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. 2023 Nov 29;71(49):19705–19716. doi: 10.1021/acs.jafc.3c04828

Diosmin Promotes Myogenesis via Activating the Akt/FOXO1 Pathway to Facilitate the Proliferation of C2C12 Myoblasts

Dingding Zhang 1, Xuan Zhang 1, Zhaojun Liu 1, Xiangfei Ma 1, Hongmin Li 1, Ming Shen 1, Jie Chen 1,*, Honglin Liu 1,*
PMCID: PMC10723065  PMID: 38029323

Abstract

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Our previous study with artificial intelligence (AI)-assisted screening found that diosmin, a natural flavonoid extracted from citrus, may affect myoblast proliferation and differentiation. At present, few studies have been conducted regarding the biological function of diosmin in muscle cells. Here, using molecular biological techniques, we found that diosmin elevated the proliferation ability of C2C12 myoblasts via activating the Akt/FOXO1 pathway to promote FOXO1 nuclear export, thus repressing p27 protein expression, increasing CDK2, CDK4, and cyclin D1 and cyclin E1 protein expression and accelerating cell cycle transformation, which contributed to myogenesis. Moreover, diosmin suppressed differentiation of C2C12 myoblasts by delaying the terminal exit of the cell cycle in early differentiated myoblasts and inhibiting autophagic flux in mature myotubes. Furthermore, diosmin promoted myogenesis by activating the Akt/FOXO1 pathway to facilitate myoblast proliferation, which had a positive biological effect on the repair of muscle injury. This study revealed the effect and mechanism of diosmin on skeletal muscle cells and simultaneously provided a new candidate drug for the treatment of myopathy.

Keywords: diosmin, myoblast proliferation, myoblast differentiation, Akt/FOXO1 pathway, muscle injury repair

Introduction

The skeletal muscle accounts for about 40% of body weight and is the main source of meat production for livestock and poultry.1 Studies have shown that the skeletal muscle has functions such as thermogenesis,2 exercise maintenance,3 immunity,4 body metabolism, and homeostasis maintenance.5 The proliferation and differentiation of skeletal muscle cells play a key role in muscle growth and development, muscle injury repair, and myopathy treatment.6 In our previous study, we identified diosmin as a candidate compound that may have a potential influence on myoblast proliferation and differentiation through AI-assisted screening.

Diosmin is a natural flavonoid compound derived from various citrus fruits with the chemical formula C28H32O15 and a molecular weight of 608.55.7 It is reported that diosmin has a wide range of biological functions such as anti-inflammatory,8 antioxidation,9 and antihyperglycemia,10 and the combination of diosmin and hesperidin effectively treats chronic venous insufficiency.11 However, the biological effects of diosmin on muscle cells remain unclear.

Skeletal muscle development is a highly ordered and complex biological process.12 Before birth, myoblasts are differentiated from the myotome and proliferate rapidly. Then, myoblasts irreversibly exit the cell cycle and express myoblast-specific genes, thus achieving myoblast differentiation, then fuse into myotubes, and finally develop into mature muscle fibers.6,13,14 Research reported that myoblast proliferation and differentiation are antagonistic events, and obstruction of the cell cycle exit inhibits myoblast differentiation.15,16 It is well-known that the proliferation and differentiation of myoblasts are regulated by various factors, such as the MAPK pathway, Akt/FOXO1 pathway, autophagy, and apoptosis.17−19 Studies have indicated that diosmin participates in the regulation of the cell cycle,20−22 induces autophagy in breast cancer cells, and inhibits autophagy flow in glioblastoma.23,24 However, it is unclear whether diosmin affects myoblast proliferation and differentiation.

Therefore, in this study, we used C2C12 myoblasts to investigate the effect and mechanism of diosmin on the proliferation and differentiation of myoblasts and explore the effect of diosmin on muscle injury repair using a muscle injury model. Our results demonstrated that diosmin induced nuclear export of forkhead box O1 (FOXO1) by activating the Akt/FOXO1 pathway, affected protein expression of cell cycle-related proteins cyclin-dependent kinase inhibitor 1B (p27), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin D1, and cyclin E1, and accelerated the cell cycle transition. Thus, this enhanced the proliferative ability of myoblasts, and diosmin repressed myoblast differentiation by delaying the end cell cycle exit and blocking autophagy flux. Moreover, diosmin simultaneously played a positive role in muscle injury repair by activating the Akt/FOXO1 pathway to promote myoblast proliferation.

Materials and Methods

Ethics Statement

In this study, all experiments and treatments were approved by the Animal Research Institute Committee of Nanjing Agricultural University, China (permit number IACUC2020132).

Cell Culture and Treatment

The C2C12 mouse myoblast cell line was purchased from the Stem Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured in growth media (GM), which contained high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Logan, UT, USA) added with 1% penicillin–streptomycin and 10% fetal bovine serum (Millipore, Sigma-Aldrich, Billerica, MA, USA) and was set at 37 °C in a 5% CO2 incubator. When the myoblast confluence reached 90%, the culture medium was replaced with high-glucose DMEM containing 2% horse serum and 1% penicillin–streptomycin for induction differentiation.

When the myoblast confluence reached 50–60%, diosmin (Selleck, Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) and finally diluted into GM at different concentrations (0, 5, 10, 50, and 100 μM) to culture C2C12 myoblasts for 24 h. When the C2C12 myoblast confluence reached 50–60%, 10 μM MK2206 (Selleck, Shanghai, China) pretreatment was performed for 2 h, and then, 50 μM diosmin treatment was performed for 24 h. When the C2C12 myoblast confluence reached 90%, it was induced to differentiate for 0, 2, 3, 4, 5, 6, and 8 days in a differentiation medium (DM) containing 0, 5, 10, and 50 μM diosmin. C2C12 myoblasts were induced to differentiate for 3 days in the DM containing 50 μM diosmin, 50 μM chloroquine (CQ) (Selleck, Shanghai, China), and 50 μM diosmin + 50 μM CQ. C2C12 myoblasts were treated with 0 and 50 μM diosmin in GM for 24 h and then treated with 0 and 50 μM diosmin in DM for 12 h. C2C12 myoblasts with a fusion degree of 50% were cultured with GM containing 50 μM diosmin for 24 h and then differentiated normally for 5 days.

CCK-8 Assay

Myoblasts were treated with different concentrations of diosmin for 24 h in GM and then incubated with a mix of cell counting kit-8 (CCK-8, Tongren, Japan) and GM (1:10) for 2 h in 37 °C according to the instructions of the CCK-8 kit and finally measured by reading the optical density at 450 nm under a microplate spectrophotometer (Tecan, Männedorf, Switzerland).

Cell Proliferation Assay

Twenty-four h after 50 μM diosmin treatment, C2C12 myoblasts were incubated in fresh GM containing 50 mM 5-ethynyl-29-deoxyuridine (EdU) obtained from a Cell-Light EdU Apollo567 in vitro kit (RiboBio, Guangzhou, China) for 2 h and then stained with the cells according to the instructions; finally, cells were observed using a confocal microscope (LSM700ME-TA; Zeiss, Oberkochen, Germany).

Cell Cycle Analysis

At 24 h after 50 μM diosmin treatment, C2C12 myoblasts were digested with trypsin, washed once with phosphate-balanced solution (PBS), and then fixed overnight with 70% ethanol. The 70% ethanol was discarded and washed once with PBS. Then, 500 μL of a propidium iodide mixture (RNase A:propidium iodide = 1:9) was added to each sample for 30 min followed by evaluation of the samples by FACSCalibur flow cytometry (Becton Dickinson, San Diego, CA, USA) and finally analysis of the samples using ModFit32 software (Verity Software House, Topsham, ME, USA).

RNA Extraction and Real-Time Quantitative PCR

The total RNA of cells was extracted with a Trizol reagent according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). cDNA for mRNA was synthesized with a PrimeScript RT Master Mix (Takara, Kyoto, Japan). qRT-PCR was carried out according to the instructions of a qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). Each reaction was repeated three times. All primers were synthesized by Qingke et al. (Nanjing, China). The sequences of the primers are listed in Table S1.

Protein Extraction and Western Blot Analysis

The total protein of the myoblasts was extracted using a radioimmunoprecipitation assay buffer (Applygen, Beijing, China). The protein concentration was determined by a BCA protein assay kit (Beyotime, Shanghai, China). The proteins were boiled for 15 min, and the denatured proteins were electrophoresed in 4 to 20% ExpressPlus PAGE gel (GenScript, Nanjing, China). Then, it was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Sigma-Aldrich). Subsequently, PVDF membranes were blocked with 5% bovine serum albumin (BSA, Millipore, Sigma-Aldrich) for 2 h at room temperature and then incubated with anti-Pcna (dilution 1:1000, no. 13110, Cell Signaling Technology, Danvers, USA), anti-Akt (dilution 1:1000, A18120, ABclonal, Wuhan, China), anti-p-Akt (Ser473) (dilution 1:1000, no. 4060, Cell Signaling Technology), anti-FOXO1 (dilution 1:1000, no. 2880, Cell Signaling Technology), anti-p-FOXO1 (Ser256) (dilution 1:1000, no. 9461, Cell Signaling Technology), anti-p27 (dilution 1:1000, no. 3686, Cell Signaling Technology), anti-CDK2 (dilution 1:1000, no. 18048, Cell Signaling Technology), anti-CDK4 (dilution 1:1000, no. 23972, Cell Signaling Technology), anticyclin E1 (dilution 1:1000, no. 20808, Cell Signaling Technology), anticyclin D1 (dilution 1:1000, no. 55506, Cell Signaling Technology), anti-MyHC (MF-20) (dilution 1:200, Developmental Studies Hybrid-oma Bank), anti-MyoG (dilution 1:1000, A17427, ABclonal), anti-MyOD (dilution 1:1000, 18943-1-AP, Proteintech, Chicago, USA), anti-LC3B (dilution 1:2000, no. L7543, Sigma-Aldrich), anti-SQSTM1 (dilution1:2000, no. ab101266, Abcam, Cambridge, USA), and anti-GAPDH (dilution 1:2000, no. 5174, Cell Signaling Technology) overnight at 4 °C. The PVDF membrane was washed thoroughly with Tris-buffered saline containing Tween-20 three times, once for 10 min, and was incubated with a horseradish peroxidase-conjugated goat antirabbit IgG antibody (1:2000, no. 7074, Cell Signaling Technology) or a horseradish peroxidase-conjugated goat antimouse IgG antibody (1:2000, no. 7076, Cell Signaling Technology) for 2 h at room temperature. The PVDF membrane was developed with a Western Bright ECL chemiluminescent HRP substrate (Advansta, San Jose, CA, USA). ImageJ software was used to quantify the protein expression.

Immunofluorescence Assay

The treated C2C12 cells were washed with PBS 3 times and then fixed with paraformaldehyde at ambient temperature for 1 h. The cells were then incubated in 0.5% Triton X-100 for 10 min at 4 °C and washed with PBS 3 times. Next, cells were blocked in 1% BSA for 1 h and incubated in antibodies against FOXO1 (1:100 dilution) and MyHC (1:20 dilution) overnight at 4 °C. Afterward, cells were incubated with rhodamine (tetramethyl rhodamine isocyanate [TRITC])-conjugated goat antimouse IgG (1:100 dilution) (ZSGB-Bio) for 1 h in the dark. The nucleus was stained with 4,6-diamidino-2-phenylindole dihydrochloride for 10 min at 37 °C in the dark. Fluorescence images were captured using a Zeiss LSM 710 Meta confocal microscope.

Animal Experiments

Eight-weeks-old male ICR mice were injected with 1.2% BaCl2 in central tibialis anterior (TA) muscles to establish a muscle injury model. Then, 0, 10, and 30 mg/kg diosmin concentrations were injected intraperitoneally on the first and second days after muscle injury, and TA muscles were collected on the third and ninth days after muscle injury.

H&E Staining

After the TA muscle was fixed in 4% paraformaldehyde for 2 days, it was then made into paraffin sections according to the traditional method and then dyed with H&E. Histological observation was performed using a Euromex BioBlue microscope.

Statistical Analysis

The gene relative expression level was analyzed using the 2–ΔΔCt method.25 The data were presented as the mean value ± SEM, t-tests were used for statistical analysis of two-group data, and an ANOVA was used for data analysis of more than two groups. * means p < 0.05, ** means p < 0.01, and *** means p < 0.001. The graphs were drawn by GraphPad Prism 6.0 software (GraphPad Software, La Jolla, CA, USA).

Results

Diosmin Promotes the Proliferation of C2C12 Myoblasts

The chemical structure formula of diosmin is shown in Figure 1A. To investigate the effect of diosmin on the proliferation of C2C12 myoblasts, we used different concentrations of diosmin in GM to culture C2C12 myoblasts. CCK-8 results showed that 10, 50, and 100 μM diosmin significantly promoted the viability of C2C12 myoblasts, and there was no difference between 50 and 100 μM diosmin treatments on cell viability (Figure 1B). Western blot results showed that 50 μM diosmin markedly increased the protein expression of the proliferating cell nuclear antigen (Pcna) (Figure 1C,D). Therefore, the subsequent proliferation-related experiments were conducted with 50 μM diosmin. Flow cytometry results showed that diosmin observably decreased the percentage of cells in the G0 and G1 phases and increased the percentage of cells in the S phase (Figure 1E,F). Western blot results showed that diosmin significantly suppressed the protein expression of p27 and facilitated the protein expression of CDK2, CDK4, cyclin E1, and cyclin D1 (Figure 1G,H). The results of the EdU assay showed that diosmin remarkably elevated the percentage of EdU-positive cells (Figure 1I,J). These results suggested that diosmin accelerated the transition of the G1/S phase, thereby expediting C2C12 cell cycle progression and ultimately enhancing the proliferation of C2C12 myoblasts.

Figure 1.

Figure 1

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Diosmin promotes the proliferation of C2C12 myoblasts. (A) Chemical structure of diosmin. (B) CCK-8 was used to detect the effect of different concentrations (0, 5, 10, 50, 100 μM) of diosmin exposed to C2C12 myoblasts for 24 h on cell viability (n = 6). (C,D) Western blot analysis of Pcna after diosmin was exposed to C2C12 myoblasts for 24 h (n = 3). GAPDH was used as a loading control in all Western blot experiments. (E,F) Effect of 50 μM diosmin exposure on C2C12 myoblasts for 24 h on the cell cycle (n = 3). (G,H) Western blot analysis of p27, CDK2, CDK4, cyclin E1, and cyclin D1 after 50 μM diosmin exposure to C2C12 myoblasts for 24 h (n = 3). (I,J) An EdU assay was used to detect the effect of diosmin on the proliferation of myoblasts. EdU-positive cells were stained red, and the nuclei were stained blue (n = 3). An ANOVA and t-test are used to analyze the difference of data. Data are expressed as mean values ± SEM; *p < 0.05, **0.01 < p < 0.05, ***p < 0.001.

Diosmin Activates the Akt/FOXO1 Pathway in C2C12 Myoblasts

It is well-documented that the Akt/FOXO1 pathway is involved in the regulation of the G1/S checkpoint of the cell cycle,26,27 so we examined the effect of diosmin on this pathway. Western blot results showed that diosmin significantly increased the phosphorylation of Akt at Ser 473 and FOXO1 at Ser 256, suggesting that diosmin activated the Akt/FOXO1 pathway (Figure 2A,B). Next, we treated C2C12 myoblasts with MK2206,28 an Akt inhibitor, to confirm the role of diosmin in the Akt/FOXO1 pathway. The optimal treatment conditions of MK2206 are shown in Figure S1. Western blot results showed that MK2206 markedly inhibited the Akt/FOXO1 pathway, and cotreatment of MK2206 and diosmin significantly suppressed the Akt/FOXO1 pathway compared with diosmin treatment alone (Figure 2C,D). These data manifested that the ability of diosmin to activate the Akt/FOXO1 pathway was repressed after inhibition of the Akt/FOXO1 pathway by MK2206, solidly confirming that diosmin activates the Akt/FOXO1 pathway in C2C12 myoblasts.

Figure 2.

Figure 2

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Diosmin activates the AKT/FOXO1 pathway in C2C12 myoblasts. (A,B) Western blot analysis of AKT, p-AKT (Ser473), FOXO1, and p-FOXO1 (Ser256) after 50 μM diosmin exposure to C2C12 myoblasts for 24 h (n = 3). (C,D) Western blot analysis of AKT, p-AKT (Ser473), FOXO1, and p-FOXO1 (Ser256) after 10 μM MK2206 pretreatment for 2 h and then 50 μM diosmin treatment for 24 h (n = 3). (E,F) C2C12 myoblasts were cultured in GM containing 10 μM MK2206 for 2 h, then treated with 50 μM diosmin treatment for 24 h, and finally collected to observe the subcellular localization of FOXO1 by the immunofluorescence assay. FOXO1 staining is red, and nucleus staining is blue (n = 3). Two-way ANOVA and t-tests are used to analyze the difference of data. Data are expressed as mean values ± SEM; *p < 0.05, **0.01 < p < 0.05, ***p < 0.001.

As a nuclear transcription factor, FOXO1 exerts biological activity by entering nuclei. The higher the phosphorylation level of FOXO1, the greater the proportion of nuclear export and the lower its biological activity. Conversely, the lower the phosphorylation level of FOXO1, the higher the proportion of nuclear import and the higher the biological activity.29,30 Next, we detected the subcellular localization of FOXO1. Immunofluorescence results of FOXO1 showed that diosmin significantly reduced the nuclear proportion of FOXO1, which was consistent with the result that diosmin markedly increased the level of phosphorylation of FOXO1 at Ser 256. The results of Akt inhibitor treatment showed that MK2206 notably increased the nuclear proportion of FOXO1, and the cotreatment of MK2206 and diosmin significantly increased the nuclear proportion of FOXO1 compared with diosmin alone (Figure 2E,F). These results demonstrated that diosmin affected the biological activity of FOXO1 through the Akt signal.

Diosmin Facilitates C2C12 Myoblast Proliferation via Activating the Akt/FOXO1 Pathway

It has been reported that p27 is one of the downstream target genes of FOXO1,31,32 and the protein encoded by this gene binds to and prevents the activation of the cyclin E-CDK2 or cyclin D-CDK4 complex, thus regulating the progress of the cell cycle in the G1 phase and affects the transition of the G1/S phase.33,34 To explore whether Akt/FOXO1 has a hand in the regulation of the cell cycle induced by diosmin, ///we examined the effect of Akt inhibitor treatment on the C2C12 cell cycle induced by diosmin. Western blot results showed that MK2206 significantly facilitated the protein expression of p27, diosmin observably inhibited the protein expression of p27, and cotreatment of MK2206 and diosmin significantly elevated the protein expression of p27 compared with diosmin alone. MK2206 markedly suppressed the protein expressions of CDK2, CDK4, Cyclin E1 and Cyclin D1, diosmin significantly promoted the protein expressions of CDK2, CDK4, Cyclin E1 and Cyclin D1, cotreatment of MK2206 and diosmin significantly inhibited CDK2, CDK4, Cyclin E1 and Cyclin D1 compared with diosmin alone (Figure 3A and B). Flow cytometry results showed that diosmin observably decreased the proportion of cells in the G0/G1 phase and increased the proportion of cells in the S phase, MK2206 significantly increased the proportion of cells in the G2/M phase and decreased the proportion of cells in the S phase, and the cotreatment MK2206 and diosmin markedly decreased the proportion of cells in the S phase and increased the proportion of cells in the G2/M phase compared with diosmin alone (Figure 3C-F). These results indicated that diosmin affected the activity of FOXO1 by stimulating the Akt signal, thus affecting the expression of its target gene p27, then participating in the regulation of C2C12 cell cycle.

Figure 3.

Figure 3

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Diosmin affects the proliferation of C2C12 myoblasts through the AKT/FOXO1 pathway. (A,B) Western blot analysis of p27, CDK2, CDK4, cyclin E1, and cyclin D1 after 10 μM MK2206 pretreatment for 2 h and then 50 μM diosmin treatment for 24 h (n = 3). (C–F) Effects of different treatment groups (control, MK2206, MK2206 + Diosmin, and Diosmin) on the cell cycle of C2C12 myoblasts (n = 3). (G,H) Western blot analysis of Pcna after 10 μM MK2206 pretreatment for 2 h and then 50 μM diosmin treatment for 24 h (n = 3). (I,J) An EdU assay was used to detect the effect of the control, MK2206, cotreatment of Diosmin and MK2206, and Diosmin on the proliferation of C2C12 myoblasts (n = 3). EdU-positive cells were stained red, and the nuclei were stained blue. Two-way ANOVA and t-tests are used to analyze the difference of data. Data are expressed as mean values ± SEM; *p < 0.05, **0.01 < p < 0.05, ***p < 0.001.

Then, we examined the effect of diosmin on the proliferation of C2C12 myoblasts after the Akt/FOXO1 pathway. Western blot results showed that MK2206 markedly inhibited the protein expression of Pcna, and the cotreatment of MK2206 and diosmin significantly suppressed the protein expression of Pcna compared with diosmin alone (Figure 3G and H), indicating that the ability of diosmin to facilitate the proliferation of C2C12 myoblasts was repressed when Akt/FOXO1 pathway inhibited by MK2206. In addition, the results of the EdU assay showed that MK2206 evidently reduced the percentage of EdU-positive cells, and cotreatment of MK2206 and diosmin obviously repressed the percentage of EdU-positive cells compared with diosmin treatment alone (Figure 3I and J). These results demonstrated that diosmin did heighten the proliferation ability of C2C12 myoblasts by activating the Akt/FOXO1 signaling pathway.

Diosmin Inhibits the Differentiation of C2C12 Myoblasts

Next, the effect of diosmin on the differentiation of the C2C12 myoblasts was investigated. Western blot results showed that the expression of myogenin (MyOG) and myosin heavy chain, cardiac muscle complex (MyHC) was visibly repressed when C2C12 myoblasts were induced to differentiate at different concentrations (5 μM, 10 μM, 50 μM) of diosmin for 3 and 5 days, except that MyOG protein expression was not affected by 5 μM and 50 μM diosmin at 5 days after differentiation (Figure 4A, B, D and E). The qRT-PCR results showed that the expression of MyOG mRNA was not affected after 3 days of diosmin-induced differentiation, while 10 and 50 μM diosmin significantly inhibited the expression of MyHC mRNA (Figure 4C). After 5 days of diosmin-induced differentiation, the expression of MyOG mRNA was suppressed, and 50 μM diosmin obviously inhibited the expression of MyHC mRNA (Figure 4F). It can be seen that the effect of 50 μM diosmin was the most obvious. Next, we found C2C12 myoblasts differentiation induced by 50 μM diosmin for 0, 2, 4, 6, and 8 days, the expression of MyHC was repressed at all differentiation time points except for D4 days compared to the control group, and the treatment of diosmin would not delay the peak expression of MyHC (G and H). Moreover, Immunofluorescence of MyHC results showed that 50 μM diosmin evidently inhibited the proportion of MyHC-positive cells compared with the control group (Figure 4I and J). These results indicated that diosmin suppressed the differentiation of C2C12 myoblasts.

Figure 4.

Figure 4

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Diosmin inhibits the differentiation of C2C12 myoblasts. Effect of C2C12 myoblast differentiation induced by different concentrations (0, 5, 10, and 50 μM) of diosmin for 3 days and 5 days on MyOG and MyHC protein expression (n = 3) (A–F). Effects of differentiation of C2C12 myoblasts induced by 50 μM diosmin on the expression of MyHC protein at 0, 2, 4, 6, and 8 days (n = 3) (G,H). Immunofluorescence analysis of MyHC expression after C2C12 myoblasts were treated with 50 μM diosmin for 5 days in DM (n = 3) (I,J). An ANOVA and t-test are used to analyze the difference of data. Data are expressed as mean values ± SEM; *p < 0.05, **0.01 < p < 0.05, ***p < 0.001.

Studies reported that permanent exit of the cell cycle is one of the necessary conditions for myoblasts differentiation.15,16 Next, we examined the effect of 50 μM diosmin on the cell cycle exit. Figure S2A shows a grouping model diagram. The results of flow cytometry showed that the proportion of S phase cells was significantly increased, and the proportion of G0/G1 phase cells was decreased after diosmin-treated C2C12 myoblast proliferation for 24 h and normal differentiation for 12 h compared with the control group (Figure S1B and S1C). After C2C12 myoblast normal proliferation for 24 h and then differentiation induced by diosmin for 12 h, the proportion of G0/G1 phase cells significantly reduced and the proportion of S phase cells raised compared with the control group, indicating that diosmin delayed the exit of myoblast cycle (Figure S2B and S2C). It was further found that the proliferation of C2C12 myoblasts treated with diosmin for 24 h and normal differentiation for 12 h evidently inhibited the protein expression of myogenic differentiation 1 (MyOD) and cyclin dependent kinase inhibitor 1A (p21) compared to the control group (Figure S2D and S2E). After C2C12 myoblast normal proliferation for 24 h and diosmin-induced differentiation for 12 h, the protein expression of MyOD and p21 markedly suppressed compared with the control group (Figure S2D and S2E). These data manifested that diosmin delayed myoblasts cycle exit and initiation of differentiation was difficult.

It is documented that myogenic differentiation is accompanied by autophagy.35 We found that 50 μM diosmin induced myoblast differentiation for 3 days and 5 days accelerated the transformation from microtubule-associated protein 1 light chain 3 alpha (LC3-I) to microtubule-associated protein 1 light chain 3 beta (LC3-II), and the expression of sequestosome 1 (SQSTM1) mRNA and protein significantly increased (Figure S3A- S3F), suggesting that autophagy flux block occurred during 50 μM diosmin inhibition of C2C12 myoblast differentiation. To further verify that diosmin blocked autophagy flux in the process of inhibiting the differentiation of C2C12 myoblasts. we challenged the C2C12 myoblasts using CQ, an autophagy blocker,36 which inhibits the fusion of autophagosome and lysosome. The results showed that cotreatment with CQ and diosmin enhanced the transformation from LC3 I to LC3 II and the expression of SQSTM1 compared with CQ alone (Figure S3G and S3H). In addition, the expression of MyHC protein was the lowest after cotreatment with CQ and diosmin compared with other groups (Figure S3G and S3H), confirming that diosmin inhibited C2C12 myoblast differentiation by blocking autophagy flux.

Proliferated C2C12 Myoblasts Treated with Diosmin Promote Differentiation

To explore the effect of C2C12 myoblasts on differentiation after growing in the GM with or without 50 μM diosmin for 24 h, we collected samples after differentiation for 5 days (Figure 5A). The results showed that after differentiation for 5 days, diosmin improved the MyoG and MyHC mRNA and protein expression (Figure 5B-E), and the immunofluorescence results of MyHC showed that the percentage of MyHC positive cells was increased compared with that of the control group (Figure 5H,I). In addition, the mRNA expression of myocyte fusion-related genes Myomaker and caveolin-3 elevated (Figure 5F,G). These results indicated that after diosmin promoted the proliferation of C2C12 myoblasts, the differentiation was enhanced.

Figure 5.

Figure 5

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Proliferated C2C12 myoblasts treated with diosmin promote differentiation. (A) Experimental scheme. C2C12 myoblasts were cultured with 0 or 50 μM diosmin in GM and then induced to differentiate for 5 days. Effects of cultured C2C12 myoblasts with 50 μM diosmin in GM, which were then induced to differentiate for 5 days on MyOG, MyHC mRNA, and protein (B–E), myomaker and caveolin-3 mRNA (F,G), and the percentage of MyHC-positive cells (H,I). A t-test is used to analyze the difference of data. Data are expressed as mean values ± SEM; *p < 0.05, **0.01 < p < 0.05, ***p < 0.001.

Diosmin Avails Muscle Injury Repair

To explore the effect of diosmin on muscle injury repair, a mouse model of TA muscle injury was constructed by using BaCl2. Different concentrations of diosmin were injected intraperitoneally for two consecutive days after injury, and TA muscles were collected after injury for 3 and 9 days (Figure 6A). After 3 days of injury, we found that diosmin increased the protein expression of Pcna and activated the Akt/FOXO1 pathway, which is consistent with the results in vitro (Figure 6B,C). The H&E staining results showed that diosmin elevated the number of central nuclei of newly formed muscle fibers, and the proportion of muscle fibers with large cross-sectional area (CSA) and the average CSA of muscle fibers both raised after injured for 9 days (Figure 6D-G), suggesting that diosmin had a positive effect on muscle repair injury.

Figure 6.

Figure 6

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Diosmin avails muscle injury repair. (A) Experimental scheme. After 1.2% BaCl2 was injected into the middle TA muscles, different concentrations of diosmin were injected intraperitoneally for 2 consecutive days, and TA muscles with muscle injury were collected after 3 days and 9 days of muscle injury. (B–C) Effects of muscle injury on Pcna and Akt/FOXO1 pathway after 3 days. (D) H&E staining cross-sectional morphology of TA muscles on 9 days after the muscle injury (n = 3). (E) New muscle fiber CSA of TA muscles (n = 3). (F) Percentage of CSA of different new muscle fibers (n = 3). (G) Percentage of different central nuclei in each new muscle fiber (n = 3). An ANOVA is used to analyze the difference of data. Data are expressed as mean values ± SEM * p < 0.05, ** 0.01 < p < 0.05, *** p < 0.001.

Discussion

Diosmin, a natural compound derived from citrus, was reported to have a good safety profile based on toxicological tests.37 Oral diosmin is converted to diosmetin, which is absorbed and esterified into glucuronic acid conjugates and dispersed throughout the body and eventually excreted in urine.38,39 At present, the anti-inflammatory, antioxidant and antidiabetes functions exhibited by diosmin have shown its medicinal value.8−10 Of note, diosmin also has anticancer characteristics, which trigger apoptosis and inhibit the proliferation of cancer cells. However, our results found that diosmin facilitated the proliferation of C2C12 myoblasts. We speculate that the biological effects of diosmin on various cells are quite different due to the different mechanisms of action, especially cancer cells and normal cells, as it is known that the growth environment and metabolism of cancer cells are different from normal cells. For example, in A431 skin cancer cells, the antiproliferation effect of diosmin is mainly reflected in the increase of apoptosis level and the inhibition of invasion;22 In glioblastoma, diosmin induces cell cycle arrest in G1 phase by inhibiting autophagy flux, while repressing GBM cell migration and invasion to achieve anticancer effects;24 In breast cancer cells, different concentrations of diosmin induce different p53 states and ERK activities, leading to different fates of senescence, apoptosis and autophagy, thus achieving antiproliferation effect.23 It can be seen that the mechanism of diosmin in these cancer cells is different from that in C2C12 myoblasts by activating the Akt/FOXO1 pathway to induce the FOXO1 export nucleus, inhibiting p27 expression, promoting the expression of CDK2, CDK4, Cyclin D1 and Cyclin E1, and accelerating the G1/S phase transition, so it is reasonable to cause different biological effects. In addition, we concluded many research results indicate that diosmin mostly have positive biological effects on normal cells, but have killing effects on cancer cells, which was similar to some natural plant compounds such as resveratrol, which shows anti-inflammatory, antioxidant, antiaging and cardiovascular protective effects on normal cells, but can induce the death of cancer cells.40

Many studies demonstrated that Akt/FOXO1 signaling pathway plays a crucial role in regulating cell cycle, which regulates G1/S checkpoint.41,42 Studies reported that FOXO1, as a nuclear transcription factor, is influenced by the phosphorylation at Ser256 and subcellular localization.29,30 Akt activation enhances FOXO1 phosphorylation at Ser256, which leads to FOXO1 nuclear translocation and represses the FOXO1 transcription activity. Conversely, inhibition of Akt activity leads to entry of FOXO1 into the nucleus and facilitates the transcription activity of FOXO1. Studies have shown that p27 is one of the downstream target genes of FOXO1,31,32 and it is a member of the Kip/Cip family of cell cycle-dependent kinase inhibitors (CKIs), and its inhibitory binding with CDK2/cyclin E complex strengthens G1 restriction point, thus participates in cell cycle regulation.33,34 Our study found that diosmin activated the Akt/FOXO1 pathway, promoted FOXO1 nuclear translocation, inhibited p27 protein expression, facilitated the expression of CDK2, CDK4, Cyclin E1 and Cyclin D1 protein, and expedited G1/S phase transition, thus heightening the proliferation ability of C2C12 myoblasts. MK2206-related (Akt inhibitor) experiments showed that the proliferation effect of diosmin on C2C12 myoblasts was significantly inhibited by MK2206, including FOXO1 function, cell cyclin-related proteins CDK2, CDK4, Cyclin E1, Cyclin D1, p27, and S phase cell proportion. These results further confirmed that diosmin promoted the proliferation of C2C12 myoblasts through the activation of Akt/FOXO1. However, we cannot rule out that diosmin may enhance the C2C12 myoblast proliferation through other pathways or mechanisms.

Skeletal muscle cell differentiation is a multistep biological process involving permanent cell cycle exit and activation of myogenic regulatory factors such as MyOD, Myf5, MRF4 and Myogein.6,12−14 Among them, MyOD plays an important role in triggering muscle differentiation program and inducing complete block of cell proliferation.43 It is documented that the expression of MyOD activates the expression of P21 in mouse muscle cell differentiation, and the terminal cell cycle arrest in skeletal muscle is correlated with MyOD induced p21 expression.44 Later, the researchers found that MyOD induced p21 expression by inhibiting the activity of cyclin-dependent kinase, thus achieving irreversible withdrawal from the cell cycle, and found that the induction and sustained expression of p21 appears to be a facilitating mechanism by which muscle cells irreversibly exit the cell cycle during terminal differentiation.45 In this study, we found that diosmin suppressed differentiation of C2C12 myoblasts, which conformed to the fact that myocyte proliferation and differentiation are mutually exclusive. In addition, since diosmin accelerated the cell cycle, we examined whether diosmin accelerated cell cycle exit would affect the initiation of cell differentiation. we found that it was difficult for differentiated myoblasts to exit the cell cycle after proliferation treatment with diosmin, and the expressions of MyOD and p21 were inhibited. Moreover, myoblasts induced by diosmin for 12 h had difficulty withdrawing the cell cycle, and the expressions of MyOD and p21 were suppressed. These results indicated that diosmin hinders the terminal exit of the cell cycle and it is difficult to initiate differentiation.

Cell differentiation is related to the change of cell morphology and protein removal.46 The remarkable cell morphology remodeling and protein removal are closely related to the degradation system. Bulk degradation of protein aggregates and redundant/damaged organelles can generally be achieved through ubiquitin-protease systems and autophagy.47−49 Among them, autophagy degrades impaired proteins and organelles into small molecules, then recycles them in the body to maintain homeostasis.50,51 Studies have shown that autophagy occurs in the differentiation process of skeletal muscle cells, and the level of autophagy first increases and then decreases to a normal level.52 Researchers found autophagy is necessary during the differentiation of skeletal muscle cells,35 and the inhibition of autophagy by 3-MA impairs the normal differentiation of skeletal muscle cells.17 Our results showed that diosmin increased the protein expression of LC3 II and SQSTM1 during the differentiation of C2C12 myoblasts, indicating that autophagy flux was inhibited. The results of CQ-related experiments suggested that diosmin repressed the differentiation of C2C12 myoblasts by inhibiting the autophagy flux. Studies have shown that diosmin inhibits autophagy flux in glioblastoma,24 our results were consistent with them. The autophagy flux is blocked for the following reasons: ① The production of autophagosomes increased while the degradation decreased or constant; ② The production of autophagosome is not affected, and the binding of the autophagosome and lysosome is hindered. However, it remains to be further explored which pathway diosmin inhibits autophagy flux during C2C12 myoblasts differentiation.

Satellite cells exist between the muscle membrane and the basement membrane. Normally they are in the rest state. When muscles are injured, satellite cells are activated, then they are proliferated, differentiated and fused to repair the injured muscle fibers.53,54 Studies showed that the first cell cycle of satellite cells from the rest state to the active state takes 36 to 48 h, and the subsequent myoblasts complete a cell cycle only 8 to 10 h.55,56 C2C12 myoblasts are considered as activated satellite cells, which are often used as a good experimental model in vitro. We found that after diosmin promoted C2C12 myoblasts proliferation, its differentiation was facilitated, suggesting that although diosmin affected the cell cycle exit in the initial stage of differentiation, the increase of the number of myoblasts eventually promoted myoblasts differentiation, so we speculated that diosmin might enhance the proliferation of myoblasts and promote myogenesis during fetal muscle fiber development. It can be seen from the muscle injury model that disomin promoted myoblast proliferation by activating the Akt/FOXO1 pathway, which is consistent with the results in vitro. Meanwhile, diosmin increased CSA and the number of central nuclei in new muscle fibers. These results indicated that diosmin accelerated muscle repair by enhancing the proliferation of myoblasts.

In conclusion, this study demonstrated that diosmin facilitated the proliferation of C2C12 myoblasts via activating the Akt/FOXO1 pathway to promote FOXO1 nuclear export, thus affecting the expression of cell cycle related proteins, participating in cell cycle regulation, which subserved myogenesis (Figure 7). And diosmin repressed the differentiation of C2C12 myoblasts by delaying the terminal cell cycle exit in early differentiated myoblasts and inhibiting autophagy flux in mature myotube. Furthermore, it had the function of repairing muscle injury. This study filled the blank on the role of diosmin in skeletal muscle cells, simultaneously provided a new candidate drug for the treatment of myopathy.

Figure 7.

Figure 7

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Graphical abstract. Diosmin promotes myogenesis via activating the Akt/FOXO1 pathway to facilitate the proliferation of C2C12 myoblasts.

Glossary

Abbreviations Used

AI

artificial intelligence

TA

tibialis anterior

CSA

cross-sectional area

p27

cyclin-dependent kinase inhibitor 1B

CDK2

cyclin-dependent kinase 2

CDK4

cyclin-dependent kinase 4

Pcna

proliferating cell nuclear antigen

FOXO1

forkhead box O1

MyOD

myogenic differentiation

p21

cyclin-dependent kinase inhibitor

GM

growth media

DM

differentiation medium

DMEM

Dulbecco's modified Eagle's medium

CCK-8

cell counting kit-8

EdU

5-ethynyl-29-deoxyuridine

PBS

phosphate-balanced solution

PVDF

polyvinylidene difluoride

BSA

bovine serum albumin

DMSO

dimethyl sulfoxide

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.3c04828.

  • (Figure S1) Effects of different concentrations of MK2206 on p-Akt/Akt (Ser 473) protein levels, (Figure S2) diosmin repressing the cell cycle exit of C2C12 myoblasts, (Figure S3) diosmin suppressing the differentiation of C2C12 myoblasts by inhibiting autophagy flux, and (Table S1) primer information (PDF)

Author Contributions

D.Z., J.C., and H.L. performed conceptualization; D.Z. performed methodologies, formal analysis, original draft preparation, and visualization; X.M. and H.L. performed software analysis; D.Z., X.Z., and Z.L. performed validation; X.Z. and Z.L. performed investigation; M.S. performed resource acquisition and data curation; J.C. and H.L. performed review and editing of the manuscript, project administration, and funding acquisition; X.Z. performed supervision. All authors have read and agreed to the published version of the manuscript.

This research was funded by the National Natural Science Foundation of China (no. U20A2052 and no. 32372853), the Jiangsu Agriculture Science and Technology Innovation Fund (no. CX(20)2011), the National Natural Science Foundation of China (no. 31972571), the modern molecular breeding technique for breeding high-fertility and high-quality lean porcine matching lines (no. T0201900482), and the National Major Project of Breeding for Transgenic Pigs (no. 2016ZX08006001-003).

The authors declare no competing financial interest.

Supplementary Material

jf3c04828_si_001.pdf (370.9KB, pdf)

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