Flavonoids and Omega3 Prevent Muscle and Cardiac Damage in Duchenne Muscular Dystrophy Animal Model

Nutraceutical products possess various anti-inflammatory, antiarrhythmic, cardiotonic, and antioxidant pharmacological activities that could be useful in preventing oxidative damage, mainly induced by reactive oxygen species. Previously published data showed that a mixture of polyphenols and polyunsaturated fatty acids, mediate an antioxidative response in mdx mice, Duchenne muscular dystrophy animal model. Dystrophic muscles are characterized by low regenerative capacity, fibrosis, fiber necrosis, inflammatory process, altered autophagic flux and inadequate anti-oxidant response. FLAVOmega β is a mixture of flavonoids and docosahexaenoic acid. In this study, we evaluated the role of these supplements in the amelioration of the pathological phenotype in dystrophic mice through in vitro and in vivo assays. FLAVOmega β reduced inflammation and fibrosis, dampened reactive oxygen species production, and induced an oxidative metabolic switch of myofibers, with consequent increase of mitochondrial activity, vascularization, and fatigue resistance. Therefore, we propose FLAVOmega β as food supplement suitable for preventing muscle weakness, delaying inflammatory milieu, and sustaining physical health in patients affected from DMD.


Introduction
Dietary supplementation is an important source of vitamins, minerals, herbs, or products made from plants, animals, algae, seafood, or yeasts, intended to complement the common diet in physiological or pathological conditions. In fact, food supplements may be useful for the maintenance of a proper nutrient supply to promote health span and improve physical performance of healthy subjects, in case of nutritional deficiencies and as an adjuvant tool for the management of multiple disease conditions. Neuromuscular disorders, such as Duchenne muscular dystrophy (DMD), present important secondary pathologic features that fulfil the requirements for dietary supplementation. DMD is an X-linked recessive disease that affects muscular function and strength. It is caused by mutations of the dystrophin gene, which result in null expression of the structural protein, leading to instability of the dystrophin-associated glycoprotein complex and sarcolemma fragility. DMD muscles are characterized by low regenerative capacity, fibrosis, fiber necrosis [1], inflammatory infiltrates, intracellular Ca 2+ dysregulation, aberrant cellular signaling, mitochondrial malfunction and overproduction of reactive oxygen species (ROS), which outweigh a physiological antioxidant response [2]. Heart complications and respiratory disorders are manifested as the pathology progresses and are the major causes of death in DMD patients [3]. Among cardiac complications, dilated cardiomyopathy (DCM) leads to increased ventricular chamber size, coupled with loss of contractile function (ejection fraction < 40%) [4], progressive myocardial fibrosis and decreased cardiac function [5].

Animal Statement
All procedures involving living animals were performed in accordance with Italian law (D.L.vo 116/92 and subsequent additions), which conforms to the European Union guidelines. The use of animals in this study was authorized by the National Ministry of Health (protocol number 10/13-2014/2015). Eight mdx mice (C57BL6/10ScSn-DMDmdx/J) 3 months of age were provided by Charles River. All animals were housed in Cells 2021, 10, 2917 3 of 19 a controlled environment (12 h light/dark cycle) at a temperature between 21 and 23 • C. Cage population was limited to a maximum of four animals each to ensure the health and welfare of animals. Mice had free access to clean water and food. Systemic administration of 100 µL of FLAVOmega β (100 µg/µL) per mouse was performed by oral gavage for 4 weeks (1 oral administration per day). Untreated aged-matched mdx mice were used as controls. After treatment, mice were deeply anesthetized and then sacrificed by cervical dislocation.

Supplement Formulations
Six different formulations of the supplement mixture were produced, as shown in Table 1. FLAVO α, FLAVO β, FLAVO γ only consist of a powder phase of curcumin, CoQ10, Scutellaria dry extract (baicalein), green tea extract (epicatechins) and aloe vera, at variable concentrations (1X, 5X and 10X, respectively). An oil phase with variable concentrations of Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) (1X, 5X and 10X, respectively) was added to FLAVO α to obtain FLAVOmega α, FLAVOmega β and FLAVOmega γ. All components were bought as pure and directly dissolved in 0.1% dimethyl sulfoxide (DMSO). DMSO is a class III solvent according to FDA classification (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidance for industry Q3C Impurities: Residual Solvents). 0.1% is the lowest concentration of DMSO that allows complete dissolution of all components in absence of cytotoxic effects. Preliminary studies confirmed that there is no difference in cytotoxicity between normal growth medium, 0.01% and 0.1% DMSO.

Optical Properties of FLAVOmega β
Photoluminescence (PL) and time-resolved PL spectra were recorded with a Cary Varian Eclipse at a 90 • -degree incidence, with a bandpass of 5 nm. Absorption spectra were recorded with a Cary Varian 50 spectrophotometer using Quartz Suprasil cuvettes with optical path of 1 cm at normal incidence with a band pass of 0.5 nm. Ethanol (EtOH) and ultrapure water (H 2 O) were used as solvents.

ROS Detection
ROS production was evaluated for the following conditions: CTRL, CTRL DMSO, C2C12 treated with formulation FLAVO α, FLAVO β, FLAVO γ, FLAVOmega α, FLAVOmega β and FLAVOmega γ. CTRL, CTRL DMSO and FLAVOmega β were also tested in conditions of high oxidative stress, via addition of H 2 O 2 (250 µM) to the medium. A total of 5.000 cells/well (n = 3 wells per condition) were seeded into a 96-well plate, as described for MTT test. The analysis was performed at 8-24-48-72-96 h after treatment by means of ROS-Glo™ H 2 O 2 Assay (Promega Corporation, Madison, WI, USA) lytic procedure, following manufacturer's instructions. Relative luminescence units were measured by GloMax Discover System (Promega Corporation, Madison, WI, USA) at 570 nm.

Fusion Index and Desmin Immunofluorescence Staining
C2C12 cells at passage 5 were seeded at a density of 20.000 cells/well (n = 3 wells per condition) and grown in DMEM supplemented with 15% Fetal Bovine Serum (FBS) and Penicillin/Streptomycin. Horse serum (HS) (2%) is normally added to the growth medium when a confluence of 80% is reached, in order to induce differentiation into myotubes. Three growth conditions were then set: culture in DMEM with 15% FBS (growth medium), in DMEM with 15% FBS and 2% HS (diff. medium) and in DMEM with 15% FBS and 2% HS plus FLAVOmega β (diff. medium + FLAVOmega β). For FLAVOmega β administration, a 2.7 mg/µL formulation was produced and diluted 1:27 (v/v) in growth medium. Fusion index was calculated after 7 days of culture. Cells were fixed with methanol-free 4% paraformaldehyde (PFA) (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at room temperature (RT), followed by 3 washings in phosphate-buffered solution (PBS) 1X, 5 min each. Then, cells were incubated with a blocking solution consisting of 5% FBS and 2% HS in PBS 1X at RT for 30 min, followed by incubation with anti-desmin primary antibody (1:100, ab15200, Abcam, Cambridge, UK) diluted in blocking solution for 90 min at RT. After 3 PBS 1X washes of 5 min, cells were incubated with goat antirabbit 488 secondary antibody diluted 1:100 in PBS 1X for 1 h. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) for 5 min at RT. Slides were mounted with PBS 1X-Glycerol (Sigma-Aldrich, St. Louis, MO, USA) at a 1:1 ratio and with coverslips. A Leica DMi8 fluorescence microscope was used for acquiring the images. Fusion index was calculated as the ratio between the number of nuclei located on desmin-positive signal and the total number of nuclei per section.

Assessing Functional Performance
Functional performance of mdx mice was evaluated by Rotarod running test to measure balance and motor coordination; treadmill test was performed to evaluate fatigue resistance.

Histological Analysis
Muscles and hearts were collected from mdx mice treated with 100 µL FLAVOmega β and untreated mice, frozen in liquid hydrogen-cooled isopentane, and cut by cryostat into 8 µm sections. Azan Mallory (AM) and Hematoxylin and Eosin (H&E) staining were performed as described in [19].

Immunofluorescence Analysis
Sections were brought at RT from −80 • C and fixed with 4% PFA (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at RT, followed by 3 washings in PBS 1X, 5 min each. PBS 1X + 0.01% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 20 min at RT was used to permeabilize tissues. Sections were then incubated with a blocking solution of 10% donkey serum in PBS 1X, for 1 h at RT. Sections were then incubated overnight at 4 • C with rat anti-mouse CD31 (1:50, Clone MEC 13.3, BD PharMingen, Franklin Lakes, NJ, USA) primary antibody, diluted in blocking solution (1:50). After 3 PBS 1X washes of 5 min,

Immunohistochemistry Staining
Endogenous peroxidase activity of tissue slices was blocked with 0.3% alcoholic hydrogen peroxide for 30 min at RT. After PBS 1X washing, antigen retrieval was performed in sodium citrate buffer [10 mM] pH 6 for 30 min at 100 • C, followed by a 15 min cooling period and 3 washings with 0.05% Tween in PBS 1X for 3 min each. Sections were incubated with a blocking solution of 5% HS and 5% FBS in PBS 1X for 1 h at RT, and then incubated with PAX7 (1:50, AB92317, Abcam, Cambridge, UK) primary antibody, diluted 1:20 in blocking solution, ON at 4 • C. After PBS 1X washing, sections were incubated with appropriate biotinylated immunoglobulin antibodies for 30 min at RT, followed by incubation with peroxidase-avidin-biotin complex (Vectastain ABC Elite kit; Vector Labs, Burlingame, CA, USA) for 30 min. 3,30-Diaminobenzidine (DAB) was used as the chromogen. Eosin 1% diluted 1:50 in dH 2 O solution was used to stain myofibers for 30 s. Dehydration was performed by immersing slides into increasing ethanol dilutions in water (Carlo Erba, Milan, Italy), for 45-60 s. 100% xylene (Sigma-Aldrich, St. Louis, MO, USA) and DPX (VWR International, Radnor, PA, USA) were used to mount slides with coverslips. Acquisition of section images was carried out using a laser microdissection microscope (LMD6000B, Leica Microsystems, Wetzlar, Germany).

Western Blot Analysis
Skeletal muscles and cardiac tissues were isolated from treated and untreated 3m mdx mice and total proteins were obtained as in [20]. Briefly, tibialis anterior muscles and hearts were homogenized and resuspended with NP40 buffer with Phostop and Complete protease inhibitors 10X. After 30 min at 4 • C, lysates were centrifuged at 13.000 rpm for 10 min. Supernatants were stored at −80 • C and the pellets discarded. Protein concentration was determined by Bradford Assay absorbance with a Promega™ GloMax ® system and software. For Western blot analysis, samples were resolved on polyacrylamide gels (ranging from 6% to 14%) and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). Filters were incubated overnight with the following antibodies: LC3B

Image Quantification and Statistical Analysis
Quantitative analyses of H&E, AM, immunochemistry and immunofluorescence staining were performed by ImageJ Software (NIH). A threshold color plug-in of ImageJ Software was used to quantify AM staining as percentage of area over a fixed grid area. Data were analyzed using GraphPad Prism TM and expressed as means ±SD. To compare multiple group's means, one-way ANOVA and two-way ANOVA followed by Tukey's multiple comparison test were used to determine significance (* p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001). To compare two groups, Student's t-test was applied assuming equal variances (* p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001).

Results
We synthetized six different formulations of our mixture (FLAVO α, FLAVO β, FLAVO γ, FLAVOmega α, FLAVOmega β, FLAVOmega γ) and performed preliminary assays on C2C12 cell cultures to evaluate their effects on proliferation and antioxidant activity, and to select the formulation with the best dose-response trait. MTT-based cell proliferation assay showed slightly increased absorbance through time, but the viability was not significantly modified ( Figure 1A). ROS detection showed a decrease of reactive oxygen species production in cell culture medium after treatment. In particular, we observed a significant decrease of ROS production in C2C12 cells treated with FLAVOmega β compared to CTRL DMSO (p = 0.0040) at 72 h after treatment, and in C2C12 treated with FLAVOmega β compared to CTRL (p = 0.0048) and CTRL DMSO (p = 0.0046) at 96 h ( Figure 1B). It should be noted that the overall decrease in ROS production observed at 48 h compared to 24 h, for all experimental groups, could be attributed to the adaptation of the cell population to the culture conditions following splitting [21]. Based on these preliminary results, we selected FLAVOmega β for further evaluations. compared to 24h, for all experimental groups, could be attributed to the adaptation of the cell population to the culture conditions following splitting [21]. Based on these preliminary results, we selected FLAVOmega β for further evaluations.

Physical Properties of FLAVOmega β
Macroscopically, FLAVOmega β appears as a clear, yellow-green solution, compared to the vehicle solution, which is colorless ( Figure 1C). The absorption spectrum of the mixture shows a peak at approximately 425nm, which corresponds to the one of curcumin [22]. Dynamic Light Scattering (DLS) analysis demonstrates that the synthesized mixture is a homogenous emulsion composed of particles with an average size of 10nm. To assess the stability of FLAVOmega β we recorded the time-resolved PL spectra at 0, 6, 24, 48 and 72 h and observed no signs of particle degradation for the whole duration of the analysis, as demonstrated by the unchanged emission decay at different time points ( Figure 1D).

FLAVOmega β Increases Proliferation and Myogenicity of C2C12 Myoblasts
MTT-based cell proliferation and ROS detection assays were repeated in conditions of high oxidative stress for CTRL, CTRL DMSO and FLAVOmega β groups. In MTT experiment we observed increased cell proliferation in C2C12 treated with FLAVOmega β

Physical Properties of FLAVOmega β
Macroscopically, FLAVOmega β appears as a clear, yellow-green solution, compared to the vehicle solution, which is colorless ( Figure 1C). The absorption spectrum of the mixture shows a peak at approximately 425 nm, which corresponds to the one of curcumin [22]. Dynamic Light Scattering (DLS) analysis demonstrates that the synthesized mixture is a homogenous emulsion composed of particles with an average size of 10 nm. To assess the stability of FLAVOmega β we recorded the time-resolved PL spectra at 0, 6, 24, 48 and 72 h and observed no signs of particle degradation for the whole duration of the analysis, as demonstrated by the unchanged emission decay at different time points ( Figure 1D).

FLAVOmega β Increases Proliferation and Myogenicity of C2C12 Myoblasts
MTT-based cell proliferation and ROS detection assays were repeated in conditions of high oxidative stress for CTRL, CTRL DMSO and FLAVOmega β groups. In MTT experiment we observed increased cell proliferation in C2C12 treated with FLAVOmega β (p = 0.0288) compared to CTRL at 72 h after treatment (Figure 2A). Evaluation of ROS production in cell culture media showed an important effect of FLAVOmega β starting from 24 h after treatment; in particular, we observed a ROS decrease in C2C12 treated with FLAVOmega β compared to CTRL DMSO at 24 h (p = 0.0273). In addition, we observed a ROS production decrease in C2C12 treated with FLAVOmega β compared to CTRL at 48 h (p = 0.0450) and at 72 h (p = 0.0460) after treatment ( Figure 2B). To assess whether FLAVOmega β can actually influence muscle homeostasis, we evaluated the differentiation capacity of C2C12 cells after FLAVOmega β treatment. In presence of FLAVOmega β, C2C12 cells showed a higher myogenic capacity, as confirmed by the increased number of desmin positive myotubes, compared to control C2C12 cultured in differentiation medium (p < 0.0001) ( Figure 2C).
(p = 0.0288) compared to CTRL at 72 h after treatment (Figure 2A). Evaluation of ROS production in cell culture media showed an important effect of FLAVOmega β starting from 24 h after treatment; in particular, we observed a ROS decrease in C2C12 treated with FLAVOmega β compared to CTRL DMSO at 24 h (p = 0.0273). In addition, we observed a ROS production decrease in C2C12 treated with FLAVOmega β compared to CTRL at 48 h (p = 0.0450) and at 72 h (p = 0.0460) after treatment ( Figure 2B). To assess whether FLAVOmega β can actually influence muscle homeostasis, we evaluated the differentiation capacity of C2C12 cells after FLAVOmega β treatment. In presence of FLA-VOmega β, C2C12 cells showed a higher myogenic capacity, as confirmed by the increased number of desmin positive myotubes, compared to control C2C12 cultured in differentiation medium (p < 0.0001) ( Figure 2C).

Treatment with FLAVOmega β Improves Skeletal Muscle Performance and Dystrophic Phenotype
We performed histological analysis in 3-month-old dystrophic mice treated with FLAVOmega β, compared to age-matched mdx, to evaluate fiber morphology and fibrotic deposition. Three-month-old dystrophic mice treated with FLAVOmega β displayed an increased myofiber area (p < 0.0001) compared to age-matched untreated mdx mice. The values of frequency distribution confirmed large area of myofibers in mice that received FLAVOmega β administration (25% Percentile 3m mdx: 1488, 8285; 25% Percentile 3m mdx + FLAVOmega β: 1305, 71,775. 75% Percentile 3m mdx: 3002, 51,825; 75% Percentile 3m mdx + FLAVOmega β 3606, 8955), displaying a considerable presence of myofibers larger than 3000 µm 2 ( Figure 3A). Moreover, AM staining of skeletal muscles showed a downregulation of fibrosis in FLAVOmega β treated mdx mice (p < 0.0001) related to untreated age-matched mdx ( Figure 3B). Reduced fibrosis deposition and large myofiber area correlated with an improved fatigue resistance, as demonstrated by treadmill outcomes, which show a reduced number of shocks (p = 0.0002) (Figure 3D), and a higher latency to fall from the rotating rod (p = 0.0001) in FLAVOmega β treated mdx mice ( Figure 3C). Based on previous studies, we expected that the amelioration of functional performances may be mediated by an augmented capillary abundance and more organized arteriole distribution [23]. Immunofluorescence staining of FLAVOmega β treated and untreated mdx muscles showed increased percentage of CD31+ cells (p < 0.0001) and augmented number of α-SMA+ vessels in 3-month-old mdx mice treated with FLAVOmega β (p < 0.0001) compared to age-matched mdx control ( Figure 4A). Western blot analysis showed an increased expression of eNOS protein in 3-month-old mdx mice treated with FLAVOmega β (p = 0.0282) compared to age-matched mdx control ( Figure 4B). Moreover, FLAVOmega β treatment induced a myofiber shift towards oxidative type, likely resulting in functional improvement. Immunofluorescence staining showed an increased expression of type IIa fibers (p < 0.0001) and a downregulation of type IIb, IIx and I fibers (IIb: p < 0.0001; IIx: p = 0.0252; I: p = 0.0134) in 3-month-old mdx mice treated with FLAVOmega β (p < 0.0001) compared to age-matched mdx control ( Figure 4C). downregulation of fibrosis in FLAVOmega β treated mdx mice (p < 0.0001) related to untreated age-matched mdx ( Figure 3B). Reduced fibrosis deposition and large myofiber area correlated with an improved fatigue resistance, as demonstrated by treadmill outcomes, which show a reduced number of shocks (p = 0.0002) (Figure 3D), and a higher latency to fall from the rotating rod (p = 0.0001) in FLAVOmega β treated mdx mice ( Figure  3C). Based on previous studies, we expected that the amelioration of functional performances may be mediated by an augmented capillary abundance and more organized arteriole distribution [23]. Immunofluorescence staining of FLAVOmega β treated and untreated mdx muscles showed increased percentage of CD31+ cells (p < 0.0001) and augmented number of α-SMA+ vessels in 3-month-old mdx mice treated with FLAVOmega β (p < 0.0001) compared to age-matched mdx control ( Figure 4A). Western blot analysis showed an increased expression of eNOS protein in 3-month-old mdx mice treated with FLAVOmega β (p = 0.0282) compared to age-matched mdx control ( Figure 4B). Moreover, FLAVOmega β treatment induced a myofiber shift towards oxidative type, likely resulting in functional improvement. Immunofluorescence staining showed an increased expression of type IIa fibers (p < 0.0001) and a downregulation of type IIb, IIx and I fibers (IIb: p < 0.0001; IIx: p = 0.0252; I: p = 0.0134) in 3-month-old mdx mice treated with FLA-VOmega β (p < 0.0001) compared to age-matched mdx control ( Figure 4C). TA of mdx mice treated with FLAVOmega β and untreated mdx mice. Boxes indicate 25th to 75th percentiles. t-test: **** p < 0.0001. (B) Representative AM staining images of mdx mice treated with FLAVOmega β and untreated mdx mice skeletal muscles (n = 3 mice; n = 186 slices/3m mdx, n = 109 slices/3m mdx + FLAVOmega β. Histogram represents the percentage of fibrotic area per section of mdx mice. Scale bar: 200 μm. t-test: *** p < 0.0001. (C,D) Evaluation of coordination, balance and fatigue resistance with the Rotarod running test and the treadmill exercise of mdx mice treated with FLAVOmega β and untreated mdx mice (n = 6 mice/3m mdx, n = 6 mice/3m mdx + FLAVOmega β). (C) Histogram represents the average latency of the 3-month-old mdx mice treated with FLAVOmega β and age-matched mdx controls. t-test: *** p = 0.0001. (D) Histogram represents the number of shocks suffered by 3-month-old mdx mice treated with FLAVOmega β and age-matched mdx controls. t-test: *** p = 0.0002. All values are expressed as the mean ± SD.

FLAVOmega β Treatment Positively Influences Muscle Regenerative Processes
Based on histological evidence, we investigated some of the proteins involved in muscle regeneration. In Western blot analysis, we found a sustained expression of PAX7 and MyoD1 protein in 3-month-old mdx mice treated with FLAVOmega β (PAX7: p = 0.0050; MyoD1: p = 0.0002) compared to age-matched mdx mice ( Figure 5A). Immunoreactivity of PAX7 staining was also quantified, confirming increased number of PAX7 immunoreactive cells in FLAVOmega β treated mice (p < 0.0001) compared to the untreated group ( Figure 5B). ± SD.

FLAVOmega β Treatment Positively Influences Muscle Regenerative Processes
Based on histological evidence, we investigated some of the proteins involved in muscle regeneration. In Western blot analysis, we found a sustained expression of PAX7 and MyoD1 protein in 3-month-old mdx mice treated with FLAVOmega β (PAX7: p = 0.0050; MyoD1: p = 0.0002) compared to age-matched mdx mice ( Figure 5A). Immunoreactivity of PAX7 staining was also quantified, confirming increased number of PAX7 immunoreactive cells in FLAVOmega β treated mice (p < 0.0001) compared to the untreated group ( Figure 5B).

Modulation of Metabolism and Downregulation of Inflammatory Pathways in Mdx Mice Treated with FLAVOmega β
We performed Western blot analysis to investigate metabolism processes and inflammatory pathways. We observed a slight, but not significant, increase of PGC1-α expression, which plays a central role in the regulation of cellular energy metabolism, stimulating mitochondrial biogenesis [24]. Moreover, the analysis confirmed an increase of the OXPHOS complexes in muscles from mdx mice treated with FLAVOmega β, compared with agematched untreated mice, in line with the abundancy of more oxidative fibers, enriched in mitochondria and capillaries. We detected increased levels of CV-ATP5A (p = 0.0007), CIII-UQCRC2 (p = 0.0100), CII-SDHB (p = 0.0069), CI-NDUFB8 (p = 0.0004) after FLAVOmega β treatment ( Figure 6A), which also modulated the inflammatory process as demonstrated by the downregulation of IL-6 (p = 0.0003) and TLR2 (p < 0.0001) in mdx mice treated with FLAVOmega β compared to mdx mice control. Similarly, Western blot analysis on S100-β and RAGE proteins showed a slightly decreased protein level in treated mdx mice compared to the control group ( Figure 6B). Finally, we investigated autophagic protein expression, but did not find any relevant modulation of P62, LC3B, and ATG7 proteins after FLAVOmega β treatment ( Figure 6C). sion, which plays a central role in the regulation of cellular energy metabolism, stimulating mitochondrial biogenesis [24]. Moreover, the analysis confirmed an increase of the OXPHOS complexes in muscles from mdx mice treated with FLAVOmega β, compared with age-matched untreated mice, in line with the abundancy of more oxidative fibers, enriched in mitochondria and capillaries. We detected increased levels of CV-ATP5A (p = 0.0007), CIII-UQCRC2 (p = 0.0100), CII-SDHB (p = 0.0069), CI-NDUFB8 (p = 0.0004) after FLAVOmega β treatment ( Figure 6A), which also modulated the inflammatory process as demonstrated by the downregulation of IL-6 (p = 0.0003) and TLR2 (p < 0.0001) in mdx mice treated with FLAVOmega β compared to mdx mice control. Similarly, Western blot analysis on S100-β and RAGE proteins showed a slightly decreased protein level in treated mdx mice compared to the control group ( Figure 6B). Finally, we investigated autophagic protein expression, but did not find any relevant modulation of P62, LC3B, and ATG7 proteins after FLAVOmega β treatment ( Figure 6C). Figure 6. (A) Evaluation of OXPHOS complex and PGC1-α expression in skeletal muscles of mdx mice treated with FLA-VOmega β and untreated mdx mice (n = 3 mice/each group). Representative blots of CV-ATP5A, CIII-UQCRC2, CII-SDHB, CI-NDUFB and PGC1-α. 8. In the lateral panel, densitometric analysis of data, expressed as the ratio of the different proteins on vinculin in arbitrary units. t-test: *** p = 0.0007, *** p = 0.0004, ** p = 0.0069, * p = 0.0100 (B) Evaluation of inflammatory markers in skeletal muscles of mdx mice treated with FLAVOmega β and untreated mdx mice (n = 3 mice/each group). Representative blots of IL-6, TLR2, S100 β and RAGE. In the lateral panel, densitometric analysis of data, expressed as the ratio of the different proteins on vinculin in arbitrary units. t-test: **** p < 0.0001, *** p = 0.0003 (C) Figure 6. (A) Evaluation of OXPHOS complex and PGC1-α expression in skeletal muscles of mdx mice treated with FLAVOmega β and untreated mdx mice (n = 3 mice/each group). Representative blots of CV-ATP5A, CIII-UQCRC2, CII-SDHB, CI-NDUFB and PGC1-α. 8. In the lateral panel, densitometric analysis of data, expressed as the ratio of the different proteins on vinculin in arbitrary units. t-test: *** p = 0.0007, *** p = 0.0004, ** p = 0.0069, * p = 0.0100 (B) Evaluation of inflammatory markers in skeletal muscles of mdx mice treated with FLAVOmega β and untreated mdx mice (n = 3 mice/each group). Representative blots of IL-6, TLR2, S100 β and RAGE. In the lateral panel, densitometric analysis of data, expressed as the ratio of the different proteins on vinculin in arbitrary units. t-test: **** p < 0.0001, *** p = 0.0003 (C) Evaluation of autophagic mediators in skeletal muscles of mdx mice treated with FLAVOmega β and untreated mdx mice (n = 3 mice/each group). Representative blots of P62, ATG7 and LC3B. In the lateral panel, densitometric analysis of data, expressed as the ratio of the different proteins on vinculin in arbitrary units. All values are expressed as mean ± SD.

Modulation of Dilated Cardiomyopathy Onset in Mdx Mice after FLAVOmega β Administration
We evaluated cardiac morphology and fibrotic process through histological analysis. As observed in skeletal muscles, AM staining showed a downregulation of fibrosis in 3-month-old mdx mice treated with FLAVOmega β (p = 0.0006) related to untreated agematched mdx ( Figure 7A) also in cardiac tissues. Vascularization was also modulated after FLAVOmega β treatment. Western blot analysis showed an increased expression of eNOS in cardiac tissue of mdx mice treated with FLAVOmega β (p = 0.0136) compared to age-matched mdx control ( Figure 7C), with an increased number of CD31+ positive cells in treated mdx mice (p < 0.0001) ( Figure 7D). Through Western blot analysis, we found an upregulation of MMP9 in 3-month-old mdx mice treated with FLAVOmega β (p = 0.0207) related to untreated age-matched mdx mice ( Figure 8A). MMP-9 enzymatically cleaves numerous extracellular matrix substrates and cytokines/chemokines including TNFα and pSMAD2-3 to facilitate cardiac remodeling [25]. Thus, MMP-9 is capable of propagating cardiac inflammatory signaling that is both necessary for cardiac wound healing and potentially deleterious in sustenance of chronic inflammation. Among cardiac inflammatory mediators, we previously described the role of alarmins in prolonging inflammation in DMD cardiomyopathy influencing the expression of PTX3. One possible mechanism through which MMP-9 may blunt cardiac inflammation could be by reducing S100-β alarmin and PTX3 protein levels. In FLAVOmega β treated muscles, we observed a low expression of TNF-α and pSMAD2-3 and the downregulation of S100-β alarmin and PTX3 expression, usually highly expressed in dystrophic cardiomyopathy [26], in 3-monthold mdx mice treated with FLAVOmega β (S100-β: p = 0.0214; PTX3: p = 0.0222) compared to age-matched mdx control ( Figure 8B). All these data suggest an anti-inflammatory effect of FLAVOmega β in dystrophic cardiac remodeling. Autophagy is a process essential for heart maintenance and adaptation, and it is usually highly sensitive to cardiac stress and failure. Although proteins associated to autophagic flux did not show major changes, FLAVOmega β treated mdx mice displayed slight P62 deposition and a considerable LC3B flux, that may suggest a modulation of the autophagic machinery ( Figure 8C).

Discussion
Food supplements are concentrated sources of nutrients or other substances with a physiological effect to support the normal diet [27]. All types of supplements have the role of balancing the physiological conditions and many of these can be used as adjuvants of drug therapies. DMD patients, lacking dystrophin expression, suffer from muscle weakness, loss of ambulation, cardio-respiratory complications, metabolic and gastrointestinal problems [28]. The molecular phenotype in dystrophic patients consist in persistent disturbance of muscle homeostasis, myofiber necrosis, fibrosis deposition, influx of inflammatory cells, and severe mitochondrial dysfunction [29]. Indeed, nutraceutical therapies targeting inflammation and oxidative stress may be helpful to counteract, or at least delay, DMD pathology by preserving physiological muscle conditions and functional perfor-

Discussion
Food supplements are concentrated sources of nutrients or other substances with a physiological effect to support the normal diet [27]. All types of supplements have the role of balancing the physiological conditions and many of these can be used as adjuvants of drug therapies. DMD patients, lacking dystrophin expression, suffer from muscle weakness, loss of ambulation, cardio-respiratory complications, metabolic and gastrointestinal problems [28]. The molecular phenotype in dystrophic patients consist in persistent disturbance of muscle homeostasis, myofiber necrosis, fibrosis deposition, influx of inflammatory cells, and severe mitochondrial dysfunction [29]. Indeed, nutraceutical therapies targeting inflammation and oxidative stress may be helpful to counteract, or at least delay, DMD pathology by preserving physiological muscle conditions and functional performance for longer time. Our group and others have identified the beneficial role of flavonoids and omega-3 fatty acids in dystrophic skeletal muscle metabolism [7]. However, the molecular mechanisms that mediate effects of these compounds in dystrophic muscle are not well known. In the present study, we identified a new food supplement formulation named FLAVOmega β, which induces a generalized improvement of the dystrophic phenotype by modulating inflammation and oxidative stress. Our preliminary in vitro studies demonstrated that FLAVOmega β is able to promote myogenic differentiation of C2C12 cells and exert a moderate ROS scavenging activity in condition of normal and high oxidative stress, in absence of evident cytotoxic effects. Oxidative stress has a relevant role in the occurrence of the dystrophic phenotype and causes free-radical induced necrosis in myoblasts, therefore interfering with their subsequent differentiation toward mature and functional fibers [30]. To evaluate the effects of the supplement in vivo, we treated mdx mice of 3 months of age, daily, for one month-at this stage of life the dystrophic features become pronounced with occurrence of necrotic or regenerating myofibers, infiltrates of inflammatory cells and high levels of pro-inflammatory cytokines. Overall, muscle cross sectional area (CSA) significantly increased in FLAVOmega β treated mice, suggesting a successful progression of regenerating fibers towards mature stages and the protection from atrophic conditions with maintenance of muscle mass [31]. Injured fibers in FLAVOmega β treated muscles were replaced by a sustained muscle regeneration that may depend on a declined rate of degenerative/regenerative myofiber cycles [32].
In fact, we showed an increased MyoD1 and PAX7 expression and higher number of satellite cells compared to untreated mdx mice, indicating a less detrimental muscle environment for myogenic cell proliferation and proper differentiation after injury.
Moreover, muscles of FLAVOmega β-treated mice showed few cells infiltrate areas, thin interstitial connective tissue between fibers, and restricted fibrosis, suggesting a modulation of the fibrotic and inflammatory trend typical of the morphological patterns of dystrophic muscles [33]. We also found a significant downregulation of IL-6 and TLR-2 [34]. These results confirmed the expected anti-inflammatory properties of the components of our mixture and explained the overall improvement observed in the muscular performance of treated mice. Indeed, the above-mentioned histological findings were accompanied by an improved resistance to fatigue, balance and motor coordination, as demonstrated by Rotarod and treadmill analyses.
The ameliorated exercise performance could also be explained by a tuning of the muscle metabolism toward an oxidative one. FLAVOmega β supplementation produced a higher percentage of oxidative myofibers, in particular the so called "fast oxidative" type IIa ones, that significantly exceeded the number of glycolytic type IIb fibers. Although we did not find evidence for enhanced mitochondrial biogenesis, this oxidative shift was supported by the augmented expression of mitochondrial oxidative phosphorylation complexes (OXPHOS) in muscle of treated mice [35][36][37]. Flavonoids and omega-3 fatty acids might impact oxidative metabolism by increasing mitochondrial activity and metabolic gene expression through activation of lipid-sensing transcription factors [38][39][40]. Improved oxidative potential likely relies on the rescue of mitochondrial dysfunctions, which originates since the early onset of DMD pathology, worsens with pathology progression [40], and is accompanied by ROS and inflammatory cytokines production. Interestingly, we also demonstrated abundance of CD31+ cells in FLAVOmega β-treated muscles with upregulation of eNOS, which is an enzyme with protective role on the vasculature branches and tone [41]. These findings correlate with a remodeling of the vascular apparatus of the muscle that may result from an attempt to support the high metabolic demand of oxidative fibers.
Dilated cardiomyopathy is the major cause of death in patients with DMD. Previous studies demonstrated a protective effect of flavonoids and omega-3 fatty acids against heart disease complications [42,43]. Experiments conducted on cardiac tissue of mdx mice treated with FLAVOmega β showed a moderately preserved cardiac tissue structure accompanied by the lack of inflammatory infiltrate population, the decrease in fibrotic tissue and the upregulation of vascular markers. These cardiometabolic benefits might be related to improved endothelial production of eNOS, as observed in skeletal muscle [44], and to a modulation of PTX3-related pathways, which are known to be predictive of myocardial damage and fibrosis in mdx mice. However, due to its late onset (starting from 8 month of age), the effective role of FLAVOmega β in the modulation of the dilated cardiomyopathy phenotype is yet to be established via additional in vivo studies on older mice [26].
The complexity of these cardiac and muscular metabolic responses highlights the rapid advances in nutritional science and the continued need to generate robust empirical evidence on the mechanistic and clinical effects of specific foods. Although the exact mechanisms that govern the broad metabolic effects of flavonoids and omega-3 fatty acids in dystrophic muscles are not yet understood, our results provide new insights into the health benefits brought by these supplements to contribute in the maintenance of muscle function in DMD and constitute a preliminary indication for translation to clinical setting. Funding: This research was funded by Novystem s.r.l. This work presents independent research funded by Ricerca corrente FR230 Policlinico Hospital and Associazione Centro Dino Ferrari. Funders of the study had no role in the study design, data analysis, data interpretation, or writing of the report.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in the article.