Fig 1: On sepsis satellite cells display altered but hyperactive mitochondria.(a–e) Quantification by RT–qPCR of nuclear genes in the time course of sepsis in the control (no injury no CLP) at 6 h, 24 h, 48 h and 4 days (d) post induction of sepsis; n=6 per time point; (a) expression of hypoxia-inducible factor1 (Hif1a); (b) NADPH oxidase 1 (Nox1); (c,d) expression of antioxidant enzymes Sod1 and Sod2; (e) peroxisome proliferator-activated receptor coactivator 1 (PGC1a). (f,g) Quantification of immunofluorescence intensity by confocal microscopy image acquisition and ImageJ analysis. The levels of carbonylated (antibody anti-dinitrophenyl) and nitrated (antibody anti-nitrotyrosine) proteins (resulting essentially from reactive oxidative and nitrosative species, respectively) were measured in controls, 6 h, 24 h, 48 h and 4 days post induction of sepsis. Representative immunostaining (three-dimensional-reconstructed cells) are displayed. Scale bar, 5 μm. (h) Confocal microscopy immunofluorescence intensity quantification of TOM22 (mitochondrial outer membrane protein) and (i) confocal microscopy intensity of MitoTracker Deep Red staining probe in control (healthy) and septic mice. Once acquired by confocal microscopy the images were analysed using the mean grey value ImageJ plugin. (j) Mitochondrial DNA content (mtDNA) in control and septic SC. (k) Agarose gel electrophoresis of long PCR amplifications on sorted SC mtDNA in controls and septic Tg:Pax7nGFP 6 h, 24 h, 48 h and 4 days post sepsis showing mtDNA size alterations. Left panel, amplification of a fragment of expected size (9,898 bp) from positions 7,998–16,299/1 (conventional end/origin of circular mtDNA) and 1–14,400 of the mtDNA; the image on this panel is intentionally highly contrasted to underscore multiple bands, and also the low intensity of the expected band (9,898 bp) in sepsis samples compared with controls; centre panel, schematic representation of genes and regulatory regions in the human mtDNA, with the coordinates of PCR-amplified regions; right panel, amplification of a fragment of expected size 9,677 bp from positions 15,377 to 5,701 of the mtDNA. Note that size alterations in cells from septic mice are present in one (left panel) but not the other PCR-amplified fragment. (l) Tetramethylrhodamine ethylamine (TMRE; mitochondrial membrane potential) levels. (m) Relative percentage of glycolytic and mitochondrial (OXPHOS) ATP in controls (no injury no CLP) and 24 h post sepsis. (n) Percentage of ATP relative to control in controls (n=6) and septic Tg:Pax7nGFP at 24 h (n=6). (o) Basal mitochondrial respiration of SC in non-septic (control) and 24 h post sepsis evaluated with Seahorse XFe96 analyser. Unless specified data are represented as mean±s.d. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; NS, not significant, compared with the respective control (Mann–Whitney test).
Fig 2: Mesenchymal stem cells improve muscle regeneration by decreasing septic state and restoring affected mitochondrial parameters in satellite cells.(a) Schematic representation of timing of sacrifice, post-caecal ligature puncture and injury (with notexin) plus MSCs grafting. (b) Haematoxylin and eosin staining of the TA 21 days (d) post sepsis and injury (n=16). Scale bar, 100 μm. (c) Haematoxylin and eosin of TA 21 days post sepsis and injury with injection 6 h post injury of mesenchymal stem cells (n=16). Scale bar, 100 μm. (d–f) Fractions of fibrotic area. (d) Fibrotic area of the muscle tissue after regeneration (21 days post injury) in control, septic and septic injected with MSC conditions. (e) Sirius Red staining of muscle tissue of septic mice 21 days post injury (n=16) Scale bar, 100 μm. (f) Sirius Red staining of muscle tissue 21 days post injury of septic mice treated with MSC injection (n=16). Scale bar, 100 μm. (g) Example of Luminex on interleukin-6 (IL-6) representing the level of protein expression in pg g−1 of muscle tissue. Control are non-injured mice (n=4), septic Tg:Pax7nGFP at 24 h (n=4) and septic Tg:Pax7nGFP injected with MSCs (n=4). (h) TMRE label in injured (n=3); injured, septic TgPax7nGFP (n=3); and injured, septic, MSC-injected TgPax7nGFP (n=3) at 24 h. (i) ATP content relative to control (n=6), septic TgPax7nGFP (n=6) and septic TgPax7nGFP injected with MSCs (n=6) at 24 h. (j) Relative glycolytic and mitochondrial ATP content in controls (no injury no CLP), 24 h post sepsis, and septic Tg:Pax7nGFP injected with MSCs. (k) Quantification by RT–qPCR of PGC1a, Nox1, Hif1a, Sod1 and Sod2 expression. (l) Relative level of intensity of MitoTracker Deep Red in controls, septic Tg:Pax7nGFP and septic Tg:Pax7nGFP injected with MSCs at 24 h (n=6). (m) Quantification of mtDNA in FACS cell-sorted satellite cells in injured, injured and septic, and injured, septic and MSC-injected Tg:Pax7nGFP mice. (n) Agarose gel electrophoresis of long PCR amplification of mtDNA in SCs from controls, septic and septic mice injected with MSCs showing mtDNA alterations only in septic mice (left panel). The coordinates of the amplified fragments are shown in Fig. 3k. MSCs rescue normal mitochondrial genome size after sepsis. (o) Maximal tension of muscle fibres of septic and injured (n=6) compared with septic, injured and MSC-injected C57Bl/6 mice (n=7) according to the Ca2+ concentration. Data are represented as mean±s.d. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; not significant, compared with the respective control (Mann–Whitney test).
Fig 3: Macrophage SAMSN1 deficiency exacerbates LPS-induced inflammation and oxidative stress in mice. (A) Samsn1Flox or Samsn1MKO mice were intratracheally injected with LPS (5 mg/kg) and sacrificed after 12 h, and then the numbers of total cells, neutrophils and macrophages in BALF were detected. (B) MPO activity was detected to evaluate infiltration of neutrophils in the lungs. (C) The levels of TNF-α and IL-6 in the lungs. (D–E) The levels of ROS, MDA and 4-HNE. (F) Total SOD activity and GSH level. (G) NLRP3 and ASC protein levels. (H) Quantification of caspase-1 activity. (I–J) The levels of IL-1β and IL-18 in the lungs. n = 6 per group, differences with P value < 0.05 were considered statistically significant.
Fig 4: Expression of Keap1, Nrf2, SOD, NQO1, HO-1, and GCLC proteins in myocardial tissue of each group. P*< 0.05 vs Control group; P#< 0.05 vs DOX group.
Fig 5: CTRP6 overexpression inhibits OGD/R-induced inflammation and oxidative stress in PC12 cells. Alteration of (A) cell viability and inflammatory cytokines (B) TNF-α, (C) IL-1β, (D) IL-6 and (E) IL-10 levels (n=3). Alteration of oxidative products (F) ROS, (G) MDA and (H) antioxidant SOD levels (n=3). ***P<0.001 vs. control, ##P<0.01 and ###P<0.001 vs. OGD/R + NC. CTRP6, C1q/tumor necrosis factor-related protein-6; OGD/R, oxygen-glucose deprivation and reperfusion; NC, negative control; TNF, tumor necrosis factor; IL, interleukin; ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase.
Supplier Page from Abcam for Superoxide Dismutase Activity Assay Kit (Colorimetric)