Introduction

Diosgenin is a steroidal compound found in various plant parts, including the rhizome of Dioscorea, a member of the Dioscoreaceae family [1, 2]. Previous studies have shown that diosgenin has various pharmacological properties, such as anti-inflammatory, antidiabetic, immunostimulatory, anti-metabolic syndrome, hepatoprotective, neuroprotective, and cardiovascular protective effects, and is effective in treating numerous diseases due to its absence of side effects [3,4,5,6]. In addition, diosgenin has been shown to induce growth arrest and apoptosis in various types of cancer cells, inhibit angiogenesis, cancer cell invasion, and metastasis, and improve anticancer drug sensitivity, which is closely related to the induction of oxidative stress by targeting reactive oxygen species (ROS) production [7,8,9]. In contrast, the antioxidant activity of diosgenin has been shown to be involved in defense mechanisms that prevent damage to normal cells caused by various stimuli. For example, in a diabetic nephropathy model, diosgenin attenuated the apoptosis of renal proximal tubular epithelial cells by suppressing ROS production through the maintenance of mitochondrial homeostasis [10]. Diosgenin also protected against type II diabetes-associated non-alcoholic fatty liver disease by reducing ROS and improving β-oxidation and mitochondrial function by enhancing the activation of antioxidant enzymes such as catalase (CAT), glutathione (GSH) peroxidase (GPx), and superoxide dismutase (SOD) [11]. Similarly, studies showing that diosgenin possesses antioxidant-mediated antiapoptotic activity can also be found in its blocking effect on doxorubicin-induced cardiac injury [12] and hydrogen peroxide (H2O2)-induced apoptosis in human vein endothelial cells [13]. A common finding regarding the antioxidant activity of diosgenin found in similar studies [14, 15], including these, is that the blockade of ROS production is mostly associated with an improvement in mitochondrial dysfunction, which is the opposite of the anticancer activity of this compound in a number of cancer cell lines. In particular, Chen et al. [16] reported that diosgenin not only attenuates Fas-dependent and mitochondria-dependent apoptosis in H2O2-exposed cardiomyocytes through activation of the insulin-like growth factor-1 survival pathway, but also inhibits H2O2-induced cytotoxicity through estrogen receptor-activated phosphorylation of phosphoinositide 3-kinase/Akt and extracellular signal-regulated kinase signaling pathways in cardiomyocytes via estrogen receptor interaction. Diosgenin also prevents or inhibits death receptor-mediated and mitochondria-initiated apoptosis in ovariectomized cardiac cells [17]. These results support those of previous studies showing that diosgenin is effective in preventing diseases such as cardiac fibrosis [18] and myocardial inflammatory damage [19].

Furthermore, it has recently been reported that diosgenin can enhance the anabolic action of myotubes by interacting with the natural steroid hormone ecdysterone [20], and can attenuate glucocorticoid-induced skeletal muscle atrophy [21]. These results correlate well with those of Kusano et al. [22], who reported that diosgenin supplementation increased the area and diameter of thigh skeletal myofibers and promoted the differentiation and strengthening of skeletal muscle by activating the catabolic pathway. They concluded that this effect of diosgenin was due to the enhancement of the catabolic pathway through the activation of the AMP-activated protein kinase (AMPK) pathway, an enzyme that induces myoblast fusion and senses and regulates the intracellular energy status. Interestingly, in skeletal muscles exposed to oxidative stress, AMPK activation is closely linked to damage to mitochondria, the source of energy production [23, 24]. Skeletal muscles consume a lot of energy for muscle contraction, and this process consumes a lot of oxygen, which continuously produces oxidant species such as ROS. Since oxidative stress plays a key role in compromising muscle homeostasis by inhibiting skeletal muscle differentiation and increasing muscle loss [25, 26], it is evident that blocking mitochondrial pathological damage caused by oxidative stress is important for maintaining skeletal muscle homeostasis. However, the potential of diosgenin to protect against oxidative stress-induced skeletal muscle injury has not been properly evaluated. Therefore, this study aimed to establish the basis that diosgenin can block the damage to skeletal muscle cells caused by oxidative stress, wherein, C2C12 murine myoblasts were used, and H2O2 was used as an oxidative stress inducer. In particular, we focused on elucidating that the blockade of oxidative stress-induced cytotoxicity by diosgenin is due to the inhibition of mitochondrial ROS following mitochondrial dysfunction.

Materials and methods

Cell culture and diosgenin treatment

C2C12 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and antibiotic mixtures at 37 °C and 5% CO2. All the materials used for cell culture were purchased from WelGENE (Gyeongsan, Republic of Korea). Diosgenin (Sigma-Aldrich Co., St. Louis, MO, USA) was solubilized in dimethyl sulfoxide (Sigma-Aldrich Co.) to prepare a stock solution, which was then diluted to various concentrations with culture medium before treating the cells.

Cytotoxicity assay

Cells were cultured in medium containing diosgenin or H2O2 at different concentrations for 48 h or pretreated with or without 25 µM diosgenin or 100 µM Mito-TEMPO for 1 h, and then treated with 0.5 mM H2O2 for 48 h. After treatment, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed as previously described [27]. The lactate dehydrogenase (LDH) release assay was performed using the LDH activity assay kit (Sigma-Aldrich Co.) according to the manufacturer’s protocol. Images were captured using an optical microscope (Carl Zeiss, Oberkochen, Germany).

Comet assay

The comet assay kit (Trevigen Inc., Gaithersburg, MD, USA) was used to assess DNA damage. Briefly, H2O2-exposed cells in the presence or absence of diosgenin were collected and the comet assay was performed as described by the manufacturer. Randomly selected images were acquired using a fluorescence microscope (Carl Zeiss).

Detection of 8-hydroxy-2’-deoxyguanosine (8-OHdG) levels

Levels of 8-OHdG, an oxidized nucleoside form of DNA were determined using an 8-OHdG ELISA kit (Abcam, Cambridge, UK). The cells were mixed with the reaction buffer and then allowed to react with the 8-OHdG antibody provided in the kit according to the manufacturer’s instructions. The cells were then washed with washing buffer and the absorbance was measured using an ELISA reader (BioTek, Winooski, VT, USA) at 405 nm, as previously reported [28].

Quantitative assessment of apoptosis

To investigate the degree of apoptosis induction, a FITC annexin V/propidium iodide (PI) apoptosis detection kit (BD Bioscience, Franklin Lakes, NJ, USA) was used. Briefly, Annexin V-FITC and PI buffer were added to cells suspended in binding buffer and it was allowed to react for 20 min, according to the manufacturer’s protocol [29]. The cell suspension was analyzed using a flow cytometer (Millipore Corporation, Hayward, CA, USA).

Protein extraction and immunoblotting

Whole cell lysates were prepared from cells cultured under various conditions as previously described [30]. Mitochondrial and cytoplasmic fractions were isolated using a Mitochondria/Cytosol Fractionation Kit (Sigma-Aldrich Co.). Equal amounts of protein extracted from the cells of each treatment group were fractionated by electrophoresis using sodium dodecyl sulfate-polyacrylamide gels and transferred to immunoblot membranes (Bio-Rad Lab. Inc., Hercules, CA, USA). The protein-transferred membranes were hybridized with primary antibodies against the target proteins and then incubated with secondary antibodies conjugated to horseradish peroxidase. Proteins were detected using an enhanced chemiluminescence detection kit (Sigma-Aldrich Co.). Antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), Cell Signaling Technology, Inc. (Danvers, MA, USA), and Abcam, Inc. Cytochrome c oxidase subunit IV (COX IV) and β-actin were probed as loading controls for mitochondrial and cytosolic proteins, respectively.

Caspase-3 activity assay

The activity of caspase-3 in cells cultured under various conditions was calculated using a caspase-3 assay kit (Abcam, Inc.) based on the hydrolysis of fluorescent substrate peptides by activated caspases. Briefly, after resuspending the cells in the cell lysis buffer provided in the kit, the supernatants were allowed to react with the substrate according to the manufacturer’s instructions. Finally, the concentration of p-nitroaniline released from the substrates was determined using a microplate reader [31].

Mitochondrial Membrane Potential (MMP) assay

To analyze the MMP, 5,5,6,6’-tetrachloro‐1,1’,3,3’‐tetraethylbenzimi‐dazoylcarbocyanine iodide (JC-1) fluorescent dye (Abcam, Inc.) was used. Cells were stained with 10 µM JC-1 for 30 min according to the manufacturer’s protocol. Images of JC-1-stained cells were monitored using fluorescence microscopy, as previously described [32].

ROS generation assay

Intracellular ROS and mitochondrial ROS (mtROS) levels were analyzed using the dichlorodihydrofluorescein diacetate (DCF-DA) and MitoSOX staining technique, respectively. Briefly, harvested cells were incubated with 10 µM DCF-DA or 5 µM MitoSOX (Thermo Fisher Scientific, Waltham, MA, USA), and intracellular ROS and mtROS levels were measured using flow cytometry [33]. Additionally, fluorescence images of the cells stained with DCF-DA or MitoSOX were acquired using a fluorescence microscope. To visualize the location of nuclei in ROS fluorescence images, cells were additionally counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, Sigma-Aldrich Co.) solution.

Assessment of the GSH/oxidized glutathione (GSSH) ratio

The antioxidant capacity of diosgenin was determined using the GSH assay kit (Sigma-Aldrich Co.), and the GSH/GSSG ratio was calculated using a standard curve containing known amounts of GSH and GSSG.

GPx and manganese SOD (MnSOD) activity measurement

The enzyme activities of GPx and MnSOD were analyzed using commercially available kits (Sigma-Aldrich Co.). Briefly, cell lysates were immunoprecipitated with antibodies against each enzyme, and the relative activity values were determined based on the measured absorbance according to the manufacturer’s protocol.

Statistical analysis

All data were statistically analyzed using GraphPad Prism 5.03 software (GraphPad Software Inc., La Jolla, CA, USA) using an unpaired two-tailed Student’s t-test and one-way analysis of variance. All results are presented as mean ± standard deviation (SD) of at least triplicate independent experiments. p was set at p < 0.05.

Results

Diosgenin reduced H2O2-induced cytotoxicity in C2C12 cells

An MTT assay was performed to study the protective ability of diosgenin against oxidative damage in C2C12 cells. According to the results shown in Fig. 1A, cell viability decreased in the 50 µM diosgenin treatment group; therefore, the pretreatment concentration of diosgenin was set to 25 µM or less. The concentration of H2O2 used to mimic oxidative stress (0.5 mM) resulted in approximately 60% cell viability as analyzed using the MTT assay (Fig. 1B). As shown in Fig. 1B, pretreatment with diosgenin concentration-dependently attenuated the H2O2-induced reduction in cell viability. Diosgenin also significantly blocked the leakage of LDH, which indicates cell damage in the H2O2-treated cells (Fig. 1C). H2O2-induced morphological changes and numerical reduction of cells were also suppressed in the presence of diosgenin (Data not shown) indicating that diosgenin attenuated H2O2-induced cytotoxicity in C2C12 cells.

Fig. 1
figure 1

Diosgenin inhibited H2O2-induced cytotoxicity in C2C12 cells. Cells were treated with different concentrations of diosgenin for 48 h (A) or treated with the indicated concentrations of diosgenin for 1 h and then treated with H2O2 for 48 h (B-D). Cell viability was analyzed using the MTT assay (A and B) or the relative level of LDH release (C) was investigated. **p < 0.01 and ***p < 0.001 vs. control cells; #p < 0.05 and ###p < 0.001 vs. H2O2-treated cells

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Diosgenin inhibited H2O2-induced DNA damage in C2C12 cells

To explore whether the reduction in H2O2-induced cytotoxicity by diosgenin was associated with the inhibition of DNA damage, the comet assay was performed and 8-OHdG levels were examined. As shown in Fig. 2A, the formation of comet tails, which indicates DNA double-helix breakage, markedly increased in the H2O2-treated cells. Additionally, the level of 8-OHdG, a marker of oxidative DNA damage, significantly increased following H2O2 treatment (Fig. 2B). However, increase in the expression of these DNA damage markers was largely offset by the presence of diosgenin, suggesting that diosgenin protects DNA from oxidative stress.

Fig. 2
figure 2

Diosgenin attenuated H2O2-induced DNA damage and apoptosis in C2C12 cells. Cells were pretreated with or without diosgenin for 1 h and then stimulated with H2O2 for 48 h. (A and B) The extent of DNA damage was examined by Comet assay (A) or changes in 8-OHdG levels (B). (C) After treatment, the degree of apoptosis was analyzed using the Annexin V/PI staining technique, and the frequency of Annexin V-positive cells in each treatment group was presented. (D) The change in expression of the indicated proteins were investigated using total proteins isolated from cells. The loading control used was β-actin. (E) Caspase-3 activity was assessed using a commercially available kit. ***p < 0.001 vs. control cells; ###p < 0.001 vs. H2O2-treated cells

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Diosgenin suppressed H2O2-induced apoptosis in C2C12 cells

To determine whether the H2O2-induced cytotoxicity suppressed by diosgenin was due to the inhibition of apoptosis, flow cytometric analysis was performed. As shown in Fig. 2C, the frequency of annexin-positive cells, indicating the induction of apoptosis, significantly increased in cells treated with H2O2, whereas it significantly reduced in the presence of diosgenin. In addition, the expression of Bcl-2 protein, an anti-apoptotic factor that reduced due to H2O2 treatment, and the increase in Bax, a pro-apoptotic protein, was maintained at control levels by pretreatment with diosgenin (Fig. 2D). In parallel, the expression of the pro-form of caspase-3, a key effector of caspase-dependent apoptosis, was downregulated in H2O2-treated cells, whereas the enzymatic activity and degradation of poly(ADP-ribose) polymerase (PARP), a representative substrate protein of caspase-3 increased (Fig. 2D and E). However, neither caspase-3 activation nor PARP degradation occurred in the presence of diosgenin (Fig. 2D and E), indicating that diosgenin inhibited the induction of apoptosis by preventing an increase in the Bax/Bcl-2 expression ratio and inactivating the caspase cascade due to H2O2 stimulation.

Diosgenin attenuated mitochondrial impairment in H2O2-treated C2C12 cells

To evaluate whether the protection of H2O2-trigerded apoptosis by diosgenin was due to the maintenance of mitochondrial homeostasis, we investigated changes in MMP, an indicator of mitochondrial stability. According to the fluorescence microscopy results after JC-1 staining, an increase in the green-to-red fluorescence ratio was observed in the H2O2-treated cells (Fig. 3A and B), indicating mitochondrial depolarization and a loss of MMP. The expression of cytochrome c, which exists in the space between the outer and inner mitochondrial membranes in normal cells, was upregulated in the cytosolic fraction of H2O2-treated cells but downregulated in the mitochondrial fraction (Fig. 3C). However, the translocation of cytochrome c into the cytoplasm was effectively blocked by diosgenin, suggesting that diosgenin blocked H2O2-induced disruption of mitochondrial membrane stability in the C2C12 cells.

Fig. 3
figure 3

Diosgenin counteracted H2O2-induced mitochondrial dysfunction in C2C12 cells. Cells were pretreated with or without diosgenin for 1 h and then exposed to H2O2 for 48 h. (A and B) After JC-1 staining, the fluorescence intensities of JC-1 aggregates (red) and monomers (green) were observed (A), and their relative intensities were indicated (B). ***p < 0.001 vs. control cells; ###p < 0.001 vs. H2O2-treated cells. (C) Change in expression of cytochrome c were investigated using mitochondrial and cytosolic fractions isolated from cells. COX IV and β-actin were used as loading controls for each fraction

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Diosgenin restored ROS production and the decreased activity of antioxidant enzymes in H2O2-treated C2C12 cells

To confirm the antioxidant activity of diosgenin, we assessed whether this compound could block the increase in ROS production by H2O2. According to the results of DCF-DA staining, the increased intracellular ROS levels in H2O2-treated cells significantly reduced by pretreatment with diosgenin, similar to the effect of NAC, a well-known ROS scavenger (Fig. 4A). Moreover, a decrease in the GSH/GSSG ratio, a marker of oxidative stress, was observed in H2O2-treated cells. This decrease was significantly alleviated in the NAC and diosgenin pretreatment groups (Fig. 4B). Therefore, we examined the changes in the expression of GPx1, which plays a major role in removing intracellular ROS, and MnSOD, which is active in the mitochondrial matrix, and confirmed that their expression and activity, which were reduced by H2O2 treatment, were neutralized by diosgenin (Fig. 4C-E). These results suggest that the antioxidant activity of diosgenin involves the activation of these enzymes.

Fig. 4
figure 4

Diosgenin removed H2O2-induced oxidative stress in C2C12 cells. Cells were pretreated with or without diosgenin or NAC for 1 h and then treated with H2O2 for 1 h (A and B) or 48 h (C-F). (A) Changes in intracellular ROS levels were examined after DCF-DA staining, and the frequency of DCF-positive cells in each treatment group was presented. (B) Intracellular GSH/GSSG ratio was measured using a GSH assay kit. (C) Expression changes of GPx1 and MnSOD were examined using total proteins isolated from cells. (D and E) Enzyme activities of GPx (E) and MnSOD (F) were measured using the corresponding assay kits. ***p < 0.001 vs. control cells; ###p < 0.001 vs. H2O2-treated cells

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Diosgenin alleviated H2O2-induced MtROS production in C2C12 cells

Because diosgenin restored the inactivation of MnSOD by H2O2, we examined whether its antioxidant activity was related to the inhibition of mtROS production. Staining with MitoSOX, a mitochondrial superoxide indicator, showed that diosgenin significantly blocked the production of mtROS induced by H2O2, similar to Mito-TEMPO, a mitochondrially targeted antioxidant (Fig. 5A). To confirm the ROS production inhibition ability of diosgenin, fluorescent images of cells stained with DCF-DA and MitoSOX were obtained. As shown in Fig. 5B-D, diosgenin significantly reduced the H2O2-induced fluorescence intensity, indicating not only the accumulation of intracellular ROS (green) but also the production of mtROS (red). Furthermore, when mtROS production by H2O2 was artificially blocked, both the increase in apoptosis and decrease in cell viability observed in H2O2-treated cells significantly improved (Fig. 6). Thus, maintenance of mitochondrial homeostasis by diosgenin significantly contributes to the inhibition of oxidative stress-mediated cytotoxicity in C2C12 cells.

Fig. 5
figure 5

Diosgenin restored H2O2-induced mtROS production in C2C12 cells. Cells were pretreated with or without diosgenin or Mito-TEMPO for 1 h and then treated with H2O2 for 1 h. (A) Changes in mtROS levels after MitoSOX staining were investigated, and the frequency of MitoSOX-positive cells in each treatment group was presented. (B-D) After treatment, cells were stained with DCF-DA (green) and MitoSOX (red), and then nuclei were additionally stained with DAPI (blue). Representative fluorescence images (B) and the relative values of each fluorescence intensity (C and D) are presented. ***p < 0.001 vs. control cells; ###p < 0.001 vs. H2O2-treated cells

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Fig. 6
figure 6

The reverse effect of H2O2-induced apoptosis and cytotoxicity by diosgenin in C2C12 cells was comparable to that of the mtROS production inhibitor. Cells were pretreated with or without diosgenin or Mito-TEMPO for 1 h and then exposed to H2O2 for 48 h. (A) The degree of apoptosis was examined by Annexin V/PI staining, and the frequency of Annexin V-positive cells in each treatment group was presented. (B) The MTT assay was performed using cells cultured under the same conditions. ***p < 0.001 vs. control cells; ###p < 0.001 vs. H2O2-treated cells

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Discussion

Recent studies have shown that diosgenin promotes skeletal muscle differentiation and inhibits muscle atrophy [20,21,22]. Oxidative stress can impair the two essential processes of skeletal muscle strengthening. Therefore, in this study, we investigated whether diosgenin protects against oxidative damage in myoblasts and cells prior to differentiation into myotubes. We first performed an MTT assay, a widely used method to measure in vitro cell viability based on changes in mitochondrial activity [34]. Since this assay reflects total mitochondrial activity as the number of viable cells, our finding that diosgenin protected against the decrease in cell viability in H2O2-treated cells may be related to the maintenance of mitochondrial homeostasis. Meanwhile, an increase in LDH, a cytosolic enzyme found in most cells in cell culture supernatants, indicates that the cell membrane is damaged, and the cells are undergoing necrosis, apoptosis, or other forms of cell death [35]. Our finding that diosgenin counteracted H2O2-induced extracellular release of LDH using this assay provides evidence that this blocked cell death. These results imply that the inhibition of cell death induced by oxidants, such as H2O2 in C2C12 cells by diosgenin is based on the preservation of mitochondrial function.

Most of the cell damage caused by oxidative stimuli is accompanied by DNA damage and unrepaired DNA damage contributes to cell transformation and death [32, 36]. Similar to our results, Son et al. [37] reported that diosgenin blocked lymphocyte DNA damage caused by H2O2 using a comet assay. The comet assay is a well-established method for assessing DNA damage based on the cleavage of the DNA double helix, and the formation of the comet tail is an indicator of apoptosis [38]. Diosgenin decreased the increase in the level of 8-OHdG, which indicates the level of nucleosides in oxidized DNA caused by H2O2, suggesting that this saponin enhanced the resistance to oxidative DNA damage in C2C12 cells. In addition, diosgenin reduced apoptosis by inhibiting the Bax/Bcl-2 expression ratio, caspase-3 activity, and PARP degradation in C2C12 cells treated with H2O2. These antiapoptotic effects of diosgenin are consistent with previously reported results [39,40,41]. Bcl-2 family proteins are major factors that regulate pore formation in the mitochondrial outer membrane and are composed of factors that can inhibit or promote apoptosis. Bax induces apoptosis by attacking mitochondria and increasing mitochondrial outer membrane permeability, whereas Bcl-2 is a pro-survival factor that supports cell survival by inhibiting apoptotic activity. Compared to Bcl-2, increased Bax weakens MMP, thereby releasing cytochrome c into the cytoplasm to activate the caspase cascade and terminate apoptosis [42, 43]. Therefore, our results suggest that diosgenin neutralizes the H2O2-induced increase in the Bax/Bcl-2 ratio, thereby maintaining mitochondrial stability and preventing cytochrome c release or caspase activation, ultimately blocking the mitochondria-mediated apoptotic pathway.

In a subsequent study, diosgenin inhibited ROS production and preserved the GSH/GSSG ratio in response to H2O2 treatment in C2C12 cells. Similar results have been reported in previous studies [12,13,14, 16] and we propose that the activities of MnSOD and GPx also contribute to the antioxidant potential of diosgenin. The results showed that the expression and activity of MnSOD, which reduced due to H2O2 stimulation, was the same in the presence of diosgenin, which may serve as evidence that it directly contributes to the preservation of mitochondrial function. This is because MnSOD is an enzyme that scavenges the mitochondrial superoxide present in the mitochondrial matrix [44], indicating that diosgenin inhibits the production of mtROS caused by H2O2 treatment. Dioscin, a natural steroid saponin composed of diosgenin, has been reported to increase the expression levels of MnSOD, CAT, and SOD to suppress oxidative injury induced by intestinal ischemia-reperfusion, thereby reducing ROS levels [45]. This is the first report to show that diosgenin itself increases the activity of MnSOD in an oxidative environment. Moreover, the alleviation of H2O2-induced apoptosis and cell viability reduction by mitochondria-targeting antioxidants in C2C12 cells was very similar to that of diosgenin, suggesting that the antioxidant capacity of diosgenin was closely associated with the inhibition of mtROS production.

In the current study, we propose that the preventive effect of diosgenin on C2C12 myoblasts in an oxidative environment mimicked by H2O2 treatment is due to the maintenance of mitochondrial homeostasis by ameliorating mtROS production. The ROS-scavenging effect of diosgenin may contribute to the inhibition of DNA damage and apoptosis during myoblast survival. However, as suggested by previous studies, various intracellular signaling pathways are involved in the antioxidant activity of diosgenin. For example, the improvement of mitochondrial dysfunction by this saponin was mediated through increased activity of AMPK, which suggests that mitochondrial biogenesis was enhanced [10, 16, 46, 47]. Nuclear factor erythroid-2-related factor 2, a regulator of redox homeostasis, plays a key role in ameliorating nonalcoholic fatty liver disease and renal inflammation by eliminating diosgenin-induced oxidative stress [48, 49]. And since diosgenin rescued H2O2-induced increase in mitochondrial superoxide and decrease in the expression and activity of MnSOD, it is difficult to predict that diosgenin completely blocked the production of mitochondrial ROS. Therefore, how the mtROS-scavenging efficacy of diosgenin is related to various signaling pathways involved in redox regulation and cell survival needs to be further studied. These include investigations of stress response pathways involving the role of the upstream transcription factors of MnSOD and studies in vivo models.

In conclusion, our results showed that diosgenin significantly suppressed DNA damage and apoptosis in H2O2-treated C2C12 myoblasts, which was related to the blockade of caspase cascade activation, and the Bax/Bcl-2 ratio increased following H2O2 treatment. In addition, diosgenin maintained the stability of mitochondrial membranes in H2O2-treated cells, as evidenced by an improvement in MMP loss and inhibition of cytochrome c efflux into the cytosol. Furthermore, diosgenin significantly maintained the GSH/GSSG ratio and GPx activity under H2O2-treated conditions, while eliminating ROS production. Diosgenin also attenuated H2O2-induced reduction in MnSOD activity and the production of mtROS, suggesting that the control of mtROS generation acts as an upstream event to preserve mitochondrial homeostasis for the suppression of oxidative damage by this saponin (Fig. 7). Therefore, the present results suggest that diosgenin has potential as a therapeutic agent to protect muscle cells from oxidative injury.

Fig. 7
figure 7

Schematic diagram of the antioxidant activity of diosgenin in C2C12 myoblasts. Diosgenin can at least rescue C2C12 cells from mitochondrial dysfunction, DNA damage, and apoptosis mediated by excessive mitochondrial ROS production due to H2O2 exposure

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