Animals and experimental design

Male Sprague–Dawley rats were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan). All rats were maintained in a pathogen-free facility under controlled environmental conditions with a 12:12-hour light:dark cycle at a stable temperature of 23 °C. The animals were provided with ad libitum access to water and a normal diet (ND) (CE-2; CLEA Japan Inc., Tokyo, Japan).

NAC administration and hindlimb I/R model

Male Sprague–Dawley rats (8–10 weeks old; body weight, 291.6 ± 23.3 g) were allocated to either control (Ctrl) or N-acetyl-L-cysteine (NAC) group without a formal randomization procedure. There was no statistically significant difference in baseline body weight between the two groups (each with n = 4, exact P-values for Ctrl vs. NAC; P = 0.782 [1d], P = 0.998 [7d], P = 0.999 [14d]). To assess outcomes at different stages, independent cohorts of animals were sacrificed at each specific time point (cross-sectional design), with an exact sample size of n = 4 per group, per time point. Some animals were excluded from final analysis due to predefined technical criteria, such as surgical failure or unexpected death. A priori sample-size estimation (power analysis) was not performed due to difficulty in reliably estimating effect sizes and variances from previous studies or preliminary data. Instead, the sample size (n = 4 per group) was determined based on numbers commonly utilized in similar I/R models [38], strictly adhering to the ethical principle of minimizing animal use. Using an anesthesia box, rats were anesthetized by inhalation of isoflurane (4.0%, ~ 2.7 MAC for induction) and intraperitoneal administration of a triad of anesthetics (medetomidine hydrochloride 0.375 mg/kg + midazolam 2.0 mg/kg + butorphanol tartrate 2.5 mg/kg). After anesthetized rats were secured in a supine position, the hair on the right lower side was shaved and the skin moistened with alcohol. A longitudinal incision, approximately 1 cm in length, was made along the inner thigh, and the femoral arteries were dissected. To investigate its effects on I/R injury, NAC was administered intravenously (150 mg/kg) 15 minutes (min) before the initiation of reperfusion. Following this initial dose, the NAC group received continuous supplementation of NAC in drinking water (600 mg/L) throughout the experimental period [39], while the Ctrl group received an equivalent volume of saline intravenously 15 min before reperfusion and regular drinking water thereafter. For the I/R procedure, ischemia was induced in the right hindlimb by clamping the femoral artery for 4 hour (h), combined with applying a proximal rubber tourniquet to block collateral blood flow. Reperfusion was initiated by removal of both the clamp and tourniquet. The contralateral left hindlimb served as internal sham control, receiving only a skin incision and partial femoral artery exposure under the same anesthetic conditions.

Animal ethics

Rats were euthanized using a humane procedure, taking into consideration alleviation of suffering. Using an anesthesia box, rats were anesthetized by inhalation of isoflurane and intraperitoneal administration of a triad of anesthetics (medetomidine hydrochloride 0.375 mg/kg + midazolam 2.0 mg/kg + butorphanol tartrate 2.5 mg/kg). After determining that pain relief was adequate, we euthanized rats by cervical dislocation. All animal experiments were carried out in accordance with the Institutional Guidelines on Animal Experimentation at Kumamoto University, and animal experiment procedures were approved by the Animal Studies Committee and the Institutional Animal Care and Use Committee at Kumamoto University, Japan. This study is reported in accordance with ARRIVE guidelines.

Hindlimb blood flow assessment

Hindlimb blood flow was measured in separate cohorts at baseline (before surgery), 4 h after ischemia, and at 1, 7 and 14 days after reperfusion using a two-dimensional laser Doppler flowmetry system (OMEGA ZONE, Omega Wave Co., Tokyo, Japan). This laser speckle blood flow (LSBF) analyzing system was used to obtain representative images of perfused tissues. During the procedure, the same I/R protocol and NAC administration (intravenous injection, followed by supplying drinking water containing NAC) were performed as described above. To avoid data variations caused by environmental factors such as ambient light and temperature, hindlimb blood flow was expressed as the ratio of values obtained from the ischemic (right) versus contralateral control (left) hindlimb.

Tissue preparation and sectioning

At indicated time points after reperfusion, rats were deeply anesthetized, and transcardial perfusion fixation was performed with 4% paraformaldehyde. The gastrocnemius (GAS) and soleus (SOL) muscles were then harvested and immersion-fixed in 4% paraformaldehyde for 24 hours at 4 °C. Tissues were then dehydrated through a graded series of ethanol, cleared in xylene, and embedded in paraffin. Paraffin-embedded tissues were then sectioned to 4 μm thickness using a microtome (REM-710; Yamato Kohki Industrial Co., Ltd., Saitama, Japan).

Immunofluorescence staining

To evaluate cell apoptosis in muscle tissues, fluorescent immunohistochemistry was performed using the MEBSTAIN Apoptosis TUNEL Kit Direct (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) according to the manufacturer’s instructions. After deparaffinization and rehydration, tissue sections were subjected to antigen retrieval by incubation with Proteinase K (provided in the kit) for 30 min at 37 °C. Sections were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature to minimize non-specific binding. Sections were then stained with the TUNEL reaction mixture, and nuclei were counterstained with propidium iodide (PI; 29037−76; Nacalai Tesque, Inc., Kyoto, Japan). TUNEL-positive cells were detected and captured using a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan). To quantify apoptotic cells, images were captured at 400 × magnification. Ten random fields from the GAS and five from the SOL were evaluated per section. The number of TUNEL-positive nuclei and the total number of PI-stained nuclei were counted in each field, and the apoptotic index was calculated as the percentage of TUNEL-positive nuclei relative to the total number of nuclei (TUNEL-positive nuclei/ total nuclei × 100).

Immunohistochemistry

After deparaffinization and rehydration, sections were subjected to antigen retrieval by microwave treatment for 10 min in 10 mM citrate buffer solution (pH 6.0). Endogenous peroxidase activity was blocked with 3% H₂O₂ for 10 min. To minimize non-specific reactions, sections were incubated with Blocking One Histo (Nacalai Tesque, Inc., Kyoto, Japan) for 10 min at room temperature. Sections were then incubated overnight at 4°C with a mouse monoclonal antibody against 8-hydroxy-2’-deoxyguanosine (8-OHdG) (ab48508, 1:160; Abcam, Cambridge, UK). Samples were then incubated 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody (ab97040, goat anti-mouse, 1:400; Abcam). Immunoreactivity was visualized using 3,3’-diaminobenzidine (DAB) chromogen, and nuclei were counterstained with hematoxylin. Sections were observed under a light microscope (BZ-X800, Keyence, Osaka, Japan). To quantify 8-OHdG-positive cells, images were captured at 400 × magnification. Ten random fields from the GAS and five from the SOL were evaluated per section. The number of 8-OHdG-positive nuclei and the total number of hematoxylin-stained nuclei were counted in each field, and the percentage of 8-OHdG-positive cells was calculated the number of 8-OHdG-positive nuclei relative to the total number of nuclei (8-OHdG-positive nuclei/ total nuclei × 100).

Masson’s trichrome staining

Muscle fibrosis was evaluated by Masson’s trichrome staining using reagents from Wako Pure Chemical Industries (Osaka, Japan) and Muto Pure Chemicals (Tokyo, Japan). After deparaffinization and rehydration, paraffin sections were stained with Weigert’s iron hematoxylin (Wako) for 5 min, followed by staining with Ponceau xylidine-acid fuchsin solution (Wako) for 30 min. Sections were then incubated in a mixture of Aniline blue and Orange G (Muto) for 8 min. Between each staining step, sections were rinsed with 1% acetic acid. Collagen fibers were stained blue, nuclei black, and muscle fibers red. Sections were observed under a light microscope (BZ-X800, Keyence, Osaka, Japan). To evaluate collagen-positive areas quantitatively, images were captured at 400 × magnification. Ten random fields from the GAS and five from the SOL were evaluated per section. The percentage of collagen-positive area was calculated as the ratio of the area stained with aniline blue to the total tissue area (aniline blue-stained area/ total tissue area × 100).

Histological assessment

Although investigators were not strictly blinded while conducting experimental procedures or image acquisition, observer bias was minimized during the subsequent histological quantification. To do so, we utilized automated image quantification software integrated into the BZ-X800 microscope system (Keyence, Osaka, Japan). Target areas and positive cells were mechanically detected and quantified across all samples based on uniform, pre-set threshold parameters.

Cell culture

For in vitro experiments, C2C12 myoblast cells were cultured in growth medium (Dulbecco’s Modified Eagle Medium (DMEM)) supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin, and 100 µg/mL streptomycin) and maintained at 37 °C in a humidified atmosphere with 5% carbon dioxide (CO₂). Cells were treated with or without 4 μM H₂O₂ (Wako) for 4 h to induce oxidative stress. For some experiments, cells were pre-incubated with various concentrations of NAC (4, 20, 100, and 500 μM) for 15 min prior to H₂O₂ stimulation. For mitogen-activated protein kinase (MAPK) inhibitor analysis, cells were cultured in the presence of 4 μM H₂O₂ with 0.4 μM U0126 (MAPK/ERK (Extracellular signal-regulated kinase) 1/2 (MEK1/2) inhibitor) (S1460; Selleck Chemicals, Houston, TX, USA), 0.4 μM SB203580 (p38 MAPK inhibitor) (S1076; Selleck Chemicals), or 0.4 μM SP600125 (c-Jun N-terminal kinase (JNK) inhibitor) (#662005; Calbiochem, San Diego, CA, USA). An equivalent volume of PBS was used as a vehicle control.

ROS assays

Intracellular ROS accumulation was evaluated using a Highly Sensitive 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA)-ROS Assay Kit (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, after washing C2C12 cells twice with Hanks’ Balanced Salt Solution (HBSS), cells were incubated with DCFH-DA dye working solution for 30 min. Fluorescence signals were observed and captured using a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan). Quantification of fluorescence intensity was performed using the BZ-X800 analyzer software (Keyence).

Glutathione disulfide (GSSG)/ Glutathione (GSH) quantification

GSSG/GSH accumulation was evaluated using a GSSG/GSH Quantification Kit (G257; Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, C2C12 cells (1 × 10⁷ cells) were washed with PBS and lysed with 10 mmol/L hydrogen chloride (HCl) through two freeze-thaw cycles. After adding 5% 5-sulfosalicylic acid (SSA) and centrifugation at 8,000 × g for 10 min, the supernatant was collected and diluted with deionized water to a final SSA concentration of 0.5%. Absorbance was then measured at 405 nm using a PowerScan HT microplate reader (DS Pharma Biomedical Co., Ltd., Osaka, Japan).

Real-time polymerase chain reaction (PCR)

Total RNA was isolated from cells using TRIzol reagent (Invitrogen, Tokyo, Japan). Complementary DNA (cDNA) was synthesized from total RNA using PrimeScript RT Master Mix (Takara Bio Inc., Shiga, Japan). Real-time PCR was performed using TB Green Premix Ex Taq II (Takara Bio Inc.) on a Thermal Cycler Dice Real Time System (Takara Bio Inc.), according to the manufacturer’s instructions. Relative expression levels were determined using a standard curve generated from serially-diluted cDNA products. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) served as internal control. Mouse-specific primer sequences used for C2C12 cell analysis were as follows:

1. Gapdh forward: 5’-GTCATGGGTGTGAACCATGAGAAG-3’;
2. Gapdh reverse: 5’-AGTCTTCTGGGTGGCAGTGATG-3’;
3. Bcl2 forward: 5’-CCTGTGGATGACTGAGTACCTG-3’;
4. Bcl2 reverse: 5’-AGCCAGGAGAAATCAAACAGAGG-3’;
5. Bax forward: 5’-AGGATGCGTCCACCAAGAAGCT-3’;
6. Bax reverse: 5’-TCCGTGTCCACGTCAGCAATCA-3’;
7. Caspase-3 (Casp3) forward: 5’-GGAGTCTGACTGGAAAGCCGAA-3’;
8. Casp3 reverse: 5’-CTTCTGGCAAGCCATCTCCTCA-3’;
9. Col1a1 forward: 5’-ACTGTCCCAACCCCCAAAG-3’;
10. Collagen type 1 chain (Col1a1) reverse: 5’-ACGTATTCTTCCGGGCAGAA-3’;
11. Col3 forward: 5’-AACCTGGTTTCTTCTCACCCTTC-3’;
12. Col3 reverse: 5’-ACTCATAGGACTGACCAAGGTGG-3’;
13. Transforming growth factor beta 1 (Tgfb1) forward: 5’-CACCGGAGAGCCCTGGATA-3’;
14. Tgfb1 reverse: 5’-TGTACAGCTGCCGCACACA-3’;
15. Tgfb2 forward: 5’-CGAGGCGAGATTTGCAGGTATT-3’;
16. Tgfb2 reverse: 5’-TTAGCAGGAGATGTGGGGTCTT-3’;
17. Fibroblast growth factor 2 (Fgf2) forward: 5’-AAGCGGCTCTACTGCAAGAACG-3’;
18. Fgf2 reverse: 5’-CCTTGATAGACACAACTCCTCTC-3’;
19. Ccn2 forward: 5’-CAAAGCAGCTGCAAATACCA-3’;
20. Ccn2 reverse: 5’-GGCCAAATGTGTCTTCCAGT-3

Western blot analysis

C2C12 cells were serum-starved (no FBS) in DMEM for 24 h and then stimulated with 4 μM H₂O₂ for various times. Cell lysates were collected, and Western blot analysis was performed using polyclonal antibodies for ERK (#9102), phosphorylated ERK (#9106), p38 MAPK (#8690), phosphorylated p38 MAPK (#9211), JNK (#9102), and phosphorylated JNK (#9255). All antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) and used at a 1:1000 dilution.

Statistical analysis

All numerical data are presented as means ± SD (standard deviation). The Shapiro-Wilk test was used to assess normality of data distribution before conducting statistical analyses. For histological comparisons between the ischemic (right) and contralateral sham (left) limbs in the same animal, a paired t-test was employed. For comparisons of the blood-flow ratio (ischemic relative to contralateral control limb) between treatment groups, we used Student’s t-test. For comparisons among three or more groups across different time points, muscles, and treatment groups, we performed one-way analysis of variance (ANOVA), followed by the Tukey–Kramer post hoc test to adequately control for multiple testing. A p-value less than 0.05 was considered statistically significant (*p < 0.05; **p < 0.01; NS, not significant, throughout the paper).

Accumulation of oxidative DNA damage in rat gastrocnemius and soleus muscles is induced after I/R

First, we established an animal model using rat hindlimbs as a limb I/R model by combining femoral artery clamping with a tourniquet applied to the thigh. As noted (see Introduction), although clinically relevant, such models tend to be inconsistent due to complexity of the surgical procedure [29,30]. In contrast, I/R injury is milder following application of vascular clamping or tourniquet methods, and these techniques offer a more stable and reproducible model [32–34,37]. Thus, here, we combined femoral artery clamping with a tourniquet applied to the thigh, releasing both after 4 hours to create an I/R model (Fig 1A). The contralateral lower limb underwent a sham procedure with a skin incision only. After reperfusion, tissue specimens were collected from the bilateral gastrocnemius and soleus muscles on days 1, 7, and 14. Tissue samples were then prepared and evaluated for oxidative DNA damaged using anti-8-OHdG antibody (Fig 1B). We confirmed significant accumulation of damaged DNA, based on 8-OHdG positivity, in both gastrocnemius and soleus muscles at all time points evaluated (Fig 1B).

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Fig 1. Oxidative stress is induced in rat hindlimb of an ischemia–reperfusion (I/R) model.

(A) Representative photos illustrating the rat hindlimb I/R model. Ischemia was induced by clamping right femoral artery (black arrow) for 4 h, combined with application of a proximal rubber tourniquet to block collateral blood flow (leftmost and second-from-left panels). The left hindlimb, which served as a Sham control, received only a skin incision and partial femoral artery exposure. Tourniquet is shown in third panel from left. Far right shows a rat with the femoral artery clamped, the wound closed, and the tourniquet applied to the thigh. (B) (Left) Paraffin sections of GAS and SOL muscles prepared 1, 7 and 14 days after reperfusion (as shown in A) and stained with a biotin-conjugated anti-8-OHdG antibody. Immunoreactivity was visualized using the streptavidin-peroxidase method with 3,3’-diaminobenzidine (DAB) chromogen, and nuclei were counterstained with hematoxylin. Representative images are shown. Scale bar: 100 μm. Right, graphs show quantification of staining 8‑OHdG–positive cells shown as a proportion of total cells. Data are presented as mean ± SD (each with n = 4, *P < 0.05, **P < 0.01 [n.s. = not significant] vs. sham limb; all P < 0.01).

https://doi.org/10.1371/journal.pone.0357829.g001

Administration of the antioxidant NAC significantly decreases oxidative DNA damage promoted by I/R in rat gastrocnemius and soleus muscle

Next, we administered the antioxidant NAC to our rat I/R model (Fig 2A). After clamping the femoral artery and applying a thigh tourniquet for 4 hours, 150 mg/kg NAC (NAC group) or an equal volume of saline (Ctrl group) was administered intravenously to rats, and 15 minutes later, the clamp and tourniquet were released to restore blood flow. Thereafter, 600 mg/L NAC water (NAC group) or normal water (Ctrl group) was administered continuously in drinking water until the end of the experiment (Fig 2A). Relative to controls, NAC administration significantly suppressed accumulation of DNA damaged by oxidation in gastrocnemius muscles across all time points, and in soleus muscles on days 1 and 7, whereas this effect was not statistically significant on day 14 following administration (Fig 2B).

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Fig 2. NAC administration reduces oxidative DNA damage in skeletal muscle in a rat limb I/R injury model.

(A) Schematic showing N-acetyl-L-cysteine (NAC) administration protocol in rat hindlimb ischemia–reperfusion (I/R) model. The NAC group received NAC intravenously (150 mg/kg) 15 min before reperfusion, followed by continuous NAC supplementation (600 mg/L) in drinking water throughout the experimental period. The control (Ctrl) group was injected intravenously an equivalent volume of saline (N/S: Normal Saline) 15 min before reperfusion, and then NAC-free drinking water was provided thereafter. (B) Rats were allocated to Ctrl (saline) or NAC groups prior to I/R without a formal randomization procedure. Following reperfusion, animals were sacrificed on days 1, 7 and 14 and paraffin sections of GAS and SOL muscles were prepared. Sections were incubated with biotin-conjugated anti-8-OHdG antibody. Immunoreactivity was visualized using a streptavidin–peroxidase system with 3,3′-diaminobenzidine (DAB) chromogen. Nuclei were counterstained with hematoxylin. Representative images are shown. Scale bar: 100 μm. (C) Quantification of 8-OHdG-findings shown in (B). Shown is percentage of 8-OHdG-positive cells relative to total cells at each time point presented as mean ± SD (each with n = 4, *P < 0.05, **P < 0.01 [n.s. = not significant] for comparisons among sham limb, Ctrl, and NAC groups; exact P-values for Ctrl vs. NAC: GAS, P = 0.015 [1d], P = 0.039 [7d], P = 0.036 [14d]; SOL, P = 0.027 [1d], P = 0.041 [7d], P = 0.068 [14d]).

https://doi.org/10.1371/journal.pone.0357829.g002

NAC administration significantly suppresses muscle cell apoptosis induced by I/R in rat gastrocnemius and soleus

To evaluate the extent of apoptotic cell death after potential I/R injury, we performed TUNEL assays of gastrocnemius and soleus tissues following treatment with or without NAC. In the Ctrl (no treatment) group, we observed significantly higher percentage of TUNEL-positive cells in both muscles at 1, 7 and 14 days after clamp and tourniquet removal compared with Sham group, and that staining was significantly suppressed by NAC treatment at all time points in gastrocnemius muscles, whereas in soleus muscles, significant suppression was observed only on day 1 (Fig 3A and 3B).

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Fig 3. NAC administration blocks skeletal muscle cell apoptosis in a rat limb I/R injury model.

(A) Prior to I/R, rats were assigned to Ctrl or NAC groups and maintained under conditions shown in Fig 2A. On days 1, 7 and 14 after reperfusion, paraffin sections of GAS and SOL muscles were prepared and labeled with biotin-dUTP using terminal deoxynucleotidyl transferase (TdT), followed by avidin-DTAF to identify apoptotic cells (TUNEL-positive, green). Nuclei were counterstained with propidium iodide (PI, red), and sections were observed under a fluorescence microscope. Scale bar: 100 μm. (B) Graphs show percentage of TUNEL-positive nuclei relative to total nuclei at each time point. Data are presented as mean percentage of TUNEL-positive nuclei relative to total nuclei ± SD (each with n = 4, *P < 0.05, **P < 0.01 [n.s. = not significant] for comparisons among sham limb, Ctrl, and NAC groups; exact P-values for Ctrl vs. NAC: GAS, P = 0.0001 [1d], P = 0.044 [7d], P = 0.046 [14d]; SOL, P = 0.049 [1d], P = 0.281 [7d], P = 0.171 [14d]).

https://doi.org/10.1371/journal.pone.0357829.g003

We also assessed blood flow in both lower limbs of the NAC and Ctrl groups using two-dimensional laser Doppler flowmetry (Fig 4). In both groups, blood flow decreased to the same level 4 hours after femoral artery clamping and femoral tourniquet application, and recovered to baseline levels 1 day after clamp and tourniquet release (Fig 4A and 4B). Seven days after clamp and tourniquet release, blood flow was slightly but significantly higher in the NAC compared to the Ctrl group (Fig 4A and B), but by 14 days there were no differences between groups (Fig 4A and 4B). These findings suggest that amelioration of I/R injury promoted by NAC administration was likely due to decreases in oxidative stress. However, although the presence of NAC did not promote sustained differences in gross hindlimb blood-flow recovery, we cannot exclude the possibility that altered microvascular perfusion or vascular protection contributed to this effect.

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Fig 4. NAC administration increases blood flow to hindlimb in a rat limb I/R injury model.

(A) Rats treated according to protocol shown in Fig 2A were subjected to two-dimensional laser Doppler flowmetry at each designated time point. Shown are representative images of hindlimb tissue at baseline, at 4 h after ischemia, and at indicated days after reperfusion. Black arrows indicate ischemic limb (right hindlimb). (B) Graph shows mean blood flow in ischemic relative to contralateral control limb. Data for both groups are presented as mean blood flow in ischemic versus control limb ± SD (each with n = 4, *P < 0.05, **P < 0.01 [n.s. = not significant] vs. Ctrl group; exact P-values for Ctrl vs. NAC: P = 0.48 [1d], P = 0.0153[7d], P = 0.69 [14d]).

https://doi.org/10.1371/journal.pone.0357829.g004

I/R induces tissue fibrosis, which in rat models is improved by NAC administration

Even when replantation via vascular anastomosis is successful after finger or limb amputation, functional impairment due to tissue contracture often persists [40]. Since it is known that tissue contracture is induced by fibrosis [41], Masson trichrome staining was performed to evaluate tissue collagen deposition and fibrosis. In the control group (no treatment), after restoring blood flow following femoral artery clamping and tourniquet application, we observed significantly higher accumulation of Masson trichrome-stained collagen fibers in both GAS and SOL muscles on days 1, 7, and 14 days after reperfusion compared with Sham group, indicative of collagen deposition and fibrosis (Fig 5A and 5B). Interestingly, NAC administration (following protocol shown in Fig 2A) significantly reduced the I/R-induced Masson trichrome-stained positive areas in gastrocnemius muscles across all time points and in soleus muscles on day 1, but not on days 7 and 14 (Fig 5A and 5B).

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Fig 5. NAC administration suppresses skeletal muscle fibrosis in a rat limb I/R injury model.

(A) Prior to I/R, rats were assigned to Ctrl or NAC groups and maintained as indicated in Fig 2A. On day 7 after reperfusion, paraffin sections of GAS and SOL muscles were prepared and stained using Masson’s trichrome protocol. Nuclei stained dark purple with iron hematoxylin; muscle fibers and cytoplasm stained red with acid fuchsin; and collagen fibers stained blue with aniline blue. Representative images are shown. Scale bar: 100 μm. (B) Quantification of collagen-positive (blue-stained) areas in GAS and SOL muscles. Graphs show proportion of the collagen-positive area relative to total tissue area. Data are presented as mean percentage of collagen-positive to total tissue areas ± SD (each with n = 4, *P < 0.05, **P < 0.01 [n.s. = not significant] for comparisons among sham limb, Ctrl, and NAC groups; exact P-values for Ctrl vs. NAC: GAS, P = 0.024 [1d], P = 0.015 [7d], P = 0.004 [14d]; SOL, P = 0.019 [1d], P = 0.117 [7d], P = 0.059 [14d]).

https://doi.org/10.1371/journal.pone.0357829.g005

Oxidative stress induces expression of apoptosis-inducing factors in myoblasts

To investigate mechanisms underlying tissue damage caused by oxidative stress induced by I/R, we conducted analysis in in vitro cultured C2C12 myoblasts, treated with and without H₂O₂, an oxidative stress agent (Fig 6). Treatment of C2C12 myoblasts for 4 hours with 4 μM H₂O₂ significantly increased ROS levels relative to untreated cells, based on by fluorescent staining with DCFH-DA. Co-treatment of cells with NAC significantly suppressed ROS levels induced by H₂O₂ (Fig 6A). Conversely, H₂O₂ treatment significantly decreased levels of the antioxidant glutathione (GSH) in C2C12 cells relative to controls, but those levels were significantly rescued by NAC co-administration (Fig 6B). H₂O₂ treatment also significantly increased expression of Bax and Caspase 3 (Casp3), both apoptosis-inducing factors [42,43], an increase significantly blocked by NAC co-administration (Fig 6C). By contrast, expression of the apoptosis inhibitor Bcl2 [44] in C2C12 myoblasts was unchanged by H₂O₂ stimulation, although its levels were significantly increased by NAC co-administration (Fig 6C).

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Fig 6. NAC suppresses H₂O₂-induced oxidative stress and expression of pro-apoptotic genes in C2C12 myoblasts.

(A) C2C12 myoblasts were treated 4 h with or without H₂O₂ (4 μM) in the presence or absence of NAC (500 μM). (Left) ROS were visualized in representative images of indicated groups using the fluorescent probe DCFH-DA. (Right) Quantification of fluorescence intensity expressed in arbitrary units. Scale bar: 200 μm. (B) Quantification of GSH levels in indicated C2C12 myoblasts using a colorimetric assay based on redox-dependent chromogenic reactions. (C) Levels of Bax, Casp3 or Bcl2 mRNA in indicated C2C12 myoblasts relative to Gapdh based on qPCR. Data are presented as fold-change relative to the control cells not treated with H₂O₂ or NAC. For all analyses, data are shown as mean indicated gene expression relative to Gapdh ± SD (n = 6, *P < 0.05, **P < 0.01 vs. sham; n.s. = not significant).

https://doi.org/10.1371/journal.pone.0357829.g006

Oxidative stress induces expression of fibrosis-related factors in myoblasts

We next analyzed effects of oxidative stress on fibrosis in C2C12 cells (Fig 7). Treating C2C12 myoblasts 4 hours with 4 μM H₂O₂ significantly increased expression of collagen genes Col1a1 and Col3 (Fig 7A), an effect significantly suppressed by NAC co-administration (Fig 7A). The expression of Tgfb1, Tgfb2, Fgf2, and Ccn2, all factors associated with tissue fibrosis [45–47], significantly increased in C2C12 myoblasts following H₂O₂ treatment (Fig 7B), and among these factors, increased Ccn2 expression was significantly suppressed when NAC was co-administered together (Fig 7B).

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Fig 7. NAC inhibits H₂O₂-induced expression of genes associated with fibrosis and related signaling pathways in C2C12 myoblasts.

(A) C2C12 myoblasts were treated 4 h with or without H₂O₂ (4 μM) in the presence or absence of NAC and assessed for levels Col1a1 (left) or Col3 (right) transcripts relative to Gapdh based on qPCR. Data are presented as fold-change relative to control cells not treated with either H₂O₂ or NAC. Data are shown as mean ± SD (n = 6, *P < 0.05, **P < 0.01 vs. sham; n.s. = not significant). (B) C2C12 myoblasts were treated as described in (A) and then assessed for transcript levels of Tgfb1, Tgfb2, Fgf2 or Ccn2 relative to Gapdh, based on qPCR. Data are presented as fold-change relative to control cells not treated with H₂O₂ or NAC. Data are shown as mean ± SD (n = 6, *P < 0.05, **P < 0.01 vs. sham; n.s. = not significant). (C) C2C12 cells were serum-starved for 24 h and stimulated with 4 μM H₂O₂ for indicated times. Then, cell lysates were collected, and phosphorylated and non-phosphorylated forms of ERK, p38 MAPK and JNK protein were examined by western blot. Representative images are shown. (D) C2C12 cells were cultured with 4 μM H₂O₂ in the presence or absence (vehicle) of a MEK1/2 inhibitor (0.4 μM U0126), a p38 MAPK inhibitor (0.4 μM SB203580), or a JNK inhibitor (0.4 μM SP600125) for 4 h, and Col1a1, and Gapdh expression was analyzed by qPCR. NC, no template control. (each with n = 6, *P < 0.05, **P < 0.01 [n.s. = not significant] for comparisons among vehicle, U0126, SB203580, and SP600125 groups; exact P-values for vehicle vs. SB203580: P = 0.036).

https://doi.org/10.1371/journal.pone.0357829.g007

Finally, we asked which signaling pathways are activated by oxidative stress to induce fibrosis in myoblasts. To do so, we treated C2C12 myoblasts after 24-hour serum-starvation with 4 μM H₂O₂ for 90 minutes and collected cell lysates at time zero and various time points thereafter for Western blot analysis (Fig 7C). For that analysis we used antibodies specific for total and phosphorylated forms of proteins in the MAPK pathway, namely p38, JNK, and ERK. Interesting, we observed that H₂O₂ treatment specifically increased p38 phosphorylation in C2C12 myoblasts (Fig 7C). We then cultured C2C12 myoblasts with or without H₂O₂ in the presence or absence (vehicle) of p38 (SB203580), JNK (SP600125) or ERK (U0126) inhibitor, and we found that H₂O₂-induced Col1a1 expression was specifically and significantly inhibited by p38 inhibitor (Fig 7D).