In this study, 4-week-old male C57BL/6 J mice were purchased and tested after undergoing a 1-week acclimation period. The room was maintained at a controlled temperature (18-22°C) and humidity (40-60%) on a 12-h light-dark cycle. Water and feed were provided ad libi-tum during the breeding period.

At 5 weeks of age, the mice were randomly assigned to two groups: sedentary (SED, n=12) and endurance training (ETR, n=12). The mice were then treated for a duration of 12 weeks with ETR. At the end of the 12-week training period, the SED group was further categorized into the SCON (SED and control, n=6) and SPM (SED and PM_2.5 exposure, n=6) groups, while the ETR group was categorized into the ECON (ETR and control, n=6) and EPM (ETR and PM_2.5 exposure, n=6) groups. After the completion of all treatments, skeletal muscle was harvested at 18 hours after the last PM_2.5 exposure. An overview of the experimental design can be found in the Fig. 1. All experimental procedures were conducted in accordance with the guidelines and regulations set by the Institutional Animal Care and Use Committee of Jeonbuk National University (IACUC approval no. CBNU-2022-0067).

The ASC system is a whole-body exposure device designed to repli-cate the inhalation of PM_2.5 in the atmosphere while maintaining a con-sistent average concentration. The PM solution was formulated by mixing 10 organic and inorganic compounds, including oxalic acid, malonic acid, glutaric acid, sucrose, 2,5-dihydroxybenzoic acid, glycine, ammonium sulfate, ammonium nitrate, acetate, and glycerol, into distilled water (Table 1). The PM was aerosolized using a nebulizer (TQ-50-C0,5; Mein-hard, USA) and, subsequent to passing through a polypropylene melt-blown filter, particles larger than 2.5 μm are sieved out and entered the chamber. The concentration of PM_2.5 within the chamber is continuous-ly monitored in real-time using a particle counter (BT-610; Met One, USA), and predetermined concentration was automatically maintained through a flow controller program. SPM and EPM group were exposed to PM_2.5 for 1 hour per day, for three consecutive days, at a concentration of 52.7±7.4 μg/m³ (Table 2). The chamber was maintained a humidity level of 55-60% and a temperature range of 23-25°C.

The organic components and inorganic salts for the preparation of artificial PM_2.5 are listed in Table 1. Eight organic compounds with car-boxylic acid, polyol, sugar, aromatic, and amino acid functional groups were investigated. Ammonium sulfate and ammonium nitrate were used as the model inorganic salts because of their abundance in air [13-16]. The 10 compounds in Table 1 were mixed at an organic to inorganic dry mass ratio of 1:1 to mimic the chemical complexity of atmospheric aerosols. The compounds were purchased from Sigma-Aldrich (purity ≥98%) and were used without further purification. The mixture of 10 components was dissolved in purified water.

Endurance training was conducted using a treadmill. Prior to the ini-tiation of the 12-week program, all mice underwent treadmill adaptation exercise for 1 week, which consisted of running at a speed of 10 m/min for 20-30 minutes, three times per week.

Following the adaptation treadmill exercise, the exercise group received a training program on the treadmill with a 0% incline. One-time exercise program included a 10-minute warm-up period at a speed of 10 m/min, a 45-minute main exercise period at a speed of 15 m/min, and a 5-minute cool-down period at a speed of 10 m/min. This program was repeated 5 times per week. The speed for the main exercise period was set at 60-70% of the maximum running speed determined through the endurance exercise capacity (EEC) test, as described [17]. EEC test for setting running speed was conducted after the mice had adapted to the treadmill.

This study confirmed the differences in fitness levels among the groups through EEC test. The progressive exercise test for EEC was conducted on all mice 24 hours after the completion of the 12-week exercise program to assess the impact of endurance training. The EEC test was performed on a treadmill with a fixed slope of 15 degrees. Initially, the treadmill speed was set at 10 m/min for the first 5 minutes, and subsequently, the speed was increased by 2 m/min every minute. During the test, if a mouse was unable to continue running despite sponge stimulation and remained on the electric shock grid for more than 3 seconds, the test was stopped. The total running time, vertical distance, and work were measured or calculated using methodologies described in previous studies [18].

The analysis of protein expression was conducted using the gastrocnemius muscle (GAS), which was rapidly frozen in liquid nitrogen upon extraction and subsequently stored at −80°C. The GAS samples were ho-mogenized in a cold lysis buffer [50 mM Tris·HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM ethylenediaminetet-raacetic acid (EDTA, pH 7.4), 1 mM Pefabloc (Roche, Basel, Switzerland), 1 mM NaF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 0.1 mM bpV(phen), and 2 mg/mL β-glycerophosphate] kept on ice.

The homogenate was solubilized in Laemmli sample buffer after de-termining the protein concentration using the Bradford (BIO-RAD, Bio-Rad Protein Assay Dye Reagent Concentrate, CA, USA) [19]. Following gel electrophoresis, each sample was transferred onto nitrocellulose membranes and blocked with skim milk at room temperature. Subse-quently, the membranes were incubated overnight at 4°C with primary antibodies [PGC-1α (GTX37356, GeneTex, CA, USA); NADH: ubiqui-none oxidoreductase subunit A9 (NADH, ab110242, Abcam, MA, USA); succinate dehydrogenase B (SDHB, ab14714, Abcam); cytochrome oxi-dase (COX) subunit 1 (COX-1, ab14705, Abcam,); mitofusin 1 (MFN-1, sc-166644, Santa Cruz Biotecnology), MFN-2 (sc-515647, Santa Cruz Biotecnology), optic atrophy 1 (OPA-1, sc-393296, Santa Cruz Biotecnology), mitochondria fission 1 protein (Fis-1, sc-376447, Santa Cruz Biotecnology), dynamin-related protein 1 (Drp-1, sc-32898, Santa Cruz Biotecnology), superoxide dismutase (SOD) type 1 (SOD-1, sc-8637, Santa Cruz Biotechnology), SOD-2 (sc-18503, Santa Cruz Biotechnology); catalase (sc-271358, Santa Cruz Biotechnology); glutathione peroxidase 4 (GPx-4, Santa Cruz Biotechnology), β-actin (MA1-140 Invitrogen, MN, USA)], and then subjected to further incubation with appropriate secondary antibodies [mouse anti-goat (sc-2354, Santa Cruz Biotechnology); mouse anti-rabbit (sc-2357, Santa Cruz Biotechnology); goat anti-mouse (sc-2005, Santa Cruz Biotechnology)] for protein detection. Protein visualization was conducted using the ECL Western Blotting Detection Reagent (GE Healthcare, Chalfont St Giles, UK), and quantification was carried out using the ChemiDoc XRS+ system (BIO-RAD, Hercules, CA, USA).

Transmission electron microscopy (TEM) was used to examine the mitochondrial structure. The soleus muscle was fixed in a solution containing 2.5% glutaraldehyde and 4% formaldehyde and a 0.1 M phosphate buffer at pH 7.4 for 2 hours immediately after extraction. The fixed muscles were post-fixed with 1% osmium tetroxide for 2 hours. The muscles were then dehydrated using a graded series of ethanol solutions and em-bedded in Epon-812 resin. Sections were obtained using a NOVA ultra-microtome (LKB, Vienna, Austria) and mounted on a 100-mesh grid. Sections approximately 80 nm thick were prepared for TEM and stained with 0.1% toluidine blue. To enhance visualization, the sections were stained with uranyl acetate and lead citrate and examined using an H7650 electron microscope (Hitachi, Japan) with an accelerating voltage of 80 kV to confirm the specimens. Samples were analyzed using a JEM-2010 TEM (JEOL) at the Center for University Wide Research Facilities at Jeonbuk National University.

All data were presented as mean±standard deviation (SD) and were analyzed using GraphPad Software (Prism 10, MA, USA). In order to verify the effects of endurance training for 12 weeks, an independent t-test was performed to compare the SED and ETR groups. Comparisons between groups (SCON, SPM, ECON, and EPM) after PM_2.5 exposed following endurance training were performed with two-way ANOVA followed by Tukey's HSD post-hoc test to confirm a significant difference (p<.05).

Order to verify whether fitness differences have a certain effect on skeletal muscle mitochondrial damage caused by short-term PM_2.5 exposure. We first subjected mice in the growth period to low to moderate-intensity ETR for 12 weeks (Fig. 1) and confirmed the EEC of each group after the exercise to clarify the differences in fitness levels between the groups.

After 12 weeks, the ETR group had significantly higher body weight (p <.01), maximum running time (p <.001), Vertical distance (p <.001), and work (p <.001) levels than the SED group (Fig. 2). Therefore, the ETR group undoubtedly had higher fitness levels.

The expression levels of PGC-1α, a regulator of mitochondrial biogenesis, and enzymes of the electron transport chain are shown in Fig. 3. The expression levels of PGC-1α and NADH are regulated by the interaction between ETR and PM_2.5 exposure (Fig. 3B and C). And the expression levels of SDHB and COX-1 are regulated solely by the independent effect of ETR (Fig. 3D and E). 6 days after the end of exercise, NADH (Fig. 3C; SCON vs. ECON, p <.01), SDHB (Fig. 3D; SCON vs. ECON, p <.05) and COX-1 (Fig. 3E; SCON vs. ECON, p <.05) expression levels in the ECON group remained significantly higher than those in the SCON group. Although short-term PM_2.5 exposure only caused a significant decrease in the expression levels of mitochondrial enzymes NADH at EPM group (Fig. 3C; ECON vs. EPM, p <.05). However, the ETR group with high fitness levels showed a significant increase in PGC-1α expression levels after short-term PM_2.5 exposure (Fig. 3B; ECON vs. EPM, p<.01). These results indicate that high fitness levels exhibit greater reactivity of biogenesis signaling in response to PM_2.5-induced mitochondrial damage stress.

The expression levels of mitochondrial dynamics-related regulatory factors are shown in Fig. 4. The expression of mitochondrial fission-re-lated factor DRP-1 was independently regulated by PM_2.5 exposure (Fig. 4B), while the expression of Fis-1 was influenced by independently regulated by ETR and the interaction between ETR and PM_2.5 exposure (Fig. 4C). Following short-term exposure to PM_2.5, there were no significant differences in the expression levels of mitochondrial fusion-related fac-tors MFN-1 (Fig. 4D), MFN-2 (Fig. 4E) and OPA-1 (Fig. 4F) among the groups. The expression level of DRP-1 was significantly increased in the SPM group (Fig. 4B; SCON vs. SPM, p <.05). However, the expression level of Fis-1 was significantly increased in the EPM group (Fig. 4C; EPM vs. SPM, p <.001; EPM vs. ECON, p <.01). Fis-1-mediated mitochondrial fission occurs more frequently at sites of mitochondrial damage to clear damaged mitochondria and maintain the health of the mitochondrial network [20]. High fitness levels rapidly enhanced the expression of Fis-1 under PM_2.5-induced mitochondrial damage stress, indicating that it has a positive effect on the maintenance of mitochondrial homeostasis.

Although the expression of most mitochondrial enzymes and dynam-ics-related factors was not affected by short-term PM_2.5 exposure (Figs. 3 and 4), the simultaneous increase in PGC-1α (Fig. 3B) and Fis-1 (Fig. 4C) expression in the EPM group suggests that skeletal muscle mitochondria in the EPM group are undergoing dynamic changes of damage and remodeling after PM_2.5 exposure.

To confirm the extent of mitochondrial damage in each group, we performed qualitative analysis of skeletal muscle using TEM, and the results are shown in Fig. 5. Although there were no significant changes in the expression levels of various mitochondrial proteins in the SPM group (Figs. 3 and 4), but the mitochondria in the SPM group suffered extensive damage (Fig. 5). The EPM group with high fitness levels demon-strated better mitochondrial dynamic regulation (PGC-1α and Fis-1 ex-pression increased) after PM_2.5 exposure, thereby avoiding an increase in mitochondrial damage.

Numerous studies have shown that mitochondrial damage leads to increased production of ROS, which in turn triggers oxidative stress [21]. Antioxidant enzymes such as SOD, catalase, and GPx-4 are the defense mechanisms that break down ROS [22].

The expression levels of each group of antioxidant enzymes are shown in Fig. 6. The expression levels of SOD-1 (Fig. 6B) and GPx-4 (Fig. 6E) were both upregulated by ETR. The expression level of SOD-2 (Fig. 6C) was independently regulated by ETR or PM_2.5 exposure. However, the changes in the expression level of catalase (Fig. 6C) reflected the interaction effects of ETR and PM_2.5 exposure. The SPM group showed a significant increase in SOD-2 expression levels in mitochondria (Fig. 6C; SCON vs. SPM, p <.01), while the expression levels of other antioxidant enzymes in the cytoplasm remained unchanged (Fig. 6B, D, and E). However, the expression levels of the SOD-2 (Fig. 6C; EPM vs. SPM/ECON, p <.001) catalase (Fig. 6D; EPM vs. SPM/ECON, p <.001) and GPx-4 (Fig. 6E; EPM vs. SPM, p <.05) pathway significantly increased in the EPM group. The EPM group with high fitness levels was able to re-spond more quickly to increased cellular stress by upregulating the expression of antioxidant enzymes in response to PM_2.5 exposure.