two dotted x-axis lines represent a Log2(FC) of -0.5 or 0.5. A total of 572 out of 20,680 genes (2.51% of the iWAT transcriptome) are differentially expressed in male Ts65Dn mice relative to euploid controls, with 430 genes up-regulated and 142 genes down-regulated. PCG, protein coding genes; NPCG, non-protein coding genes. The adjacent table shows the top 20 up- and down-regulated genes. G) The top 20 most significant Gene Ontology (GO) and KEGG categories based on differentially expressed genes (DEGs) seen in the iWAT of Ts65Dn. The stalk color represents the category the title belongs to (KEGG pathway, MF = molecular function, BP = biological process, CC = cell component). The size of the circle represents how many genes (or counts) are altered in the group. The color of the circle represents the direction of change, and the magnitude of the gene ratio. A high gene ratio means more genes in that gene family are affected in this group. H) Heat maps of genes associated with inflammation, fibrosis, and oxidative stress. The size of the box indicates statistical significance in trisomic gene expression between male Ts65Dn mice relative to euploid controls. Full-size box denotes significantly different expression (Adjusted p-value =0.05), medium size box denotes significantly different expression (non-Adjusted p-value =0.05), and small box size denotes non-significant expression between genotypes (P value > 0.05). The color gradient indicates Log2(FC) of each gene in Ts65Dn relative to euploid controls, with up-regulated genes in the red and down-regulated genes in the blue color spectrum. Tissue sample size for iWAT RNA sequencing (Euploid, n = 6; Ts65Dn, n = 6).
Fig 3: Proposed mechanism of the effect of DTP, digested albumin, digested glutelin or pure peptides in the prevention of adipogenesis (experiments I and II, the red symbols), in the prevention of inflammation (experiments III, the green symbols) and inhibition of establishing inflammation (experiments IV, the blue symbols). Red symbols: On prevention of adipogenesis, DTP, digested albumin, glutelin and pure peptides reduced the expression of PPAR?, SREBP1, FAS, lipoprotein lipase (LPL), nuclear factor-kappa B (NF-?B), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) and the secretion of PGE2, TNFa and albumin and pure peptides decrease the levels of IL-6. Green symbols: On prevention of inflammation, DTP, digested albumin and glutelin decreased the expression of PPAR?, COX-2 and iNOS and the NO, PGE2, TNFa secretion. DTP reduce the NF-?B expression. Digested albumin and glutelin reduced the MCP-1 secretion. Blue symbols: On inhibition of inflammation, DTP, digested albumin and glutelin decreased the expression of PPAR? and SREBP1. DTP reduce the expression of LPL, FAS, iNOS, COX-2, the triacylglycerol content, lipase activity, NO secretion. Digested albumin reduced the lipase activity and the secretion of NO, PGE2 and TNFa. Digested glutelin decreased the expression of LPL and FAS and TNFa secretion. DTP: digested total protein; PPAR?: peroxisome-proliferator-activated receptors gamma, SREBP1: sterol regulatory element-binding protein-1, FAS: fatty acid synthase, LPL: lipoprotein lipase, NF-?B: factor nuclear kappa B, COX-2: ciclooxigenase-2, iNOS: inducible nitric oxide synthase, MCP-1: Monocyte chemoattractant protein-1, NO: nitric oxide, PGE2: prostaglandin E2, IL-6: Interleukin 6.
Fig 4: Effect of digested total protein and digested albumin and glutelin from chia seeds to prevent and inhibit the inflammation in mature adipocytes stimulated by inflamed macrophages. NF-?B expression on prevention (a) and inhibition (b); MCP-1 secretion on prevention (c) and inhibition (d); TNF-a secretion on prevention (e) and inhibition (f). Untreated receive any treatment, PC receive only the CM. All experiments were performed in at least two independent trials run with triplicate data points. Different letter per column means statistically different among the proteins by ANOVA and post-hoc Tukey-test (p < 0.05). CM: conditioned media; NF-kB: Factor nuclear kappa B; GAPDH: glyceraldehyde 3-phosphate; MCP-1: monocyte chemoattractant protein 1; TNF-a: Tumor necrosis factor alpha; PC: Positive control.
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