**4. Discussion**

In the present study, we demonstrated the protective effects of fenofibrate on RF/6A cells under oxidative stress. Fenofibrate inhibited ROS accumulation, mitochondrial dysfunction, and modulated the apoptosis and stress signaling pathway in oxidative stress-induced RF/6A cells.

Increasing evidence supports the idea that oxidative stress plays an important role in the pathogenesis of DR. PPAR-α is a regulator of inflammation and oxidative stress that induces the activation of antioxidant enzymes [45–47]. Evidence suggests that fenofibrate may modulate anti-oxidant pathways. For example, fenofibrate inhibits the production of ROS in streptozotocin-induced diabetic rats and reduces nephropathy development [48]. In the present study, the mRNA expression of anti-oxidant enzymes Prx, Trx1, and Trx-2 decreased in PQ-stimulated RF/6A cells, whereas the mRNA levels of these enzymes increased after fenofibrate treatment. This finding indicated that fenofibrate may induce the expression of anti-oxidant proteins and protect cells from oxidative stress. Endothelial cell apoptosis has been linked to oxidative damage through the production of 8-OHdG, nitrotyrosine, and MDA [49,50]. In the present study, the results showed that fenofibrate suppressed MDA production and protected vascular endothelial cells from lipid peroxidation. We also observed that fenofibrate suppressed 8-OHdG adduct formation but only inhibited protein oxidation at higher concentrations. Previous studies have also revealed that fenofibrate could suppress MDA production in rat models for low-density lipoprotein-induced endothelial dysfunction and Parkinson's disease [51,52]. Taken together, the results from the present study suggested that fenofibrate could induce the expression of anti-oxidant enzymes, reduce the production of ROS and decrease the generation of oxidant products, thus protecting endothelial cells from oxidative stress-induced damage.

Mitochondria are a major source of oxidative stress in DR because oxidative stress in the inner membrane leads to imbalance in the electron transport chain and generates superoxide and hydrogen peroxide, thereby damaging the membrane proteins. Furthermore, mitochondrial dysfunction activates the apoptosis-related signaling pathway [53]. Fenofibrate has been reported to decrease apoptosis in high-glucose-stimulated microvascular endothelial cells [54] and decrease the apoptotic rate of the ganglion cells in the mouse model for type 2 diabetes [55]. In the present study, we observed that fenofibrate reduced the apoptotic rate and could preserve mitochondrial function in PQ-stimulated RF/6A cells. Our findings suggested that fenofibrate could inhibit cell death and DR progression by preventing mitochondrial dysfunction.

Trxs belong to a group of small redox proteins that can be found in most cells. The anti-oxidative activity of Trxs is indispensable for cells [56]. Trxs exert most anti-oxidant properties in cells through thioredoxin peroxidase [18]. Niso-Santano et al. observed that PQ induces the phosphorylation of Ask-1 and suppress Trx expression in SH-SY5Y cells (human neuroblastoma cells) [57]. Trx-1 levels are also reduced in mycophenolic acid-induced apoptosis in pancreatic β-cells [58]. Fiuza et al. demonstrated that the protective effects of diphenyl diselenide on endothelial cells against oxidative stress are through the expression of different isoforms of Prx [59]. In our study, we found that the mRNA and protein expression of Prx, Trx-1, and Trx-2 decreased, and phosphorylated Ask-1 increased in PQ-stimulated RF/6A cells. In addition, thioredoxin-interacting protein (TRXIP) was reported to be significantly up-regulated in DR. TRXIP may interact with Trx, block its anti-oxidant activity, and then cause mitochondrial dysfunction and inflammation in DR [60,61]. The expression of Trx increased after fenofibrate treatment in our experiments. Our results were consistent with that of the study conducted by Billiet et al., in which PPAR-α activation induced Trx-1 expression [28]. The addition of PPAR-α antagonist could attenuate but not completely abolish the effects of fenofibrate, indicating that the effects of fenofibrate were not all PPAR-α dependent. In summary, our study suggested that the anti-oxidative activity and anti-apoptotic effects of fenofibrate could be attributed to the increase of Trx expression and the inhibition of Ask-1 phosphorylation.

We then investigated the effects of fenofibrate on the regulation of Trx-related signaling pathways. Trx binds to Ask-1 in the mitochondria and cytosol, thereby blocking the initiation of the cellular apoptotic process and inhibiting the activation of JNK/p38 MAP kinase [26]. In the cytosol, Ask-1 is required for the activation of JNK/p38 MAP kinases. Bcl-2 and Bcl-xl are known to regulate mitochondrial dynamics and play essential roles in anti-apoptosis; however, Bax promotes apoptosis [62,63]. JNK/p38 MAP kinase also regulates mitochondrial-mediated apoptosis [64] and facilitates the release of mitochondrial cytochrome c to the cytosol. Our study revealed that p-JNK and Bax expression were elevated in PQ-stimulated RF/6A cells and fenofibrate treatment suppressed their expression. Conversely, the expression of Bcl-2 and Bcl-xl increased after fenofibrate treatment. In mitochondria, Trx-2 inhibits Ask-1-mediated apoptosis, which in turn causes the inhibition of cytochrome c release to the cytosol [65]. Our findings showed that pretreatment of fenofibrate in PQ-stimulated cells increased Trx-2 expression, decreased the formation of p-Ask-1 and inhibited cytochrome c release. Cytochrome c release is an initiator of the main apoptotic pathway [66]. When cytochrome c is released from the mitochondria to cytosol, it binds to Apaf-1 and activates an apoptosis-related caspase cascade, consequently inducing PARP-1 cleavage leading to apoptosis [67]. We observed that fenofibrate treatment reduced the levels of cytosolic cytochrome c and the related caspase cascade in PQ-stimulated cells. In summary, our results indicated that fenofibrate could protect against oxidative stress-induced retinal/choroidal endothelial cell apoptosis by enhancing Trx-1 and Trx-2 expression, thereby suppressing Ask-1 activity, which in turn inhibits the activation of the subsequent apoptotic signaling pathways.

Our study has some limitations. It is an in vitro analysis, and the protective effects of fenofibrate and the underlying mechanisms need to be demonstrated with animal models. However, two large randomized controlled trials (FIELD and ACCORD study) have shown significant benefits of fenofibrate in patients with DR. Our results supported the assertion that fenofibrate can slow the progression of DR by modulating apoptosis- and stress-related signaling pathways.
