Fig 1: FMN inhibits myostatin-mediated dephosphorylation on the PI3K/Akt/FoxO3a signalling pathway in the muscle of CKD rats and C2C12 myotubes. (A) Protein levels of p-PI3K, PI3K, p-Akt, Akt, p-FoxO3a and FoxO3a in gastrocnemius muscle analysed using Western blotting (n = 3/group). (B) Protein levels of MAFbx and MuRF-1 in gastrocnemius muscles analysed using Western blotting (n = 3/group). (C) C2C12 myotubes were treated with FMN (50 µmol/L) in the presence or absence of TNF-a (40 ng/mL) for 48 h following 24 h of incubation with si-myostatin or si-NC. The myotubes were divided into four groups: si-NC, si-NC + TNF-a, si-NC + TNF-a + FMN (50 µmol/L) and si-myostatin + TNF-a. Protein levels of p-PI3K, PI3K, p-Akt, Akt, p-FoxO3a and FoxO3a in C2C12 myotubes (n = 3/group). (D) Protein levels of MAFbx and MuRF-1 in C2C12 myotubes (n = 3/group). (E) Myostatin OE transfection was used to overexpress myostatin in C2C12 myotubes, and they were incubated with FMN (50 µmol/L) and TNF-a for another 48 h. The myotubes were divided into four groups: vector NC, vector NC + TNF-a, vector NC + TNF-a + FMN (50 µmol/L) and myostatin OE + TNF-a + FMN (50 µmol/L). The protein levels of p-PI3K, PI3K, p-Akt, Akt, p-FoxO3a and FoxO3a in the myotubes were analysed using Western blotting. (F) Protein levels of MAFbx and MuRF1 in C2C12 myotubes analysed by Western blotting (n = 3/group). *P < .05, **P < .01
Fig 2: FMN ameliorates muscle atrophy induced by TNF-a in C2C12 myotubes. (A) Cytotoxicity of TNF-a in C2C12 myotubes. (B) The protein expressions of MAFbx and MuRF1 were detected in C2C12 myotubes treated with TNF-a (20 or 40 ng/mL) for 48 h. (C) The cytotoxic effects of FMN assessed using a CCK-8 assay. (D) The protein and mRNA expressions of myostatin were detected in C2C12 myotubes transfected with si-NC or si-myostatin. *P < .05, **P < .01 compared to the si-NC group (n = 3/group). (E) C2C12 myotubes were treated with FMN (25 µmol/L and 50 µmol/L) in the presence or absence of TNF-a (40 ng/mL) for 48 h following 24 h of incubation with si-myostatin or si-NC. The expressions of MAFbx and MuRF1 in C2C12 myotubes were determined by immunofluorescence staining (200×) with anti-MAFbx and anti-MuRF1 (green), and the nuclei were detected with DAPI staining (blue). (F) Relative fluorescence intensities of MAFbx and MuRF-1 compared between groups. *P < .05, **P < .01
Fig 3: The conclusion supports the mitochondrial mitophagy (i.e., increase in DRP1/MuRF1 ratio) in the CBS+/- mice skeletal muscle. The H2S treatment normalized the mitochondrial bioenergetics, successfully.
Fig 4: The levels of gene writer (DNMT2), skeletal muscle musclin, and the histone 3 lysine 9 methylation (H3K9) were measured. The levels of gene eraser, fat obese associated protein (FTO), ten-eleven translocate (TET2), growth arrest and DNA damage-45 protein (GADD45b), associated matrix metalloproteinase-13 (MMP13) were determined. The levels of mitochondrial mitophagy were also determined by calculating the ratios of the fission protein (DRP1), and fusion protein (MuRF1), *p < 0.05; n = 5–12.
Fig 5: PLK1 participated in regulating the apoptosis pathway and E3 ubiquitin ligases. (A) C2C12 cells were transfected with PLK1 siRNA or non-silencing control siRNA and then induced to form myotubes. Apoptosis was analysed by Annexin V-FITC/7-AAD double-labelling assays. The proportion of apoptotic cells was quantified. (B) Total-Caspase3 and cleaved-Caspase3 levels were measured after the above treatments. The graph shows the relative band densities. (C) The expression levels of the indicated proteins were measured after the above treatments. The graph shows the relative band densities. Each result was replicated in three independent experiments, and the values are the means ±SD. *p < 0.05. (D) Diagram representation of PLK1-AKT signalling in sepsis-induced muscle atrophy. Sepsis/LPS induces the downregulation of PLK1 expression in myofibres, thus reducing the activity of the AKT pathway. This reduction results in apoptosis of myotubes and inhibits the proliferation of myoblasts, ultimately leading to myofibre atrophy. Meanwhile, PLK1 also targets the Caspase3-dependent apoptosis pathway and inhibits the activity of the E3 ubiquitin ligases MURF1 and Atrogin-1, which may be another mechanism of sepsis-induced muscle atrophy
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