**3. Results**

#### *3.1. Fenofibrate Treatment Decreased PQ-Induced RF*/*6A Cell Death*

MTT assay was used to evaluate cell viability. After exposure to several concentrations of PQ for 24 h, the viability of RF/6A cells reduced to 88%, 77%, and 60% at PQ concentrations of 0.6 mM, 0.8 mM, and 1.0 mM, respectively (Figure 1a). Viability decreased substantially after exposure to 1.0 mM PQ. Therefore, we chose 1.0 mM as the concentration of PQ in the following experiments. When the cells were pretreated with fenofibrate and then exposed to 1.0 mM PQ, the survival rate increased in a dose-dependent manner (from 65% in only PQ-stimulated group to 83% at 100 μM fenofibrate) (Figure 1b).

**Figure 1.** Effects of fenofibrate on cell viability in paraquat (PQ)-stimulated RF/6A cells assessed using MTT assay. (**a**) Cell viability after exposure to different concentrations of PQ for 24 h. (\*\* *p* < 0.01 among the control group and 0.6, 0.8, and 1.0 mM PQ-stimulated groups using Kruskal–Wallis test with post hoc Dunn's test; *n* = 6 in each group) (**b**) Cell viability in PQ-stimulated RF/6A cells with fenofibrate pre-treatment. RF/6A cells were pretreated with different concentration of fenofibrate for 1 h, then exposed to 1.0 mM PQ for 24 h. (\*\* *p* < 0.01 between the control group and 1.0 mM PQ-stimulated group using Mann–Whitney U-test; # *p* < 0.05, ## *p* < 0.01 compared to only 1.0 mM PQ-stimulated group using Kruskal–Wallis test with post hoc Dunn's test; *n* = 6 in each group).

#### *3.2. Fenofibrate Treatment Suppressed PQ-Induced Apoptosis in RF*/*6A Cells*

We investigated the effects of fenofibrate on PQ-stimulated cell apoptosis by flow cytometry. After exposure to 1.0 mM PQ, the level of cell apoptosis was significantly increased compared to that in the control group. Prior treatment with fenofibrate before PQ stimulation protected RF/6A cells and dose-dependently decreased the levels of cell apoptosis (Figure 2).

**Figure 2.** Effects of fenofibrate on apoptosis in paraquat (PQ)-stimulated RF/6A cells assessed by flow cytometry. (**a**) RF/6A cells were pretreated with different concentrations of fenofibrate for 1 h and then exposed to 1.0 mM PQ for 24 h. The *x*-axis and *y*-axis represent annexin V-FITC and propidium iodide (PI) staining, respectively. PQ: 1.0 mM PQ; F25: 1.0 mM PQ with 25 μM fenofibrate; F50: 1.0 mM PQ with 50 μM fenofibrate; F75: 1.0 mM PQ with 75 μM fenofibrate; F100: 1.0 mM PQ with 100 μM fenofibrate. (**b**) Percentage of apoptotic cells treated with different concentrations of fenofibrate. (\*\* *p* < 0.01 between the control group and 1.0 mM PQ-stimulated group using Mann–Whitney U-test; # *p* < 0.05, ## *p* < 0.01 compared to only 1.0 mM PQ-stimulated group using Kruskal–Wallis test with post hoc Dunn's test; *n* = 3 in each group).

#### *3.3. Fenofibrate Treatment Suppressed PQ-Induced ROS, 8-OHdG, Malondialdehyde, and Protein Carbonyl Content Production in RF*/*6A Cells*

PQ stimulation can induce oxidative stress by overproducing ROS in RF/6A cells. PQ stimulation led to an increased ROS production, which was reduced by pretreatment with fenofibrate (Figure 3a,b). To further investigate the effects of fenofibrate on oxidative stress, the levels of 8-OHdG (oxidative DNA adduct), malondialdehyde (MDA, lipid peroxidation product), and protein carbonyl content (protein oxidative marker) were evaluated. The levels of 8-OHdG, MDA and protein carbonyl content were significantly increased after exposure to PQ. The levels of 8-OHdG and MDA decreased with fenofibrate pretreatment in a dose-dependent manner (Figure 3c,d). The levels of protein carbonyl content were reduced with higher concentration of fenofibrate (75 and 100 μM) (Figure 3e).

**Figure 3.** Effects of fenofibrate on reactive oxygen species (ROS) production and oxidative stress indicators in paraquat (PQ)-stimulated RF/6A cells assessed by flow cytometry. RF/6A cells were pretreated with different concentration of fenofibrate for 1 h, then exposed to 1 mM PQ for 24 h. (**a**) PQ-induced ROS production under fenofibrate treatment. The *x*-axis represents 2-,7--dichlorodihydrofluorescein diacetate (2-,7--DCFDA) staining, and the *Y*-axis represents cell numbers. PQ: 1 mM PQ; F25: 1 mM PQ with 25 μM fenofibrate; F50: 1 mM PQ with 50 μM fenofibrate; F75: 1 mM PQ with 75 μM fenofibrate; F100: 1 mM PQ with 100 μM fenofibrate. Dose-dependent effect of fenofibrate treatment on (**b**) ROS production; (**c**) the expression of 8-hydroxydeoxyguanosine (8-OHdG), a DNA oxidation indicator; (**d**) the expression of malondialdehyde (MDA), a lipid peroxidation indicator; (**e**) the expression of protein carbonyl content, a protein oxidation indicator. (\*\* *p* < 0.01 between the control group and 1 mM PQ-stimulated group using Mann–Whitney U-test; # *p* < 0.05, ## *p* < 0.01 compared to only 1 mM PQ-stimulated group using Kruskal–Wallis test with post hoc Dunn's test; *n* = 3 in each group).

#### *3.4. Fenofibrate Treatment Diminished Mitochondrial Damage in PQ-Induced RF*/*6A Cell*

To determine whether fenofibrate can protect mitochondrial function, the extent of mitochondrial damage was analyzed using a JC-1 assay. JC-1 spontaneously formed J-aggregates in healthy cells. Our results showed that PQ stimulation significantly decreased the ratio of J-aggregates compared to that in control group. Fenofibrate treatment dose-dependently increased the expression of J-aggregates in RF/6A cells (Figure 4a). JC-1 remained in the monomeric form in apoptotic or unhealthy cells. After PQ exposure, the expression of JC-1 monomers had a 1.58-fold increase compared to that in control

group. Fenofibrate treatment decreased the expression of JC-1 monomers in a dose-dependent manner (Figure 4b). The fluorescence signal revealed a high level of JC-1 monomers (FITC) in PQ-stimulated cells; conversely, a high level of J-aggregates (Texas Red) was detected in the control group. Fenofibrate pretreatment decreased the level of JC-1 monomers and increased the level of J-aggregates in a dose-dependent manner (Figure 4c).

**Figure 4.** Effects of fenofibrate on mitochondrial damage in RF/6A cells assessed by JC-1 staining. RF/6A cells were pretreated with different concentrations of fenofibrate for 1 h, then exposed to 1 mM paraquat (PQ) for 24 h. Dose-dependent effect of fenofibrate treatment on (**a**) the expression of J-aggregates in PQ-stimulated RF/6A cells, and (**b**) JC-1 monomers in PQ-stimulated RF/6A cells. (\*\* *p* < 0.01 between the control group and 1 mM PQ-stimulated group using Mann–Whitney U-test; # *p* < 0.05, ## *p* < 0.01 compared to only 1 mM PQ-stimulated group using Kruskal–Wallis test with post hoc Dunn's test; *n* = 4 in each group) PQ: 1 mM PQ; F25: 1 mM PQ with 25 μM fenofibrate; F50: 1 mM PQ with 50 μM fenofibrate; F75: 1 mM PQ with 75 μM fenofibrate; F100: 1 mM PQ with 100 μM fenofibrate. (**c**) Fluorescence microscopy images showing the expression of JC-1 monomers (FITC) and J-aggregates (Texas Red).

#### *3.5. E*ff*ects of Fenofibrate on PQ-Induced Oxidative Stress-Related mRNA Levels in RF*/*6A Cells*

The mRNA levels of Prx, Trx-1, Trx-2, Bcl-2, Bcl-xl, and Bax were determined using semi-quantitative PCR analysis (Figure 5). Compared to those of the control group, the expression levels of Prx, Trx-1, Trx-2, Bcl-2, and Bcl-xl mRNA were significantly lower in the PQ-stimulated group. Fenofibrate treatment significantly enhanced the expression of Prx, Trx-1, Bcl-2, and Bcl-xl mRNA levels in a dose-dependent manner (Figure 5a–e). However, the increase of Trx-1 expression was not concentration-dependent (Figure 5c). The mRNA level of Bax was significantly higher in the PQ-stimulated group than that in control group. Only high-dose fenofibrate reduced Bax mRNA level (Figure 5f). To further confirm the effects of fenofibrate, a PPAR-α antagonist, GW6471, was added to the medium before fenofibrate treatment. The results revealed that 10 μM GW6471 could attenuate the effect of fenofibrate on Prx, Trx-1, Trx-2, Bcl-2, Bcl-xl, and Bax mRNA expression (Figure 5a–f).

**Figure 5.** mRNA expression of peroxiredoxin (Prx), thioredoxin-1 (Trx-1), Trx-2, B-cell lymphoma 2 (Bcl-2), Bcl-xl, and B-cell lymphoma 2-associated X protein (Bax) in RF/6A cells detected using semi-quantitative PCR. RF/6A cells were pretreated with a high or low dose of fenofibrate or 1 h, then stimulated with 1 mM paraquat (PQ) for 24 h. In GW6471 (GW) treated groups, the cells were incubated with 10 μM GW6471 for 1 h before fenofibrate treatment. (**a**) Relative expression of Prx. (**b**) Relative expression of Trx-1. (**c**) Relative expression of Trx-2. (**d**) Relative expression of Bcl-2. (**e**) Relative expression of Bcl-xl. (**f**) Relative expression of Bax. (\* *p* < 0.05, \*\* *p* < 0.01 between the control group and 1 mM PQ-stimulated group using Mann–Whitney U-test; # *p* < 0.05, ## *p* < 0.01 compared to only 1 mM PQ-stimulated group using Kruskal–Wallis test with post hoc Dunn's test; † *p* < 0.05, †† *p* < 0.01 between GW6471 treated group and fenofibrate treated group (the same concentration of fenofibrate) using Mann–Whitney U-test; *n* = 3 in each group; β-actin was used as an internal control.).

#### *3.6. E*ff*ects of Fenofibrate on PQ-Induced Apoptosis and Stress-Signaling Pathway-Related Proteins in RF*/*6A Cells*

We evaluated the effects of fenofibrate on PQ-induced apoptosis and stress-signaling pathway-related proteins in RF/6A cells. PQ stimulation decreased the expression of PPAR-<sup>α</sup>, Prx, Bcl-2, and Bcl-xl compared to that of the control group. The expression of PPAR-<sup>α</sup>, Prx, Bcl-2, and Bcl-xl increased with fenofibrate pretreatment. The expression of p-JNK and Bax increased after PQ exposure and was suppressed by fenofibrate pretreatment. The effects of fenofibrate were partially counteracted by 10 μM of GW6471 (Figure 6).

We then assessed protein expression in mitochondria and cytosol. In mitochondria, PQ stimulation enhanced p-Ask-1 expression but reduced cytochrome c and Trx-2 expression compared to that of the control group. Fenofibrate treatment enhanced cytochrome c and Trx-2 expression and suppressed p-Ask-1 expression. Stimulation of PQ facilitated cytochrome c release from the mitochondria into cytosol, and fenofibrate treatment inhibited the release of cytochrome c. In addition, PQ stimulation enhanced p-Ask-1 expression but reduced Trx-1 expression in cytosol. Fenofibrate treatment enhanced Trx-1 expression and suppressed p-Ask-1 expression in cytosol. The effects of fenofibrate were also partially counteracted by 10 μM of GW6471 (Figure 7).

**Figure 6.** Effects of fenofibrate on the expression of paraquat (PQ)-induced apoptosis and stress-signaling pathway-related proteins assessed by western blot analysis. RF/6A cells were pretreated with a high or low dose of fenofibrate for 1 h, then exposed to 1 mM PQ for 1 h (for phospho-c-Jun amino-terminal kinase (p-JNK)) or 24 h. In GW6471 (GW) treated groups, the cells were incubated with 10 μM GW6471 for 1 h before fenofibrate treatment. The expression levels of peroxisome proliferator-activated receptor type α (PPAR-α), peroxiredoxin (Prx), p-JNK, JNK, B-cell lymphoma 2 (Bcl-2), Bcl-xl, and B-cell lymphoma 2-associated X protein (Bax) are shown and the fold changes compared to those in control group are presented under the protein bands. β-actin was used as an internal control.

**Figure 7.** Effects of fenofibrate on the expression of paraquat (PQ)-induced thioredoxins (Trxs), apoptosis signal-regulated kinase-1 (Ask-1), and cytochrome c assessed by western blot analysis. RF/6A cells were pretreated with a high or low dose of fenofibrate for 1 h, then exposed to 1 mM PQ for 1 h (for phospho-Ask-1 (p-Ask-1)) or 24 h. In GW6471 (GW) treated groups, the cells were incubated with 10 μM GW6471 for 1 h before fenofibrate treatment. Mitochondrial proteins and cytosolic proteins were isolated and analyzed separately. The expression levels of mitochondrial Trx-2, Ask-1, p-Ask-1, and cytochrome c and cytosolic Trx-1, Ask-1, p-Ask-1, and cytochrome c are shown, and the fold changes compared to those in control group are presented under the protein bands. In cytosol, β-actin was used as an internal control. In mitochondria, VDAC-1 was used as an internal control.

PQ stimulation enhanced the expression of Apaf-1, cleaved caspase-9, and caspase-7 compared to that in control group, and the expression levels of these proteins were suppressed by fenofibrate treatment. The effects of fenofibrate were partially counteracted by 10 μM of GW6471. PARP-1 was cleaved in PQ-stimulated cells, and the level of cleavage form of PARP-1 was diminished by fenofibrate treatment (Figure 8a). We also assessed the activity of caspase-3 and the results demonstrated that PQ stimulation significantly increased caspase-3 activity. The activity of caspase-3 was inhibited by fenofibrate treatment in a dose-dependent manner. The effects of fenofibrate were also partially counteracted by the addition of 10 μM GW6471 (Figure 8b).

**Figure 8.** Effects of fenofibrate on the expression of paraquat (PQ)-induced apoptosis-related proteins assessed by western blot analysis. RF/6A cells were pretreated with a high or low dose of fenofibrate for 1 h, then exposed to 1 mM PQ for 24 h. In GW6471 (GW) treated groups, the cells were incubated with 10 μM GW6471 for 1 h before fenofibrate treatment. (**a**) The expression levels of anti-apoptotic protease activating factor-1 (Apaf-1), cleaved caspase-9, caspase-7, and poly (ADP-ribose) polymerase-1 (PARP-1) are shown. The fold changes compared to those in control group are presented under the protein bands. β-actin was used as an internal control. (**b**) Caspase-3 activity. (\*\* *p* < 0.01 between the control group and 1 mM PQ-stimulated group using Mann–Whitney U-test; # *p* < 0.05, ## *p* < 0.01 compared to only 1 mM PQ-stimulated group using Kruskal–Wallis test with post hoc Dunn's test; † *p* < 0.05 between GW6471 treated group and fenofibrate treated group (the same concentration of fenofibrate) using Mann–Whitney U-test; *n* = 3 in each group.).
