Biomolecules & Therapeutics 2024; 32(3): 349-360  https://doi.org/10.4062/biomolther.2024.012
Morroniside Protects C2C12 Myoblasts from Oxidative Damage Caused by ROS-Mediated Mitochondrial Damage and Induction of Endoplasmic Reticulum Stress
Hyun Hwangbo1,†, Cheol Park2,†, EunJin Bang1, Hyuk Soon Kim3, Sung-Jin Bae4, Eunjeong Kim5, Youngmi Jung6, Sun-Hee Leem3, Young Rok Seo7, Su Hyun Hong1, Gi-Young Kim8, Jin Won Hyun9 and Yung Hyun Choi1,*
1Basic Research Laboratory for the Regulation of Microplastic-Mediated Diseases, Department of Biochemistry, College of Korean Medicine, Dong-eui University, Busan 47340,
2Department Division of Basic Sciences, College of Liberal Studies, Dong-eui University, Busan 47340,
3Department of Biomedical Sciences, College of Natural Science and Department of Health Sciences, The Graduate School of Dong-A University, Busan 49315,
4Department of Molecular Biology and Immunology, Kosin University College of Medicine, Busan 49267,
5BK21 Plus KNU Creative BioResearch Group, School of Life Sciences, College of National Sciences, Kyungpook National University, Daegu 41566,
6Department of Biological Sciences, College of Natural Science, Pusan National University, Busan 46241,
7Institute of Environmental Medicine, Department of Life Science, Dongguk University Biomedi Campus, Goyang 10326,
8Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju 63243,
9Department of Biochemistry, College of Medicine, and Jeju Research Center for Natural Medicine, Jeju National University, Jeju 63243, Republic of Korea
*E-mail: choiyh@deu.ac.kr
Tel: +82-51-890-3319, Fax: +82-51-893-3333
The first two authors contributed equally to this work.
Received: January 12, 2024; Revised: March 5, 2024; Accepted: March 13, 2024; Published online: April 11, 2024.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Oxidative stress contributes to the onset of chronic diseases in various organs, including muscles. Morroniside, a type of iridoid glycoside contained in Cornus officinalis, is reported to have advantages as a natural compound that prevents various diseases. However, the question of whether this phytochemical exerts any inhibitory effect against oxidative stress in muscle cells has not been well reported. Therefore, the current study aimed to evaluate whether morroniside can protect against oxidative damage induced by hydrogen peroxide (H2O2) in murine C2C12 myoblasts. Our results demonstrate that morroniside pretreatment was able to inhibit cytotoxicity while suppressing H2O2-induced DNA damage and apoptosis. Morroniside also significantly improved the antioxidant capacity in H2O2-challenged C2C12 cells by blocking the production of cellular reactive oxygen species and mitochondrial superoxide and increasing glutathione production. In addition, H2O2-induced mitochondrial damage and endoplasmic reticulum (ER) stress were effectively attenuated by morroniside pretreatment, inhibiting cytoplasmic leakage of cytochrome c and expression of ER stress-related proteins. Furthermore, morroniside neutralized H2O2-mediated calcium (Ca2+) overload in mitochondria and mitigated the expression of calpains, cytosolic Ca2+-dependent proteases. Collectively, these findings demonstrate that morroniside protected against mitochondrial impairment and Ca2+-mediated ER stress by minimizing oxidative stress, thereby inhibiting H2O2-induced cytotoxicity in C2C12 myoblasts.
Keywords: Morroniside, Oxidative stress, Mitochondrial damage, ER stress, Ca2+
INTRODUCTION

Reactive oxygen species (ROS) are mainly generated during mitochondrial metabolic reactions and cause oxidative stress when they accumulate, due to imbalances in the intracellular antioxidant defense systems (Kowalczyk et al., 2021; Jomova et al., 2023). Excessive ROS induce functional and structural abnormalities in the electron transport chain within mitochondria and can induce cell death by acting as a signal to initiate the mitochondria-mediated intrinsic apoptotic pathway (Zhang et al., 2022a). Additionally, ROS contribute to the initiation of apoptosis through endoplasmic reticulum (ER) stress (Liu et al., 2022; Wang et al., 2023). In particular, oxidative stress caused by insufficient removal of ROS, which occurs when muscles consume large amounts of oxygen for contractile activity, directly or indirectly affects muscle cell function and damages DNA and intracellular organelles (Foreman et al., 2021; Chen et al., 2023). For example, ROS inhibits muscle differentiation from myoblasts to myotubes, promotes muscle atrophy through catabolism of differentiated muscle cells, and acts as a pathological cause of various muscle disorders (Abrigo et al., 2019; Alizadeh Pahlavani et al., 2022).

Recently, interest in natural bioactive ingredients with high antioxidant capacities as a means to reduce oxidative stress and prevent related diseases, has been increasing. Morroniside, a natural iridoid glycoside, is a major bioactive ingredient present in medicinal herbs including Cornus officinalis Sieb. et Zucc. (Huang et al., 2018). The beneficial effects of this herb, which has long been used to alleviate various pathological phenomena, have been shown to be due to the antioxidative activity of these glycosides (Zhang et al., 2022b). Morroniside isolated from C. officinalis was first reported to be an antioxidant by Xu et al. (2006). They showed it blocked ROS production while activating superoxide dismutase (SOD) and glutathione (GSH) peroxidase in rat renal mesangial cells treated with advanced glycation end products. In addition, Yokozawa and her colleagues (Yokozawa et al., 2010; Park et al., 2011) suggested that morroniside acts as a regulator of liver lipid metabolism and the inflammatory response, along with antioxidant activity, in a type 2 diabetes mouse model. Their results suggest that morroniside can suppress the development of liver complications in diabetic mice by minimizing hepatocyte apoptosis while ameliorating inflammatory responses and oxidative stress. As an antioxidant, morroniside has also been reported in several earlier studies to act as a neuroprotective agent. For example, Wang et al. (2008) revealed that the antioxidant and anti-apoptotic efficacy of morroniside in hydrogen peroxide (H2O2)-treated SH-SY5Y neuroblastoma cells, was due to the elevation of GSH and by blocking ROS formation. They also reported that the anti-cytotoxic effect of morroniside in response to H2O2 treatment was associated with preservation of SOD activity, inhibition of calcium (Ca2+) accumulation, and blockade of mitochondrial membrane potential (MMP) reduction (Wang et al., 2009). Morroniside also prevented brain injury following focal cerebral ischemia by protecting oxidative stress and apoptosis (Wang et al., 2010), and was proven to have similar efficacy in models of Alzheimer’s (Chen et al., 2023) and Parkinson’s disease (Li et al., 2023). Recently, we reported that heme oxygenase-1 activation by morroniside plays a crucial role in minimizing oxidative and inflammatory damage in lipopolysaccharide-challenged macrophages (Park et al., 2021). However, to date, the potential of morroniside as an antioxidant in muscle cells has not been investigated. In particular, the role of morroniside in influencing endoplasmic reticulum (ER) stress, which is responsible for disruption of cellular Ca2+ homeostasis regulation by oxidative damage (Daverkausen-Fischer and Pröls, 2022; Hu et al., 2023), has not been explored. Moreover, although loss of mitochondrial function due to H2O2 in muscle is closely related to the generation of mitochondrial ROS (mtROS) in muscle (Li et al., 2022; Cai et al., 2023), existing studies were limited to intracellular ROS regulation. Therefore, in this study, we evaluated whether morroniside confers protection against H2O2- evoked mitochondrial oxidative damage and ER stress-mediated Ca2+ homeostasis disturbances using the C2C12 myoblast model.

MATERIALS AND METHODS

Cell culture and experiments

C2C12 cells (ATCC® Cat. No. CRL-1772™) were maintained in at 37°C under an atmosphere with 5% CO2 as described in previous studies (Park et al., 2023). Supplies needed for cell culture were purchased from WelGENE, Inc. (Gyeongsan, Korea). Stock solutions of morroniside and H2O2 (Thermo Fisher Scientific, Waltham, MA, USA) were prepared by dissolving in dimethyl sulfoxide (Sigma-Aldrich Co., St. Louis, MO, USA) and distilled water, respectively, immediately before use and then diluting with culture medium. To examine protective role of morroniside on H2O2-treated C2C12 cells, sub-confluent (~70-80%) cells were exposed to morroniside (100 μM) for 1 h before H2O2 (0.8 mM) treatment for 1 h or 24 h.

Measurement of cell viability and cytotoxicity

Cell viability and cytotoxicity assays were assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Co.) and Lactate Dehydrogenase (LDH) Colorimetric Assay Kit (Cat. No. ab102526, Abcam, Inc., Cambridge, UK) based on previously published study as a reference (Lee et al., 2023).

Apoptosis quantification

Quantitative analysis of apoptosis or cell death was identified by Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (Cat. No. BMS500FI-20, Thermo Fisher Scientific). Cells were cultured under various conditions for 24 h were harvested and reacted with annexin V-FITC and propidium iodide according the manufacturer’s assay protocol. The percentage of apoptosis was determined for each treatment group based on flow cytometry program (BD Biosciences, San Jose, CA, USA) installed at TRCORE, Dong-eui University (Busan, Korea). In addition, we observed morphological criteria of apoptosis after staining the nuclei with 4’,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich Co.). To provide a brief description, cells grown on coverslips were pretreated with morroniside and post-treated with H2O2 for 24 h. Afterwards, cells were put to fixation with 4% paraformaldehyde (Sigma-Aldrich Co.), stained with DAPI fluorescent dye solution, and images were acquired under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Western blot analysis

Total cellular protein, mitochondrial and cytosolic fractions from cells under experimental condition were isolated using RIPA Lysis and Extraction Buffer (Cat. No. 89900, Thermo Fisher Scientific) and analyzed by Mitochondria/Cytosol Fractionation Kit (Cat. No. ab65320, Abcam, Inc.). Appropriate primary antibodies obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), Cell Signaling Technology, Inc. (Beverly, MA, USA), and Thermo Fisher Scientific (Table 1) were used for the analysis and protein bands were visualized using Amersham™ ECL™ Western Blotting Detection Reagent (Cat. No. RPN2209, Amersham Bioscience, Piscataway, NJ, USA). Peroxidase-labeled anti-rabbit and anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. The protein expression levels were then normalized to those of β-actin or cytochrome c oxidase IV (COX VI), which were used as internal controls.

Table 1 List of antibodies used in this study

AntibodySpecies raisedDilutionProduct codeSource

Bax

Bcl-2

Caspase-3

PARP

γH2AX

Cytochrome c

p-PERK

PERK

GRP78

CHOP

Calpain 1

Calpain 2

COX IV

β-actin

Mouse monoclonal

Mouse monoclonal

Rabbit polyclonal

Mouse monoclonal

Rabbit polyclonal

Mouse monoclonal

Rabbit polyclonal

Mouse monoclonal

Rabbit polyclonal

Rabbit polyclonal

Mouse monoclonal

Rabbit polyclonal

Rabbit polyclonal

Mouse monoclonal

1:1000

1:1000

1:1000

1:1000

1:500

1:1000

1:500

1:1000

1:1000

1:1000

1:1000

1:1000

1:500

1:1000

sc-7480

sc-509

#9662

sc-8007

#2577

sc-13560

#3179

sc-377400

PA5-34941

#5554

MA1-13079

PA5-17494

#4844

sc-47778

Santa Cruz Biotechnology, Inc.

Santa Cruz Biotechnology, Inc.

Cell Signaling Technology, Inc.

Santa Cruz Biotechnology, Inc.

Cell Signaling Technology, Inc.

Santa Cruz Biotechnology, Inc.

Cell Signaling Technology, Inc.

Santa Cruz Biotechnology, Inc.

Thermo Fisher Scientific

Cell Signaling Technology, Inc.

Thermo Fisher Scientific

Thermo Fisher Scientific

Cell Signaling Technology, Inc.

Santa Cruz Biotechnology, Inc.



Caspase-3 activity measurement

To detect caspase-3 activity, the Caspase-3 Assay Kit (Cat. No. ab39401) purchased from Abcam, Inc. was used following the manufacturer’s assay protocol. In brief, cells were lysed and the extracted cellular proteins were reacted to the substrate by incubation in reaction buffer. The caspase-3 activity was examined by measuring absorbance at 405 nm with a microplate reader (BioTek Instruments, Winooski, VT, USA) (Cao et al., 2023).

DNA damage analysis

After culturing the cells with H2O2 for 24 h with or without morroniside, oxidative DNA damage was measured according to the instructions of the Comet Assay® Kit (Cat. No. 4250-050-K, Trevigen, Inc., Gaithersburg, MD, USA). As well, the content of 8-hydroxyguanosine (8-OHdG), an RNA nucleoside that is an oxidative derivative of guanosine, was assessed by 8-OHdG ELISA Kit (Cat. No. ab201734, Abcam, Inc.). Fluorescence images of comet tails were determined under a fluorescence microscope, and the levels of 8-OHdG/8-oxoguanine (8-oxoG) were measured at absorbance at 450 nm using a plate reader.

Immunofluorescence

The expression of γH2AX (DNA double-strand break marker protein) and levels of MitoSOX and Rhod-2-AM were examined using immunofluorescence. In brief, cells on coverslips, were treated with H2O2 and morroniside as described above, and then probed with γH2AX antibody (Cat. No. BLR053F, Thermo Fisher Scientific), MitoSOX™ Mitochondrial Superoxide Indicator (Cat. No. M36008, Thermo Fisher Scientific), and Rhod-2 AM, fluorescent mitochondrial Ca2+ indicator (Cat. No. ab142780, Abcam, Inc.) in accordance with each manufacturer’s recommendations (Duan et al., 2023).

GSH measurement

Cells cultured under experimental condition were measured for the intracellular GSH content of each treatment group was measured by Glutathione Fluorescent Detection kit (Cat. No. EIAGSHF, Thermo Fisher Scientific) in accordance to the manufacturer’s assay protocol.

ROS production measurement

To measure total ROS and mitochondrial superoxide formed within cells, 2′,7′-dichlorofluorescin diacetate (DCF-DA, Cat. No. 4091-99-0, Sigma-Aldrich Co.) and MitoSOX™ were used. In brief, the treated cells were incubated with DCF-DA and MitoSOX™ as recommended by each manufacturer, and then the frequencies of DCF- and MitoSOX-positive cells were quantitatively evaluated using flow cytometry. We also used fluorescence microscopy to detect visible images (green-colored cells) of intracellular ROS by DCF-DA.

Mitochondrial membrane potential (MMP)

MMP levels in cells cultured under various conditions were examined using 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), a cationic carbocyanine dye. Briefly, cells culture media was replaced with culture medium containing JC-1 (Cat. No. T3168, Thermo Fisher Scientific). Subsequently, cells that had converted JC-1 oligomers to monomers by flow cytometry were considered as the cell population with disrupted MMP (Hong et al., 2022).

ER stress and mitochondrial Ca2+ level measurement

To measure blocking effect of morroniside on H2O2-induced ER stress was assessed using ER-Tracker™ Green reagent (Cat. No. E34251, Thermo Fisher Scientific). Cells cultured as described above were reacted with ER-Tracker™ reagent and then analyzed by flow cytometry following the experimental method suggested by the manufacturer. For mitochondrial Ca2+ measurements, Rhod-2 AM, fluorescent Ca2+ indicator (Cat. No. ab142780, Abcam, Inc.) was used following previous study (Le and Kimata, 2021). Cells were then collected and resuspended in phosphate-buffered saline containing Rhod2 AM. The frequency of Rhod-2-AM-stained cells was then evaluated using flow cytometry.

Statistical analysis

Results were collected and statistically analyzed to determine for data interpretation. The GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA) were used to analyze three independent replicates with a p-value of less than 0.05 with a cut-off value for significance. The data are shown as mean ± standard deviation (*p<0.05, **p<0.01, ***p<0.001 vs. corresponding control group; ##p<0.01, ###p<0.001 vs. H2O2 group).

RESULTS

Morroniside rescues C2C12 cells from H2O2-induced cytotoxicity

Fig. 1A and 1B show the results of C2C12 cell viability analyzed by MTT assay, upon treatment with increasing concentrations of H2O2 and morroniside for 24 h. Based on these results, 0.8 mM H2O2 was set as a concentration that mimics oxidative stress, since it indicated a cell proliferation percentage of approximately 60% in comparison to the control group. To investigate any inhibitory effects of morroniside on H2O2-induced oxidative damage, 50 mM and 100 mM were chosen because no significant inhibition on the survival of C2C12 cells was observed at these concentrations. As can be seen in Fig. 1C, after pretreatment with morroniside for 1 h, the H2O2 (0.8 mM)-induced decline in cell proliferation rate was remarkably suppressed. As well, the release of LDH was greatly increased by treatment with H2O2 alone, but was significantly suppressed in the morroniside pretreatment condition (Fig. 1D). These results show that morroniside significantly blocked H2O2-mediated cytotoxicity in C2C12 cells.

Figure 1. Effect of morroniside on H2O2-induced cytotoxicity in C2C12 myoblasts. Cells were treated with H2O2 (A) or morroniside (MOR, B) at various concentrations for 24 h, or cultured in medium containing morroniside for 1 h and then exposed to H2O2 for 24 h (C, D). After treatment, cell viability was analyzed by MTT assay (A-C) or the relative amount of LDH released into the culture medium was determined (D).

Morroniside blocks apoptosis in H2O2-induced C2C12 cells

We examined whether the suppressive action of morroniside on H2O2-induced cytotoxicity was a result of blocking apoptosis. In the evaluation of apoptosis based on flow cytometry and nuclear morphological changes (Fig. 2A-2D), the induction of apoptosis was markedly increased in H2O2-induced cytotoxic condition, but this was remarkably blocked in cells pretreated with morroniside. In addition, in H2O2-treated cells, the level of Bax protein expression, an apoptosis inducer, was not induced compared to the control group, but the expression of Bcl-2, an apoptosis inhibitor, was reduced. Furthermore, the level of pro-caspase-3 protein expression was clearly inhibited in H2O2-induced cytotoxic conditioned cells, while its enzymatic activity was significantly enhanced, and cleavage of poly (ADP-ribose) polymerase (PARP) was also shown (Fig. 2E-2G). However, these H2O2-induced changes were completely blocked in the presence of morroniside, indicating that the cytoprotective ability of morroniside against H2O2 was due to blocking apoptosis in C2C12 cells.

Figure 2. Effect of morroniside on H2O2-induced apoptosis in C2C12 myoblasts. Cells exposed to H2O2 for 24 h with or without morroniside (MOR) were collected and the inhibitory efficacy of morroniside on H2O2-induced apoptosis was evaluated through flow cytometry (A, B) and observation of nuclear morphological changes (C, D). Scale bar=50 μm. After extracting total proteins from cells, changes in expression of apoptosis-regulating proteins (E) and activity of caspase-3 (G) were examined using immunoblotting and a commercially available caspase-3 activity, assay kit. (F) Protein expression levels were normalized to those of β-actin, which was used as an internal control (pro-cas-3, pro-caspase-3; c-PARP, cleaved PARP).

Morroniside reduces DNA damage in H2O2-induced C2C12 cells

To assess whether the suppression of cytotoxicity by morroniside against H2O2 was related to blocking DNA damage. To this end we analyzed comet assays, 8-OHdG levels, and γH2AX expression. As shown in Fig. 3A, an elongation in comet tail length was detected in H2O2-stimulated cells, but not in cells under co-presence of H2O2 and morroniside. Levels of 8-OHdG were measured to quantify H2O2-induced oxidative damage to nucleic acids, and there was an approximately 3-fold increase in H2O2-treated cells compared to the untreated cells. However, a significant decrease of approximately 1.5-fold was observed in cells pretreated with morroniside (Fig. 3B). Additionally, we analyzed the expression of γH2AX, another DNA damage biomarker, and immunofluorescence results showed that morroniside attenuated the H2O2-induced increase in nuclear γH2AX expression (Fig. 3C). Additionally, the H2O2-induced increase in γH2AX level detected by immunoblotting was blocked by morroniside treatment (Fig. 3D, 3E). These findings imply that morroniside could inhibit H2O2-stimulated DNA damage in C2C12 cells.

Figure 3. Reduction of H2O2-induced DNA damage by morroniside in C2C12 myoblasts. Comet assay (A) and 8-OHdG content analysis (B) were performed to investigate DNA damage in cells cultured under different conditions, and γH2AX expression was examined through immunofluorescence (C) and immunoblotting (D). Scale bar=50 μm. (E) Densitometric analysis of γH2AX relative to β-actin expression was shown.

Morroniside alleviates oxidative stress in H2O2-induced C2C12 cells

To investigate the antioxidant function of morroniside, we first analyzed the content of GSH which plays an important antioxidant role in cells. Based on our findings, intracellular levels of GSH to GSSG were significantly decreased in H2O2-treated cells, but morroniside significantly offset this decline (Fig. 4A). To investigate whether this result was directly related to changes in the level of ROS, we applied DCF-DA and MitoSOX reagents and found that H2O2 treatment dramatically increased the levels of mitochondrial superoxide as well as cellular ROS (Fig. 4B-4F). Simultaneously, a clear increase in their levels was also detected by fluorescence microscopy (Fig. 4D, 4G). However, the increase in DCF- and MitoSOX-fluorescence intensity by H2O2 was thoroughly attenuated by morroniside, suggesting that morroniside acts as a strong ROS scavenger.

Figure 4. Alleviation of H2O2-induced oxidative stress by morroniside in C2C12 myoblasts. Cells were cultured in medium containing morroniside (MOR) for 1 h and then treated with H2O2 for 24 h (A) or 1 h (B-G). (A) Changes in GSH content in each treatment group were evaluated using the GSH detection kit. (B-D) Changes in cellular ROS production were examined through flow cytometry (B, C) and fluorescence microscopy analysis (D) using DCF-DA. Scale bar=50 μm. (E-G) Changes in mitochondrial superoxide production were investigated through flow cytometry (E, F) and fluorescence microscopy (G) using MitoSOX Red, a mitochondrial superoxide indicator. Scale bar=50 μm.

Morroniside attenuates mitochondrial damage and ER stress under H2O2 in C2C12 cells

Subsequently, the inhibitory potential of morroniside on H2O2-induced mitochondrial and ER damage was assessed investigated using JC-1, an MMP fluorescent probe, and ER-Tracker, a highly selective dye for ER. Flow cytometry allows evaluation of the frequency of JC-1 monomer and ER-Tracker-positive cells, which are indicators of mitochondrial and ER damage respectively; and these were greatly increased in cells treated with H2O2 alone. In addition, the expression of cytochrome c upregulated in the cytosolic compartment while diminishing in the mitochondrial compartment upon H2O2 treatment, indicating that the mitochondrial membrane was disrupted due to the loss of MMP, as evidenced by an increase in JC-1 monomers. Furthermore, the expression of ER stress response proteins, such as phosphor (p)-protein kinase RNA-like ER kinase (PERK), C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78), was also upregulated by H2O2 treatment, without changes in the expression of total PERK protein (Fig. 5). However, all of these H2O2-induced changes were clearly reduced by morroniside pretreatment, suggesting that the antioxidant activity of morroniside contributed to maintaining mitochondrial and ER homeostasis under conditions of oxidative stress.

Figure 5. Attenuation of H2O2-induced mitochondrial impairment and ER stress by morroniside in C2C12 myoblasts. (A-C) Changes in MMP in cells stimulated with H2O2 for 24 h in the presence or absence of morroniside (MOR) were investigated through flow cytometry by staining with JC-1 (A, B), and cytochrome c expression in mitochondrial (Mito) and cytosolic (Cyto) proteins were examined by immunoblotting (C). (D-F) Cells cultured under the same conditions were examined for ER stress through cell analysis using ER-Tracker staining (D, E), and changes in the levels of ER stress marker proteins were evaluated by immunoblotting (F). (G) Protein expression levels were normalized to those of β-actin or COX VI, which were used as internal controls for cytosolic or mitochondrial proteins, respectively.

Morroniside blocks H2O2-induced Ca2+ production in C2C12 cells

Finally, we used the mitochondrial Ca2+ probe Rhod-2 AM to examine whether Ca2+ signaling plays a part in in the blockade of H2O2-stimulated mitochondrial and ER damage by morroniside. According to the flow cytometry and immunofluorescence results in Fig. 6A-6C, the intensity of Rhod-2 AM red fluorescence, indicating an increase in mitochondrial Ca2+, was significantly greater in H2O2-treated cells. However, H2O2–mediated increases in Ca2+ levels were clearly abrogated by morroniside pretreatment. In addition, the expression of calpains, Ca2+-activated cysteine proteases family present in the cytosol, were not upregulated by H2O2 in cells pretreated with morroniside (Fig. 6D, 6E).

Figure 6. Blockade of H2O2-induced mitochondrial Ca2+ production by morroniside in C2C12 myoblasts. Cells were cultured in medium with or without morroniside (MOR) for 1 h and then treated with to H2O2 for 24 h. (A-C) The collected cells were reacted with Rhod-2 AM, and the levels of mitochondrial Ca2+ in each treatment group were evaluated through flow cytometry (A, B) or immunofluorescence (C). Scale bar=50 μm. (D) Total protein from cells was isolated and subjected to immunoblotting using the indicated antibodies. (E) Densitometric analysis of Calpain-1 and Calpain-2 relative to β-actin expression was shown.
DISCUSSION

Morroniside, the primary bioactive substance of C. officinalis, has been shown to be a therapeutically interesting component that is able to protect cells from oxidative stimuli. Previously published studies have provided evidence that the ability of morroniside to be prophylactic against various diseases, a characteristic that may be related to its antioxidant effects. In contrast, few studies have specifically investigated the underlying mechanisms on protection of muscle cells against oxidative damage. Therefore, to explore the beneficial potential of morroniside in muscle, we evaluated its mechanism of action against oxidative injury induced by H2O2, which can penetrate through cell membranes and is widely used for modeling oxidative stress in vitro. The results showed that morroniside protected C2C12 cells from cytotoxicity caused by H2O2 while maintaining redox balance and reducing Ca2+-mediated mitochondrial impairment and ER stress.

To conduct this study, we first established conditions for inducing oxidative cytotoxicity by H2O2 stimulation, and confirmed that morroniside efficiently blocked H2O2-induced cytotoxicity through MTT assay and LDH release analysis. We also showed that the inhibitory effect of morroniside on cytotoxicity in C2C12 cells exposed to H2O2 was due to counteracting apoptosis, as seen with a reversal of apoptosis-specific nuclear morphological changes. Meanwhile, the Bcl-2 family proteins, which consist of apoptosis inhibitors, for example, Bcl-2 and their antagonist proteins such as Bax, play a critical role in regulating apoptosis (Wolf et al., 2022). According to several previous studies, H2O2-stimulated apoptosis is mediated by attenuation of the Bcl-2/Bax ratio in many types of cell lines, and restoration of this ratio by morroniside was accompanied by inhibition of apoptosis by H2O2 treatment (Ma et al., 2022; Chen et al., 2023). This is consistent with the results shown here. In most cells, including C2C12 cells, the reduction in Bcl-2/Bax ratio caused by H2O2 was linked to the PARP cleavage by activated caspase-3, but many natural products with antioxidant activity appear able to reverse this (You et al., 2021; Park et al., 2023). Therefore, caspase activation and PARP degradation can be used as representative biomarkers of apoptotic induction (Wen et al., 2012; Srivastava and Saxena, 2023), and the suppressive effect of morroniside on H2O2-mediated apoptosis of C2C12 cells appears responsible for their blockade. Beyond PARP’s role in apoptosis, it also belongs to a family of proteins involved in genome stability and DNA repair, and cleaved PARP is in an inactive form that has lost the ability to repair damaged DNA (Kaur et al., 2022). Therefore, the blockade of H2O2-induced PARP degradation by morroniside suggests that this compound may prevent DNA damage as well as apoptosis caused by oxidative stress. We convincingly demonstrated the protective capacity of morroniside under DNA damage caused by H2O2, by several widely used DNA damage assays such as, 8-OHdG content, γH2AX expression and comet assay. Therefore, we conclude that H2O2 causes cell death by making the DNA of C2C12 myoblasts vulnerable to damage, but morroniside can effectively alleviate this. Our results correspond well with recent results by Dey et al. (2023) showing that activation of PARP by morroniside is involved in the suppression of DNA damage by alloxan, a glucose analog that destroys β cells while causing oxidative damage.

Increasing number of studies have demonstrated that excessive ROS accumulation, resulting from an imbalance between ROS generation and scavenging rates, serve as key mediators linking the induction of cell death with numerous pathological events (Kowalczyk et al., 2021; Jomova et al., 2023).

In particular, excessive mtROS produced in skeletal muscles with high oxygen consumption for contractile activity causes mitochondrial damage, which can lead to loss of muscle function. Therefore, preservation of mitochondrial homeostasis is essential in terms of sustained energy generation for muscle exercise (Pang et al., 2021; Espinosa et al., 2023). GSH, a ubiquitous cellular thiol compound involved in ROS scavenging and antioxidant defense against electrophiles, serves as a key regulator of redox status within cells. Since GSH is oxidized to GSSG when reacting with ROS or electrophiles, the GSH/GSSG ratio reflects the oxidation state, and a decrease in this ratio is related to the accumulation of excessive ROS (Hanko et al., 2019; Chai and Mieyal, 2023). It stands to reason therefore that increasing GSH levels may be an effective strategy to protect cells from oxidative damage. Similar to results seen previously (Ceci et al., 2022a, 2022b), we show that reductions to GSH levels in H2O2-treated C2C12 cells can be significantly blocked by morroniside pretreatment. Analysis of cellular ROS levels using DCF-DA showed that intracellular ROS production was significantly increased by H2O2 stimulation, and that these were clearly restored to more normal levels through co-treatment of H2O2 with morroniside. Retention of intracellular GSH levels may be a critical factor in the ROS scavenging effect of morroniside against H2O2. GSH exists not only in the cytoplasm but also in mitochondria, and protects mitochondria from oxidative stress by scavenging ROS generated during electron transport and oxidative phosphorylation within mitochondria (Jacobs and Riemer, 2023). Recently, Li et al. (2023) found that morroniside can reduce ROS production by restoring the mitochondrial respiratory chain in a Parkinson’s disease model. Our results using MitoSOX showed that morroniside effectively attenuated H2O2-induced mitochondrial superoxide production, corresponding to major mtROS (Fujii et al., 2022; Jomova et al., 2023). Therefore, the presence of morroniside improved the viability of C2C12 myoblasts by restoring the redox state through blunting of H2O2-mediated negative effects on intracellular GSH/GSSG ratios.

Intracellular GSH content is inversely proportional to apoptotic induction, and intracellular redox imbalance can stimulate the intrinsic apoptotic pathway mediated by mitochondrial dysfunction (Su et al., 2023; Xu et al., 2023). As well, increased intracellular ROS levels may be an indicator of mitochondrial damage, and collapse of MMP is positively correlated with mitochondrial dysfunction. The Bcl-2/Bax proportion reflects the susceptibility of mitochondrial membrane stability, and cytosolic release of cytochrome c following MMP loss initiates the mitochondria-mediated intrinsic apoptotic pathway following activation of caspases (Wolf et al., 2022; Srivastava and Saxena, 2023). Our results showed that morroniside maintained MMP and Bcl-2/Bax proportion at control levels and downregulated the expression of cytoplasmic cytochrome c in H2O2-treated C2C12 cells. This suggests that morroniside attenuated initiation of the mitochondria-mediated intrinsic apoptotic pathway, as was seen in studies of its neuroprotective effects (Wang et al., 2009; Srivastava and Saxena, 2023). With regard to apoptosis, ER stress appears to be tightly coupled to mitochondrial dysfunction (Liu et al., 2022; Ong and Logue, 2023). In consistent with the results of Pierre et al. (2014), flow cytometric analyses using ER-Tracker confirmed that H2O2 exposure induced ER stress in C2C12 cells, in parallel with upregulated level of ER stress marker proteins such as p-PERK, GRP78, and CHOP. Although GRP78 is a PERK regulator, which confers drug resistance and has anti-apoptotic functions, the PERK signaling pathway functions in ROS-induced ER stress-induced apoptosis, and CHOP acts as an initiator of ER stress-related apoptosis (Lu et al., 2023). However, in the presence of morroniside, the upregulation of these proteins was alleviated, clearly demonstrating that blockade of ER stress mediates the suppression of H2O2-induced oxidative damage.

It is well known that the ER plays an important role in controlling intracellular Ca2+ homeostasis and that intracellular Ca2+ overload causes cytotoxicity and consequently the initiation of apoptosis (Liu et al., 2022). ER stress-induced release of Ca2+ from the ER into the cytosol causes mitochondrial Ca2+ loading, and mitochondrial Ca2+ overload is paralleled by cytoplasmic release of apoptosis activators such as cytochrome c following MMP loss (Morris et al., 2021). Recently, Spina et al. (2022) demonstrated that the induction of oxidative cytotoxicity by arsenite in C2C12 cells occurs by a positive feedback amplification, which results in Ca2+ release from ER and increased Ca2+ supply to mitochondria for cation accumulation, which is important for mitochondrial superoxide formation. In addition, Karthikeyan et al. (2017) suggested that increased intracellular Ca2+ caused by H2O2-induced ER stress was transferred to mitochondria, resulting in elevated ROS generation, and that disruption of Ca2+ homeostasis directly intervenes in the induction of apoptosis in mouse retinal pigment epithelial cells. These results imply that increasing mitochondrial Ca2+ due to ER Ca2+ efflux serves as a potential cause of mitochondrial dysfunction via generation of ROS, including superoxide anion. Our results using Rhod-2 AM, a probe that detects mitochondrial Ca2+, showed that morroniside clearly counteracted the increased mitochondrial Ca2+ levels in H2O2-treated C2C12 cells. At the same time, the expression of calpains, which are Ca2+-dependent non-lysosomal cysteine proteases ubiquitously present in the cytoplasm, was increased by H2O2 treatment but significantly attenuated by morroniside administration. As is well known, calpains activated in response to increased cytosolic Ca2+ destroy cell membranes and intracellular matrix, ultimately contributing to apoptosis (Hua et al., 2023). Although it remains to be confirmed whether Ca2+ released from the ER directly leads to an increase in cytosolic and mitochondrial Ca2+, our findings suggest that the decrease in calpain expression in cells co-treated with H2O2 and morroniside may be due to reductions in cytosolic Ca2+ levels. Therefore, the antioxidant activity of morroniside in C2C12 cells may have affected Ca2+ fluxes between ER and mitochondria, which ultimately inhibited the mitochondria-mediated apoptotic pathway (Fig. 7). Shedding more light on the mechanistic role of morroniside in affecting the close coupling between ER stress and mitophagy, and the associated molecular pattern changes, would clearly seem to deserve further investigation.

Figure 7. Overview of the inhibitory effect of morroniside on H2O2-induced cytotoxicity in C2C12 myoblasts.

In conclusion, morroniside markedly suppressed H2O2-mediated oxidative injury by attenuating DNA damage, mitochondrial impairment and apoptosis in C2C12 myoblasts. Additionally, maintenance of functional mitochondrial integrity by morroniside in H2O2-treated cells was associated with several mechanistic routes, highlighting that morroniside can overcome ER stress-mediated Ca2+ homeostasis disorder by blocking mtROS production under oxidative stress conditions. These data may provide new insights into the mechanisms by which morroniside contributes to the protection of muscle disorders caused by oxidative stress.

ACKNOWLEDGMENTS

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00217899 and RS2023-00270936).

CONFLICT OF INTEREST

The authors have no conflicts of interest relevant to this study to disclose.

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