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.

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.

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.

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.

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.

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.

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 H₂O_2, 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.

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).

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).

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.

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.

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.

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).

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.

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.

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).

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.

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).