Baicalin inhibits C2C12 myoblast apoptosis and prevents against skeletal muscle injury
- Authors:
- Published online on: May 27, 2019 https://doi.org/10.3892/mmr.2019.10298
- Pages: 709-718
Abstract
Introduction
Exercise, eccentric contractions, acute trauma and disease are all causal mechanisms of skeletal muscle injury (1–5). Although the type of injury differs, the general injury and repair mechanisms are similar (1–5). When skeletal muscle is injured, it undergoes sequential phases of degeneration, inflammation, regeneration and fibrosis (1–5). For the repair and regeneration of skeletal muscle, it is important to induce the proliferation and differentiation of skeletal myoblasts and to inhibit the apoptosis of myoblasts (2). Therefore, anti-inflammatory and anti-apoptotic treatments are important for skeletal muscle regeneration and to promote healing following the occurrence of injury (1,4–6).
Baicalin is a flavonoid glycoside extracted from Scutellaria baicalensis, which is used in traditional Chinese medicine, has been reported to possess significant anti-inflammatory and anti-apoptotic properties, and is widely used in the treatment of injury and inflammatory diseases (7–12). For example, Lin et al (7,8) reported that baicalin inhibited H2O2-induced cell cytotoxicity in a human renal proximal tubular epithelial cell line, and attenuated renal ischemia-reperfusion injury by suppressing inflammation and apoptosis. A study by Cao et al (9) demonstrated that baicalin attenuated global cerebral ischemia/reperfusion injury in gerbils through anti-oxidative and anti-apoptotic pathways. In addition, Zhu et al (10) reported that baicalin increased survival in a murine model of polymicrobial sepsis through inhibiting the inflammatory response and lymphocyte apoptosis, and Xiping et al (11) demonstrated that baicalin offered protection to the thymus rats with severe acute pancreatitis. Our previous study also demonstrated that baicalin significantly inhibited oxidative stress damage induced by H2O2 and decreased cell apoptosis in endplate chondrocytes, which may provide potential therapeutic benefits for patients with osteoarthritis (13).
Therefore, in order to further investigate the underlying role and mechanism of baicalin in injured skeletal muscle, the present study focused on H2O2-stimulated C2C12 myoblasts in vitro to investigate the role of baicalin on cell apoptosis, and established an animal model of injured skeletal muscle to observe the influence of baicalin on the uptake of β-2-[18F]-fluoro-2-deoxy-D-glucose (18F-FDG) in lesions in vivo via small animal positron emission tomography (PET) imaging.
Materials and methods
Cell viability
C2C12 mouse myoblast cells (ATCC, cat. no. CRL-1772) were grown in 96-well plates (BD Falcon; BD Biosciences, San Jose, CA, USA) at a density of ~1×105/ml (100 µl/well) in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The C2C12 myoblasts were then washed with PBS buffer (Gibco; Thermo Fisher Scientific, Inc.) and incubated with medium containing different concentrations of H2O2 (0, 50, 100, 150, 200, 300, 500, 600, 800 and 1,000 µM; Lingfeng, Shanghai, China) at 37°C for 4 h, respectively. Untreated cells were referred to as the normal control. The cell viability of C2C12 myoblasts was determined using a cell counting kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). The C2C12 myoblasts were subsequently incubated with H2O2 (500 µM) under the co-existence of baicalin at various treatment time points (−1, 0, 1 and 2 h) at 37°C for 4 h, respectively. In addition, to judge the optimal incubation time, the appropriate treated concentration of baicalin on C2C12 myoblasts was also investigated. The C2C12 myoblasts were pretreated with baicalin at different concentrations (5, 15, 25, 50, 100, 150, 200, 250 and 300 µM) for 1 h, and were then incubated with H2O2 (500 µM) at 37°C for 4 h, respectively. Untreated cells and cells treated with H2O2 only were referred to as the control groups. Cell viability was determined using a 1:10 dilution of CCK-8 reagent and incubated at 37°C for 1 h. All the above data are presented as the mean of at least three independent experimental repeats.
Cell apoptosis
The apoptosis of C2C12 myoblasts incubated in the three groups (treated with 500 µM H2O2, treated with 500 µM H2O2 + 100 µM baicalin pre-treatment for 1 h, and the normal control) for 4 h was measured using an Annexin V/PI assay kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) for flow cytometric analysis. Briefly, the cells from each group were washed with PBS three times, harvested with 0.25% trypsin and centrifuged at 400 × g for 5 min at room temperature. The supernatant was discarded, cells were resuspended in 500 µl binding buffer and then immediately mixed with 5 µl Annexin V-FITC and 5 µl propidium iodide and incubated for 10–15 min at room temperature. Finally, the mixture was analyzed using a BD FACS Aria II flow cytometry (BD Biosciences). All data are presented as the mean of at least three independent experimental repeats.
Oxidative activity
The oxidative activity was assessed in the three aforementioned groups following incubation with H2O2 for 4 h. Following treatment, cells from each of the groups were washed with PBS three times, harvested with 0.25% trypsin and centrifuged at 400 × g for 5 min at room temperature. The supernatant was discarded, and the cells were incubated with ROS working fluid (cat. no. KGT010-1; Nanjing KeyGen Biotech Co., Ltd.) at 37°C for 15 min. The ROS working fluid was then removed, fresh PBS was added, and detection was performed with BD FACS Aria II flow cytometry (BD Biosciences) with an excitation wavelength of 488 nm, an emission wavelength of 525 nm and the channel of fluorescein isothiocyanate (FITC). Finally, the results of flow cytometry were analyzed using Flowjo 10.1 software (Flowjo, LLC, Ashland, OR, USA). All the above data are presented as the mean of at least three independent experimental repeats.
In addition, the cells from each group were washed with cold PBS three times, harvested with 0.25% trypsin, and centrifuged at 400 × g for 5 min at room temperature. The supernatant was discarded, and the cells were lysed with radio immunoprecipitation assay (RIPA) buffer and centrifuged at 12,000 g for 10 min at 4°C. Following this, 100 µl supernatant and 200 µl malondialdehyde (MDA) working solution (cat. no. KGT003; Nanjing KeyGen Biotech Co., Ltd.) were mixed and added to the 96-well plate. The absorbance of each well was then measured at a wavelength of 532 nm in an enzyme-linked immunometric meter (FlexStation 3; Molecular Devices, LLC, Sunnyvale, CA, USA). All the above data are presented as the mean of at least three independent experimental repeats.
Mitochondrial function
Rhodamine 123 (cat. no. KGA217; Nanjing KeyGen Biotech Co., Ltd.) were analyzed in above three groups following the incubation with H2O2 for 4 h. Following treatment, cells from each group were washed with PBS three times, harvested with 0.25% trypsin, and centrifuged at 400 × g for 5 min at room temperature. The supernatant was discarded, and the cells was incubated with Rhodamine 123 working fluid at 37°C for 15 min. The Rhodamine 123 working fluid was then removed, fresh PBS was added, and detection was performed using BD FACS Aria II flow cytometry with an excitation wavelength of 488 nm, an emission wavelength of 525 nm and the channel of FITC. Finally, the results of flow cytometry were analyzed using Flowjo 10.1 software. All the above data are presented as the mean of at least three independent experimental repeats.
Tetramethylrhodamine methyl ester staining (JC-1; cat. no. KGA602; Nanjing KeyGen Biotech Co., Ltd.) were analyzed in the above three groups following incubation with H2O2 for 4 h. Following treatment, the cells from each group were washed with PBS three times, harvested with 0.25% trypsin, and centrifuged at 400 × g for 5 min at room temperature. The supernatant was discarded, and the cells was incubated with JC-1 working fluid at 37°C for 15 min. Then JC-1 working fluid was then removed and fresh PBS was added. The analysis of JC-1 was detected with the channel of BD FACS Aria II flow cytometry via P-phycoerythrin and analyzed using Flowjo 10.1 software. All the above data are presented as the mean of at least three independent experimental repeats.
Mitochondrial apoptogenic factors
The protein levels of cytochrome c oxidase (cyto-C) and apoptosis-inducing factor (AIF) from cytosolic fractions were detected in the three experimental groups by western blotting. The cells were collected and lysed by RIPA buffer for 15 min and centrifuged at 15,000 × g for 30 min at 4°C. The protein concentrations were analyzed using a NanoDrop instrument, and 40 µg protein from each sample were run on 10% salt-polyacrylamide gel electrophoresis gels, and then transferred onto polyvinylidene fluoride membranes. Following blocking with 5% nonfat dry milk, the membranes were incubated overnight at 4°C with following primary mouse monoclonal antibodies: Anti-cyto-C (cat. no. AC908; Beyotime Institute of Biotechnology, Haimen, China], anti-AIF (cat. no. AB32516; Abcam, Cambridge, UK) or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cat. no. AB181602; Abcam) at a dilution of 1:500. The membranes were washed and incubated in a 1:1,000 dilution of goat anti-mouse/rabbit HRP (cat. no. 115-035-003/111-035-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h at room temperature. The membranes were then washed and visualized using Pierce™ ECL western blotting substrate (Thermo Fisher Scientific, Inc.) followed by exposure to Tanon 5200 (Tanon Science and Technology Co., Ltd.). Anti-cyto-C, anti-AIF and GAPDH were detected as bands of approximately 15, 67 and 36 kDa, respectively. The data were normalized to the GAPDH content of the same sample and analyzed using Image J v1.8.0 (National Institutes of Health, Bethesda, MD, USA). Similar to the above methods, the levels of cyto-C and AIF from mitochondrial fractions were also analyzed in the aforementioned three groups using western blotting following incubation with H2O2 for 24 h. The extracted proteins were incubated with the following primary mouse monoclonal antibodies: A 1:1,000 dilution of anti-cyto-C, anti-AIF or anti-cyclooxygenase-4 (COX-4; cat. no. AB33958; Abcam) for 12 h at 4°C, followed by incubation in a 1:1,000 dilution of goat anti-mouse/rabbit HRP (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Anti-cyto-C, anti-AIF and COX-4 were detected as bands of approximately 15, 67 and 17 kDa, respectively. Bands were visualized according to the aforementioned procedure. Data were normalized to the content of COX-4 in the same sample and analyzed using Image J (National Institutes of Health). All the above data are presented as the mean of at least three independent experimental repeats.
Caspase proteins
The activation of caspase-3 and caspase-9 was evaluated in the three experimental groups by western blot analysis at the time point of 24 h. The extracted proteins were incubated with 1:1,000 dilutions of the following primary mouse monoclonal antibodies: Anti-caspase-3 (cat. no. AB2302; Abcam), anti-caspase-9 (cat. no. AB32539; Abcam) or anti-GAPDH for 12 h at 4°C, followed by incubation in a 1:1,000 dilution of goat anti-mouse/rabbit HRP (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Caspase-3 and caspase-9 were detected as bands of approximately 17 and 46 kDa, respectively. Bands were visualized according to the aforementioned procedure. The data were normalized to the GAPDH content of the same sample and analyzed by ImageJ (National Institutes of Health). The above data are presented as the mean of at least three independent experimental repeats.
Animal models of injured skeletal muscle induced by H2O2
All procedures were approved by the Animal Ethics Committee at Shanghai East Hospital, Tongji University School of Medicine (Shanghai, China). In total, five Female BALB/C mice of 6-8-weeks of age (18–20 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Mice were kept in a controlled environment (temperature range, 22–24°C; relative humidity, 40–60%) with a 12 h light/dark cycle and had free access to food and water. Mice were fasted for at least 24 h before the experiment and anesthetized using isoflurane inhalation. Subsequently, 500 µM H2O2 was intramuscularly administered into the right hind leg. The left hind leg was intramuscularly administered with 500 µM H2O2, and the relevant group was pre-treated with 100 µM baicalin for 1 h. At 3 days post-treatment, the animal models were used for small animal PET imaging.
Small animal PET imaging
PET imaging was performed using a small animal PET scanner (Siemens Inveon). Briefly, the mice (n=4) were injected with ~3.7–5.55 MBq (100–150 µCi) of 18F-FDG through the tail vein, and were anesthetized with 2% isoflurane for imaging experiments at 1 h p.i. The PET images were reconstructed and analyzed using the vendor-supplied software Inveon Research Workspace (Preclinical Solutions, Siemens Healthcare Molecular Imaging). Three-dimensional regions of interest (ROIs) were drawn over the organs and tissues on decay-corrected whole-body PET images. The results were divided by the injected dose, in order to obtain an image ROI-derived percent injected dose per gram of tissue (14).
Pathology
The BALB/C mice were sacrificed by cervical dislocation following anesthetization with isoflurane. Bilateral muscle lesions were separately fixed using 4% paraformaldehyde for 24 h and dehydrated using an ethanol series at room temperature. Following fixation and dehydration, the muscle samples were embedded in paraffin. Paraffin specimens were cut to a 5 µm thickness and stained with hematoxylin and eosin (H&E) for 1 h at room temperature. All stained samples were observed under a light microscope (Leica Microsystems GmbH, Germany), and images were acquired under the same conditions and displayed at the same scale for comparison (magnification, ×100).
Statistical analysis
The quantitative data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA for the comparison of multiple groups and Student's t-test for two groups. A 95% confidence level was selected, and P<0.05 was considered to indicate a statistically significant difference.
Results
Baicalin increases cell viability of H2O2-stimulated c2c12 myoblasts
As illustrated in Fig. 1A, with increasing H2O2 concentrations, the cell viability of the C2C12 myoblasts was gradually decreased. The H2O2 concentration of 500 µM was selected as an optimal dose for the subsequent experiments. As shown in Fig. 1B, it was observed that cell viability was highest when the C2C12 myoblasts under H2O2 exposure were pre-treated with baicalin for 1 h. Cell viability subsequently decreased with a gradual trend when the C2C12 myoblasts were treated with baicalin no earlier than the start of incubation with H2O2. As baicalin concentrations increased, C2C12 myoblast viability was initially gradually elevated, and then decreased under co-treatment with 500 µM H2O2. Cell viability was highest when the baicalin concentration reached 100 µM (Fig. 1C). Therefore, a baicalin concentration of 100 µM was selected as the model dose for the subsequent experiments.
Baicalin inhibits C2C12 myoblast apoptosis induced by H2O2
The Annexin V/PI staining analysis revealed that, compared with the group treated with H2O2 alone for 4 h, the apoptosis of C2C12 myoblasts was significantly decreased in the group that had been pre-treated with baicalin for 1 h at the 4-h time point (Fig. 2A and B), which suggested that baicalin significantly suppressed the apoptosis of C2C12 myoblasts induced by H2O2.
Baicalin decreases oxidative activity in C2C12 myoblasts induced by H2O2
As a biomarker of oxidative stress, the ROS levels of C2C12 myoblasts were increased following treatment with H2O2 for 4 h, which were markedly reversed by pre-treatment with baicalin for 1 h (Fig. 3A and B). Similarly, pre-treatment with baicalin for 1 h effectively reversed the abnormal MDA levels in C2C12 myoblasts exposed to H2O2 for 4 h (Fig. 3C).
Baicalin reverses mitochondrial dysfunction in C2C12 myoblasts induced by H2O2
Mitochondrial dysfunction was also observed in C2C12 myoblasts following exposure to H2O2 for 4 h, which was reflected by the loss of mitochondrial activity, determined by Rhodamine 123 staining (Fig. 4A and B), and the decrease in mitochondrial membrane potential (ΔΨm) determined via JC-1 staining (Fig. 4C and D). However, as shown in Fig. 4, pre-treatment with baicalin for 1 h noticeably reversed the aforementioned effects and protected mitochondrial function.
Baicalin inhibits the release of mitochondrial apoptogenic factors induced by H2O2
The levels of cyto-C and AIF from cytosolic fractions were identified following the treatment of C2C12 myoblasts with H2O2 for 24 h, and these were marginally lower than those in the normal control (Fig. 5A). Pre-treatment with baicalin was observed to marginally upregulate the expression of cyto-C and AIF (Fig. 5A). This effect was corroborated by the quantification of the western blotting results, which are illustrated in Fig. 5B. The levels of cyto-C and AIF from mitochondrial fractions were also identified, and these were markedly lower than those in the normal control group (Fig. 5C). In addition, pre-treatment with baicalin significantly upregulated the levels of cyto-C and AIF (Fig. 5C). The quantification results of western blotting are presented in Fig. 5D.
Baicalin inhibits the activation of caspases in C2C12 myoblasts induced by H2O2
As presented in Fig. 6A, the protein expression levels of the caspase-3 and caspase-9 were identified following the incubation of C2C12 myoblasts with 500 µM H2O2 for 24 h, which were markedly higher than those in the normal control group. Pre-treatment with baicalin significantly downregulated the expression of caspase-3 and caspase-9, as shown from the quantification of western blotting results in Fig. 6B (P<0.05).
Small animal PET imaging of injured skeletal muscle
As illustrated in Fig. 7A, a representative coronal small animal 18F-FDG PET image of an animal model of skeletal muscle injury induced by H2O2 is presented. The radioactivity uptake in the muscle lesion of the right leg was observed at 1 h p.i. However, there was no obvious FDG accumulation in the left leg lesion. Further quantification analysis demonstrated that the radioactivity uptake by the right and left muscle lesions were 3.20±0.52% ID/g and 1.09±0.22% ID/g, respectively, and the ratio of right-to-left lesion was 2.94±0.25 (Fig. 7B). In addition, the physiological radioactivity accumulation of normal muscle tissues was observed. In the other examined normal organs and tissues, including the brain, lung, heart, liver and kidneys, relatively low radioactivity accumulation was also observed, and the majority were <2% ID/g.
Pathological results
The H&E staining revealed that the muscle tissues of the right leg were injured and infiltrated with masses of inflammatory cells following the induction of H2O2 (Fig. 8A, ×100 magnification). The muscle tissues of the left leg exhibited a lesser degree of injury and less inflammatory cell infiltration following pre-treatment with baicalin for 1 h (Fig. 8B, ×100 magnification).
Discussion
As a flavonoid glycoside extracted from Scutellaria baicalensis, a type of traditional Chinese medicine, baicalin has been reported to possess significant anti-inflammatory and anti-apoptotic properties, and is widely used in the treatment of injuries and inflammatory diseases (7–12). In addition, the results of our previous study confirmed the protective effects of treatment with baicalin on H2O2-induced apoptosis in endplate chondrocytes (13). Therefore, the aim of the present study was focused on the protective effects of baicalin on H2O2-stimulated C2C12 myoblasts in vitro and animal models with skeletal muscle injury in vivo.
Initially, the present study confirmed the protective effects of baicalin on the viability of C2C12 myoblasts using a CCK-8 assay. H2O2 was observed to decrease the viability of the C2C12 myoblasts, and the effects of H2O2 on C2C12 myoblasts were dose-dependent. With increased H2O2 dose, the viability of the C2C12 myoblasts was significantly decreased. The potential protective effects of baicalin against H2O2, with an obvious increase in cell viability, were observed using the CCK-8 assay, with baicalin pre-treatment for 1 h exhibiting an optimal protective effect against the H2O2-induced loss of C2C12 myoblasts. In addition, the results under different concentrations of baicalin pre-treatment were analyzed to identify an appropriate concentration with the most effective protective effect; the results revealed that 100 µM baicalin had the highest efficacy, and was selected as a model dose for further in vitro and in vivo experiments.
The present study subsequently observed that pre-treatment with baicalin suppressed the activation of the apoptotic C2C12 cell death pathway triggered by H2O2, which was similar to the results obtained in previous studies (7,8,13). Decreased apoptosis was found to be associated with the suppression of H2O2-stimulated oxidative activity in C2C12 myoblasts by baicalin, via effectively reducing the levels of ROS and MDA, which demonstrated that the protective effects of baicalin on C2C12 myoblasts were associated with reduced oxidative stress (15–17). Mitochondrial dysfunction was also observed in C2C12 myoblasts following exposure to H2O2, which was reflected by the loss of mitochondrial activity (Rhodamine 123 staining) and ΔΨm (JC-1 staining). Baicalin also reversed the effect of H2O2 and preserved mitochondrial function. One of the important functions of the mitochondria is the regulation of apoptosis (18). The present study demonstrated that pre-treatment with baicalin significantly upregulated the expression levels of cyto-C and AIF from the cytosolic and mitochondrial fractions of C2C12 myoblasts exposed to H2O2. In addition, caspase-3 and caspase-9 are commonly accepted indicators of apoptosis (15,19), and pre-treatment with baicalin in the present study downregulated the activities of caspase-3 and caspase-9 in the C2C12 myoblasts induced by H2O2 stimulation. In summary, these pathophysiological processes demonstrated the protective effects of baicalin on C2C12 myoblasts via inhibiting activation of the intrinsic apoptotic pathway, which was consistent with previously reported findings (13,18,20).
The present study further investigated the potential protective role of baicalin in vivo on the basis of animal models of skeletal muscle injury. Baicalin was pre-administered intra-muscularly 1 h prior to H2O2 injection at the same position, with the contralateral muscle receiving H2O2 injection only. At present, numerous imaging agents have been used for inflammation PET imaging, including 18F-FDG, 68Ga-Citrate and 64CuCl2 (21–27). 18F-FDG is produced by the cyclotron, which is easy and convenient to access, and has been widely used in a clinical setting (27). Therefore, in the present study, the animal models of skeletal muscle injury underwent small animal 18F-FDG PET imaging, which revealed that baicalin decreased ~65.9% of the FDG accumulation in the skeletal muscle injury induced by H2O2. In addition, baicalin inhibited and prevented the physiological uptake in normal leg muscle. These pathological findings confirmed the protective effect of baicalin on skeletal muscle injury. Therefore, baicalin was confirmed to effectively protect against the skeletal muscle injury induced by H2O2 in vivo.
However, the primary limitation of the present study was the mechanism of baicalin-mediated protection against apoptosis and inflammation, which was not fully elucidated and may be associated with molecular signaling, including nuclear factor erythroid 2-related factor 2 signaling, AMP-activated protein kinase, peroxisome proliferator-activated receptor-γ, proteasome, macrophages and notch signaling. These possibilities were not examined in the present study, and warrant further investigation (28–32).
In conclusion, the results of the present study suggested that pre-treatment with baicalin has the potential to protect myoblasts from apoptosis via decreasing the production of ROS and MDA, preserving mitochondrial function and reversing caspase protein expression, which was further verified by small animal PET imaging and pathological analysis. These results indicate the potential benefits of baicalin for patients with skeletal muscle injury.
Acknowledgements
Not applicable.
Funding
This study was supported by the National Science Foundation for Young Scholars of China (grant no. 81601675 to YP).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
ZL, YP and DS designed the study. YP, DS, WZ, XL and HW carried out the experiments and collected the data. ZL, YP and DS wrote and edited the manuscript.
Ethics approval and consent to participate
All procedures were approved by the Animal Ethics Committee at Shanghai East Hospital, Tongji University School of Medicine.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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