Activation of Sirtuin3 by 6,4′-Dihydroxy-7-methoxyflavanone Against Myoblasts Senescence by Attenuating D-Galactose-Induced Oxidative Stress and Inflammation

DMF (BBP05065) was obtained from BioBioPha (Kunming, China). DMF was dissolved in Dimethyl sulfoxide (DMSO, D8371, Solarbio, Beijing, China) before used. D-gal (G100367) was obtained from Aladdin (Shanghai, China). D-gal was dissolved in double-distilled water (DW) before used. The final vehicle content was less than 0.01% in each group. DMF and D-gal were used in this study to investigate the effect of DMF on D-gal-induced myoblasts senescence and myogenesis inhibition model. The workflow was show in Figure 1.

C2C12 myoblasts (a murine cell line, SCSP-505, obtained from Cell Bank/Stem Cell Bank, Chinese Academy of Sciences, Shanghai) were cultured in Dulbecco's Modified Eagle Medium (DMEM, 12800082, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, 10099141, Gibco) and 1% penicillin/streptomycin (P/S, VC2003, Vicmed, XuZhou, China). C2C12 myoblasts were maintained in differentiation media [DMEM, containing 2% horse serum (HS, 26050070, Gibco) and 1% P/S]. Cells were kept at 37 °C in 5% CO₂ humidified chambers (Haier, Qingdao, China).

Proliferation rates and compound toxicity were evaluated via [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] test solution (MTS, G3581, Promega, Madison, Wisconsin, USA). Myoblasts (1 × 10⁵ cells/well) in 96-well plates underwent treatment with concentration gradients of D-gal/DMF for varying durations. Fresh MTS working solution (1:20 v/v) was introduced before 1 h incubation. Absorbance at 490 nm was recorded using a Synergy 2 spectrophotometer (BioTek, Winooski, VT, USA) with Gen5 v2.0 software.

Cellular senescence was examined using a commercial SA-β-gal staining kit (C0602, Beyotime, Shanghai, China). Briefly, cells in six-well plates were fixed and then incubated with staining working solution in a dry, CO₂-free incubators at 37 °C for 6 h. Image acquisition and analysis: Blue-stained SA-β-gal-positive cells were imaged using an Olympus IX73 (Tokyo, Japan) inverted microscope (10×, Japan). For each well, three random, non-overlapping fields were captured. The percentage of SA-β-gal-positive cells was quantified by counting the number of blue cells versus the total number of cells (determined by bright-field imaging) using ImageJ2 2.16.0/1.54p software (NIH) by an investigator blinded to the treatment groups.

Collected cells were resuspended in cold RIPA lysis buffer (P0013B, Beyotime) containing 1mM Phenylmethanesulfonyl fluoride (PMSF, ST506, Beyotime). Protein concentration was determined using a bradford proteins assay kit (5000205, Bio-Rad, Hercules, CA, USA). Equal amounts of protein (50 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel and were transferred to Polyvinylidene Fluoride (PVDF) membranes (162-0177, Bio-Rad). Each membrane was incubated with primary antibody at 4 °C overnight, followed by 2 h in secondary antibody at room temperature. Proteins bands were visualized using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (34578, Termo Fisher Scientific, Waltham, MA, USA). Results shown were quantitated using Image J software. Primary antibodies as follows: anti-p53 (60283-2-lg, Research Resource Identifier: AB_2881401, 1:5000, Proteintech, Wuhan, China); anti-p21^Waf1/Cip1 (28248-1-AP, 1:4000, Proteintech); anti-p16^INK4a (ab211542, 1:2000, abcam); anti-phospho-Rb (Ser780) (p-Rb, 8180S, 1:1000, Cell signaling Technology, Beverly, MA, USA); anti-Rb (ab181616, 1:2000, abcam); anti-myoblast determination protein 1 (MyoD, 18943-1-AP, 1:6000, Proteintech); anti-myogenin (Myog, ab124800, 1:1000, abcam); anti-myosin heavy chain (MyHC, sc-376157, 1:2000, Santa Cruz Biotechnology, Inc. Heidelberg, Germany); anti-muscle-specific regulatory factor 4 (MRF4, 11754-1-AP, 1:2000, Proteintech); anti-SIRT3 (10099-1-AP, 1:500, Proteintech); anti-ac-SOD2 (ab137037, 1:10,000, abcam, Cambridge, MA); anti-SOD2 (24127-1-AP, 1:50,000, Proteintech); anti-acetylated-lysine (HA723074, 1:2000, HuaBio, Hangzhou, China); anti-phospho-IκBα (Ser32) (p-IκBα, S0B6000, 1:5000, STARTER, Shanghai, China); anti-IκBα (10268-1-AP, 1:50,000, Proteintech); anti-acetylated nuclear Factor kappa B p65 (ac-NF-κB p65,ab16502, 1:1000, abcam); NF-κB p65 (10745-1-AP, 1:6000, Proteintech); anti-GAPDH (60004-1-Ig, 1:10,000, Proteintech). Secondary antibodies as follows: goat anti-mouse secondary antibody (AS003, 1:5000, Abclonal, Wuhan, China); mouse anti-rabbit secondary antibody (sc-2357, 1:1666, Santa Cruz Biotechnology).

C2C12 myoblasts were grown on gelatin-coated coverslips (22 × 22 mm, China) in six-well plates. After treatments, cells were fixed with 4% paraformaldehyde (VIH100, Vicmed) for 30 min at room temperature (RT). After permeabilization with 0.2% Triton X-100 (T8787, Sigma, St. louis, MO, USA) for 5 min and blocking with 3% Goat Serum Albumin (GSA, WLA067a, Wanleibio, Wuhan, China) for 30 min, the samples were incubated with primary antibodies overnight at 4 °C. The following day, coverslips were incubated with secondary antibodies for 1 h at RT in the dark. Nuclei were counterstained with 1 μg/mL 4′6-diamidino-2-phenylindole (DAPI, S9119, OriLeaf, Suzhou, China). Images were captured using an epifluorescence microscope (Olympus BX43, Tokyo, Japan) and a confocal laser scanning microscope (CLSM, Leica STELLARIS 5, Wetzlar, Germany). Primary antibodies were as follows: anti-MyHC (sc-376157, 1:2000, Santa Cruz); anti-phospho-H2A.X (Ser139) (p-H2A.X, 9718T, 1:250, Cell Signaling Technology); NF-κB p65 (10745-1-AP, 1:100, Proteintech). Secondary antibodies were as follows: SA-ALEXA FLUOR 488 GOAT Anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody (A-11001, 1:1000, Invitrogen); SA-ALEXA FLUOR 568 GOAT Anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (A-11011, 1:1000, Invitrogen).

p-H2A.X: Images were captured using a confocal laser scanning microscope (Leica STELLARIS 5). For quantification of DNA damage foci, at least 50 cells per group from three independent experiments were analyzed. The number of discrete p-H2A.X foci per nucleus was counted using ImageJ software (NIH).

MyHC: Images were captured using an epifluorescence microscope (Olympus BX43) with a 20× objective. For myotube analysis, three random fields per group were analyzed. The fusion index was calculated as (number of nuclei within MyHC-positive myotubes containing ≥ 10 nuclei/total number of nuclei) × 100%. Myotube diameter was measured at three random points along the length of each MyHC-positive myotube using Image J.

After treatment, cells in six-well were incubated with 10 μM 2′,7′-Dichlorofluorescin diacetate (DCFH-DA, H31224, Aladdin, Shanghai, China) diluted in serum-free DMEM for 30 min at 37 °C in the dark. Following three washes with serum-free DMEM, fluorescence images were immediately captured using an Olympus IX73 inverted microscope (10× objective). Mitochondrial superoxide generation was assessed using MitoSOX Red reagent (G1734, Servicebio, Wuhan, China). Cells were plated in black-walled 96-well plates (Servicebio, China) at a density of 1 × 10³ cells per well. Following two washes with 1Xphosphate buffer saline (PBS), cells were loaded with 5 µM MitoSOX Red in recording medium and incubated for 20 min at 37 °C under 5% CO₂ in darkness. Fluorescence measurements were conducted at 37 °C using a Synergy 2 multi-mode microplate reader (BioTek, USA) equipped with Gen5 v2.0 software, configured for excitation/emission wavelengths of 510/580 nm. The mean fluorescence intensity from three random fields per well was quantified using Image J.

Myoblasts were transfected with 16 pmol of either SIRT3 small interfering RNAs (SIRT3 siRNA, sc-61556, Santa Cruz Biotechnology) or a fluorescently labeled control siRNA (sc-36869, Santa Cruz Biotechnology) using Lipofectamine 2000 (11668019, Invitrogen) in Opti-MEM reduced-serum medium (31985070, Gibco), following the manufacturer's instructions. Transfection Efficiency: Transfection efficiency was assessed 24 h post-transfection by quantifying the percentage of cells exhibiting fluorescence from the control siRNA. This analysis consistently revealed a transfection efficiency greater than 80%. The efficacy of SIRT3 knockdown was subsequently validated 72 h post-transfection by Western blot analysis.

SIRT3 activity was measured using the manufacturer's instructions using a SIRT3 activity assay kit (ab156067, abcam). Briefly, recombinant SIRT3 was incubated with a reaction mixture composed of assay buffer, Fluoro-Substrate peptide, NAD, developer, and the test compounds. Fluorescence intensity (excitation/emission = 360/460 nm) was recorded at 2-min intervals for 30 min using a microplate reader (Synergy 2 multi-mode microplate reader, BioTek, USA).

Mitochondrial membrane potential was evaluated using a JC-1 assay kit (G515, Servicebio, China) following the manufacturer's protocol. In brief, after treatment, cells were collected, washed twice with ice-cold PBS, and resuspended in a solution containing 1 mL culture medium and 1 mL JC-1 staining reagent. The suspension was incubated for 20 min at 37 °C in the dark. Following incubation, cells were washed three times with chilled staining buffer and analyzed by flow cytometry (Olympus, Tokyo, Japan). Cellular fluorescence was observed under an Olympus IX73 inverted microscope at 20× magnification.

Cellular ATP content was quantified with a firefly luciferase-dependent ATP detection kit (G4309, Servicebio). Measurements were performed on a Synergy 2 multi-mode microplate reader (BioTek, USA) running Gen5 v2.0 software, in accordance with the manufacturer's established procedure.

The supernatant from C2C12 myoblasts was collected and assayed for IL-6 and TNF-α levels using the ElaBoXTM Mouse ILterleukin-6 (IL-6) ELISA Kit (SEKM-0007, Servicebio) and the ElaBoXTM Mouse TNF-α ELISA Kit (SEKM-0034, Servicebio), respectively, according to the manufacturer's protocol. Subsequently, the absorbance was measured at a wavelength of 450 nm using a Synergy 2 multi-mode microplate reader.

All data are expressed as the mean ± standard error of the mean (SEM). The sample size (n) represents the number of independent biological replicates and is specified in the figure legends. All experiments were performed independently at least three times. Statistical analyses were performed using GraphPad Prism v10.2.3 software. For comparisons between two groups, an unpaired two-tailed Student's t-test was used. For comparisons among three or more groups under a single factor, one-way analysis of variance (ANOVA) was used, followed by Tukey's multiple comparison test. For the analysis of cell proliferation across multiple time points, two-way ANOVA followed by Šídák's multiple comparisons test was applied. A value of p < 0.05 was considered statistically significant. Effect sizes with 95% confidence intervals are reported in Supplementary Table S1.

To investigate the potential therapeutic effects of DMF on aged skeletal muscle, we established an in vitro model of senescent C2C12 myoblasts induced by D-gal. Cells were treated with varying concentrations and different time durations of D-gal (Figure S1). Notably, exposure to 222 mM D-gal for 48 h significantly inhibited cell proliferation (Figure S1A–C). p53, p21^Cip1/WAF1 and p16^INK4a, hallmarks of cellular senescence, were upregulated in a concentration- and time-dependent manner after D-gal treatment (Figure S1D,E,H,I). Conversely, p-Rb, hallmarks of cell growth factor, were markedly reduced under these conditions (Figure S1D,E,H,I). Furthermore, the senescent state was corroborated by a significant increase in SA-β-gal-positive cells (Figure S1F,G). Collectively, these data demonstrated that treatment with 222 mM D-gal for 48 h represents an optimal approach for establishing a robust cellular senescence model. The p-H2A.X, a DNA damage marker, provided additional validation of this model (Figure S1J,K).

To evaluate the potential cytotoxicity of DMF, myoblasts were pre-treated varying concentrations of DMF for 12 h (Figure 2A) prior to exposure to 222 mM D-gal (Figure 2B), followed by assessment with an MTS assay. As shown in Figure 2A, 0–30 μM DMF have no significant cytotoxicity. Pretreatment with 0.5–5 μM DMF ameliorated the growth inhibitory effect induced by D-gal treatment (Figure 2B). Consistent with these findings, the expression levels of aging-related markers and cell growth factor showed comparable responses following DMF treatment (Figure 2C,E). Meanwhile, DMF pretreatment resulted in a concentration-dependent reduction in SA-β-gal positive area (Figure 2D,F). These results suggest that DMF exerts an anti-aging effect and 5 μM DMF treatment have a best effect. The expression of p-H2A.X further verified these observations (Figure 2G,H).

To assess myogenic differentiation, we examined the expression of key markers, including the early factors (MyoD and MyoG) and late factors (MyHC and MRF4). We found that these differentiation markers were suppressed following D-gal-induced senescence in myoblasts (Figure 3B–E). In contrast, pre-treatment with DMF significantly up-regulated their expression (Figure 3B–E). Moreover, DMF pre-treatment led to an increase in myotubes diameter and a higher fusion index (Figure 3F–H). These findings suggest that DMF exerts an anti-aging effect by attenuating senescence in myoblasts and facilitating myotubes development.

To investigate the regulatory mechanism underlying the anti-aging effect of DMF, we examined its impact on SIRT3, a key mitochondrial deacetylase implicated in aging. C2C12 myoblasts were treated with varying concentrations of DMF for 12 h. DMF up-regulated SIRT3 protein expression and activity in a concentration-dependent manner (Figure 4A–C). Optimum SIRT3 induction was achieved at 5 μM DMF. After treatment with 5 μM DMF, the protein expression and activity of SIRT3 peaked at 12 h and declined by 24 h (Figure 4D–F). To determine whether DMF attenuates SOD2 acetylation via SIRT3, we performed siRNA-mediated knockdown of SIRT3 in the context of D-gal-induced stress. As shown in Figure 4A–C, Pre-treatment with DMF attenuated SOD2 acetylation through up-regulation of SIRT3. This effect was markedly attenuated upon SIRT3 knockdown using siRNA in C2C12 myoblasts (Figure 5A–C). Consistent results were observed in the overall levels of acetylated lysine in total cellular proteins (Figure 5D,F). These data indicated that DMF enhanced the deacetylase activity of SIRT3 (Figure 5D,F). DCFH-DA fluorescent staining demonstrated that DMF significantly suppressed ROS generation compared to the D-gal-treated group (Figure 5E,G). However, the protection effect of DMF was abolished upon SIRT3 inhibition (Figure 5E,G). Mitochondrial-specific ROS, as measured by MitoSOX Red fluorescence, followed the same pattern (Figure 5H). Furthermore, DMF improved mitochondrial function, as indicated by the recovery of mitochondrial membrane potential (JC-1 assay, Figure 5I) and increased ATP production (Figure 5J). These beneficial effects were also SIRT3-dependent. Collectively, these results indicate that DMF enhances SIRT3 expression and activity, leading to SOD2 deacetylation, reduced mitochondrial ROS accumulation, and improved mitochondrial function.

NF-κB represents a central transcription factor involved in the inflammatory response. Activation of the NF-κB pathway induces the transcription of multiple genes, including those encoding pro-inflammatory cytokines, which have been closely linked to the pathogenesis of age-related diseases [3]. To investigate the effect of D-gal exposure on NF-κB pathway, we assessed the ac-NF-κB p65 and p-IκBα in C2C12 myoblasts. Treatment with 222 mM D-gal resulted in a time-dependent increase in p-IκBα, with a significantly elevation observed between 1–12 h (Figure 6A,C). NF-κB p65 acetylation peaked around 0.5–3 h and gradually declined thereafter (Figure 6B,D). Pre-treatment with DMF markedly attenuated these increases (Figure 5E–G). Notably, this inhibitory effect of DMF was substantially abolished following SIRT3 knockdown via siRNA in myoblasts (Figure 6E–G). Moreover, DMF significantly suppressed D-gal-induced nuclear translocation of NF-κB p65, an effect that was also reversed by SIRT3 knockdown (Figure 6H,I). Consistent with these findings, DMF pretreatment reduced the secretion of the pro-inflammatory cytokines IL-6 and TNF-α induced by D-gal (Figure 6J,K). Collectively, these results demonstrate that DMF suppressed D-gal-induced NF-κB activation by suppressing IκBα phosphorylation and NF-κB p65 acetylation in a SIRT3 dependent manner.