Fig 1.
Myotube morphology evaluation.
Scanning electron microscopy photomicrographs showing myotubes treated with hydrogen peroxide—H2O2 (A, B), untreated control myotubes—CTR (C), myotubes pre-incubated with carnosinol—CNS (D, G, J), carnosine—CAR (E, H, K) or anserine—ANS (F, I, L) at the concentrations of 10 mM (D-F), 20 mM (G-I) and 30 mM (J-L), myotubes pre-incubated with carnosinol followed by hydrogen peroxide treatment—CNS + H2O2 (M, P, S), pre-incubated with carnosine followed by hydrogen peroxide treatment—CAR + H2O2 (N, Q, T) or pre-incubated with anserine followed by hydrogen peroxide treatment—ANS + H2O2 (O, R, U) at the concentration of 10 mM (M-O), 20 mM (P-R) and 30 mM (S-U). Bar equal: 10 μm.
Fig 2.
Photomicrographs of TUNEL assay (green staining) showing myotubes treated with hydrogen peroxide—H2O2 (A-C), untreated control myotubes—CTR (D-F), myotubes pre-incubated with carnosinol (30 mM) followed by hydrogen peroxide treatment—CNS + H2O2 (G-I), pre-incubated with carnosine (30 mM) followed by hydrogen peroxide treatment—CAR + H2O2 (J-L) and pre-incubated with anserine (30 mM) followed by hydrogen peroxide treatment—ANS + H2O2 (M-O). DAPI (blue staining) is used to locate the nuclei of the cells. Bar equal: 20 μm. The graphs (P and Q) summarize the quantitative analysis of TUNEL positive myotubes of all the experimental cell groups. * p≤0.05 vs CTR; # p≤0.05 vs H2O2; § p≤0.05 vs CAR + H2O2 10 mM; §§ p≤0.05 vs CAR + H2O2 20 mM; §§§ p≤0.05 vs CAR + H2O2 30 mM; + p≤0.05 vs ANS + H2O2 10 mM; ++ p≤0.05 vs ANS + H2O2 20 mM and +++ p≤0.05 vs ANS + H2O2 30 mM.
Fig 3.
Immunofluorescence photomicrographs of sirtuin3 (SIRT3—green staining) of myotubes treated with hydrogen peroxide—H2O2 (A), untreated control myotubes—CTR (B), myotubes pre-incubated with carnosinol followed by H2O2-treatment—CNS + H2O2 (C, F, I), pre-incubated with carnosine followed by H2O2-treatment—CAR + H2O2 (D, G, J), pre-incubated with anserine followed by H2O2-treatment—ANS + H2O2 (E, H, K) at the concentrations of 10 mM (C-E), 20 mM (F-H) and 30 mM (I-K) and myotubes incubated with AGK7 then with carnosinol (30 mM) and followed by H2O2-treatment—AGK7 + CNS + H2O2 (L). Bar equal: 20 μm. (M) Representative western blot showing SIRT3 level in myotubes treated with hydrogen peroxide—H2O2, untreated control myotubes—CTR, myotubes pre-incubated with carnosinol (30 mM) followed by H2O2-treatment—CNS + H2O2, myotubes pre-incubated with carnosine (30 mM) followed by H2O2-treatment—CAR + H2O2, myotubes incubated with AGK7 and then with hydrogen peroxide—AGK7 + H2O2, myotubes incubated with only AGK7—AGK7 and myotubes incubated with AGK7 then with carnosinol (30 mM) and followed by H2O2-treatment—AGK7 + CNS + H2O2. (N) The graph summarizes the quantitative analysis of SIRT3 immunopositivity. * p≤0.05 vs CTR; # p≤0.05 vs H2O2; § p≤0.05 vs CAR + H2O2 10 mM; §§ p≤0.05 vs CAR + H2O2 20 mM; §§§ p≤0.05 vs CAR + H2O2 30 mM; + p≤0.05 vs ANS + H2O2 10 mM; ++ p≤0.05 vs ANS + H2O2 20 mM and +++ p≤0.05 vs ANS + H2O2 30 mM.
Fig 4.
Peroxisome proliferator-activated receptor gamma coactivator-1α expression.
Immunofluorescence photomicrographs of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α - red staining) of myotubes treated with hydrogen peroxide—H2O2 (A), untreated control myotubes—CTR (B), myotubes pre-incubated with carnosinol followed by H2O2-treatment—CNS + H2O2 (C, F, I), pre-incubated with carnosine followed by H2O2-treatment—CAR + H2O2 (D, G, J), pre-incubated with anserine followed by H2O2-treatment- ANS + H2O2 (E, H, K) at the concentrations of 10 mM (C-E), 20 mM (F-H) and 30 mM (I-K) and myotubes incubated with AGK7 then with carnosinol (30 mM) and followed by H2O2-treatment—AGK7 + CNS + H2O2 (L). Bar equal: 20 μm. (M) Representative western blot showing PGC-1α level in myotubes incubated with AGK7 and then with hydrogen peroxide—AGK7 + H2O2, myotubes incubated with only AGK7—AGK7, myotube pre-incubated with carnosinol (30 mM) followed by H2O2-treatment—CNS + H2O2 and myotubes incubated with AGK7 then with carnosinol (30 mM) and followed by H2O2-treatment—AGK7 + CNS + H2O2. (N) The graph summarizes the quantitative analysis of PGC-1α immunopositivity. * p≤0.05 vs CTR; # p≤0.05 vs H2O2; § p≤0.05 vs CAR + H2O2 10 mM; §§ p≤0.05 vs CAR + H2O2 20 mM; §§§ p≤0.05 vs CAR + H2O2 30 mM; + p≤0.05 vs ANS + H2O2 10 mM; ++ p≤0.05 vs ANS + H2O2 20 mM and +++ p≤0.05 vs ANS + H2O2 30 mM.
Fig 5.
Superoxide dysmutase2 expression.
Immunofluorescence photomicrographs of superoxide dysmutase2 (SOD2—green staining) of myotubes treated with hydrogen peroxide—H2O2 (A), untreated control myotubes—CTR (B), myotubes pre-incubated with carnosinol followed by H2O2-treatment—CNS + H2O2 (C, F, I), pre-incubated with carnosine followed by H2O2-treatment—CAR + H2O2 (D, G, J) and pre-incubated with anserine followed by H2O2-treatment—ANS + H2O2 (E, H, K) at the concentrations of 10 mM (C-E), 20 mM (F-H) and 30 mM (I-K) and myotubes incubated with AGK7 then with carnosinol (30 mM) and followed by H2O2-treatment—AGK7 + CNS + H2O2 (L). Bar equal: 20 μm. (M) Representative western blot showing SOD2 level in myotubes incubated with AGK7 and then with hydrogen peroxide—AGK7 + H2O2, myotubes incubated with only AGK7—AGK7, myotube pre-incubated with carnosinol (30 mM) followed by H2O2-treatment—CNS + H2O2 and myotubes incubated with AGK7 then with carnosinol (30 mM) and followed by H2O2-treatment—AGK7 + CNS + H2O2. (N) The graph summarizes the quantitative analysis of SOD2 immunopositivity. * p≤0.05 vs CTR; # p≤0.05 vs H2O2; § p≤0.05 vs CAR + H2O2 10 mM; §§ p≤0.05 vs CAR + H2O2 20 mM; §§§ p≤0.05 vs CAR + H2O2 30 mM; + p≤0.05 vs ANS + H2O2 10 mM; ++ p≤0.05 vs ANS + H2O2 20 mM and +++ p≤0.05 vs ANS + H2O2 30 mM.
Fig 6.
Immunofluorescence photomicrographs of catalase (CAT—green staining) of myotubes treated with hydrogen peroxide—H2O2 (A), untreated control myotubes—CTR (B), myotubes pre-incubated with carnosinol followed by H2O2-treatment—CNS + H2O2 (C, F, I), pre-incubated with carnosine followed by H2O2-treatment—CAR + H2O2 (D, G, J) and pre-incubated with anserine followed by H2O2-treatment—ANS + H2O2 (E, H, K) at the concentrations of 10 mM (C-E), 20 mM (F-H) and 30 mM (I-K) and myotubes incubated with AGK7 then with carnosinol (30 mM) and followed by H2O2-treatment—AGK7 + CNS + H2O2 (L). Bar equal: 20 μm. (M) Representative western blot showing CAT level in myotubes incubated with AGK7 and then with hydrogen peroxide—AGK7 + H2O2, myotubes incubated only with AGK7—AGK7, myotube pre-incubated with carnosinol (30 mM) followed by H2O2-treatment—CNS + H2O2 and myotubes incubated with AGK7 then with carnosinol (30 mM) and followed by H2O2-treatment—AGK7 + CNS + H2O2. (N) The graph summarizes the quantitative analysis of CAT immunopositivity. * p≤0.05 vs CTR; # p≤0.05 vs H2O2; § p≤0.05 vs CAR + H2O2 10 mM; §§ p≤0.05 vs CAR + H2O2 20 mM; §§§ p≤0.05 vs CAR + H2O2 30 mM; + p≤0.05 vs ANS + H2O2 10 mM; ++ p≤0.05 vs ANS + H2O2 20 mM and +++ p≤0.05 vs ANS + H2O2 30 mM.
Fig 7.
Immunofluorescence photomicrographs of cyclooxygenase2 (COX2—green staining) of myotubes treated with hydrogen peroxide—H2O2 (A), untreated control myotubes—CTR (B), myotubes pre-incubated with carnosinol followed by H2O2-treatment—CNS + H2O2 (C, F, I), pre-incubated with carnosine followed by H2O2-treatment—CAR + H2O2 (D, G, J) and pre-incubated with anserine followed by H2O2-treatment—ANS + H2O2 (E, H, K) at the concentrations of 10 mM (C-E), 20 mM (F-H) and 30 mM (I-K). Bar equal: 20 μm. (L) The graphs summarize the quantitative analysis of COX2 immunopositivity. * p≤0.05 vs CTR; # p≤0.05 vs H2O2; § p≤0.05 vs CAR + H2O2 10 mM; §§ p≤0.05 vs CAR + H2O2 20 mM; §§§ p≤0.05 vs CAR + H2O2 30 mM; + p≤0.05 vs ANS + H2O2 10 mM; ++ p≤0.05 vs ANS + H2O2 20 mM and +++ p≤0.05 vs ANS + H2O2 30 mM.
Fig 8.
Carnosinol therapeutic potentials.
Schematic representation of the therapeutic protective antioxidative effects of carnosinol against hydrogen peroxide-mediated alterations at myotube level showing that carnosinol may act through the mitochondrial PGC-1α/SIRT3 signaling pathway (black arrows), but if SIRT3 is inhibited carnosinol’s effects are not blocked (blue arrows). H2O2: hydrogen peroxide; CAT: catalase; COX2: cyclooxygenase2; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator-1alpha; SIRT3: sirtuin3; SOD2: superoxide dysmutase2.