Oxidative stress in the retina plays a major role in the pathogenesis of dry AMD. While antioxidant defense systems in the retinal cells are appropriate under normal states, strong oxidative stress disintegrates the normal antioxidant systems and result in irreparable damage to the retina. It has been reported that the use of additional antioxidants reduces oxidative stress and preserves retinal function while avoiding oxidative damage [25, 26]. In addition, experimental and clinical studies suggest that consuming high doses of antioxidants, such as lutein, β-carotene, vitamins, and zinc supplements, possibly protect against and curtail the progression of AMD and vision loss . In the present study, we demonstrated the improved protective effects of TPP-Niacin, a mitochondrial targeting compound, for the first time against oxidative damage in human RPE cells. At the mitochondrial level, the TPP-Niacin exerts improved protective effects via mediation of the MMP and its related effector genes, including OXPHOS, mitochondrial dynamics, and mitochondrial DNA replication and transcription. Notably, TPP-Niacin is capable of prevention against oxidative damage by incrementing the expression levels of antioxidant enzymes, mainly HO-1 and NQO-1, via upregulation of PGC-1a and NRF2 in the ARPE-19 cells. Furthermore, TPP-Niacin provides better protection than niacin against oxidative damage in ARPE-19 cells, therefore underscoring the potential use of TPP-Niacin as a possible therapeutic agent for AMD, a disease that initiated by cell death from oxidative stress and RPE dysfunction.
RPE cells are one of the types of cells that consume high amounts of energy, exist in the back of the photoreceptor cells, and have the most commonplace oxidative-damaged compositions in the retina. H2O2 is a critical factor in producing oxidative damage and cell deaths in various cell types, including retinal cells . In the present study, H2O2 was used to test ARPE-19 cells to generate oxidative stress and cell cytotoxicity to imitate the onset of dry AMD. As noted in the viabilities and LDH assays, pretreatment with TPP-Niacin in the ARPE-19 cells significantly increased the cell viability against H2O2-induced cell death, whereas TPP-Niacin reduced cell death by oxidative damage. Intriguingly, TPP-Niacin treatment alone was able to slightly increase the growth of the ARPE-19 cells compared to the parent compound (Supplementary Figure 1A).
Intracellular accumulation of ROS is related with oxidative stress and dysfunction of RPE cells . Reduction of intracellular ROS may protect the RPE cells from oxidative damage [5, 29]. The results of this study confirmed that TPP-Niacin markedly diminished H2O2-induced intracellular ROS levels in RPE cells, as observed via H2DCF-DA and DHE staining. Major antioxidant enzymes exist, including Cu/Zn-superoxide dismutase (cytosolic SOD, SOD1), manganese superoxide dismutase (mitochondrial SOD, SOD2), catalase, and glutathione peroxidase (GPx). The SOD converts superoxide to oxygen and hydrogen peroxide, whereas catalase and GPx transform hydrogen peroxide into H2O and O2 [28, 29]. The present study demonstrated that pre-incubation with TPP-Niacin increased SOD1 and SOD2 compared to the H2O2 group, thus suggesting that TPP-Niacin could combat oxidative stress. Additionally, the TPP-Niacin significantly increased catalase and GPx activities that were decreased by H2O2 in the ARPE-19 cells. These data indicate that TPP-Niacin may retain the ability to indirectly scavenge oxygen free radicals. Consequently, TPP-Niacin may reduce H2O2-induced oxidative stress in ARPE-19 cells by decreasing the intracellular ROS status and by eliminating oxygen free radicals. In addition, we observed that ARPE-19 cells pretreated with niacin and TPP-Niacin markedly reduced the H2O2-induced ROS production. As expected, the TPP-Niacin exerted a somewhat higher preventive effect against oxidative damage, as shown by a 10% increment in cell viability and 17% decrement in ROS level compared to niacin-treated cells, respectively. The antioxidant activities of TPP-Niacin are at a slightly higher level compared to the parent compound, suggesting that the mitochondria-targeting TPP-Niacin is an effective derivative of the parent compound.
The pathological changes of mitochondrial-related dysfunctions, including accumulation of ROS and superoxide in the mitochondria and MMP (△Ψm) reduction, were discovered in AMD . In other mitochondrial targeting compounds [10, 14, 31], we observed that pretreatment with TPP-Niacin significantly enhanced the MMP and improved the mitochondrial ultrastructure in a phenotypic analysis by EM, compared to H2O2 alone. Based on these data, we next analyzed the expressions of mitochondria-related genes, such as OXPHOS subunits, mitochondrial dynamics, and mitochondrial DNA replication and transcription genes. Our results showed that TPP-Niacin significantly upregulated COX4I1, COX5B, NDUFB as well as MFN1, MFN2, TFAM, and POLG genes; thus, these mitochondrial specific effects of TPP-Niacin could lead to improved mitochondrial function and biogenesis against oxidative stress by H2O2.
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and -beta (PGC-1β) are transcriptional coactivators that control mitochondrial metabolism and functions in various tissues , including the retina [27, 33, 34]. To intermediate their functions, the PGC-1α isoforms cooperate with transcription factors, such as ESRRA, peroxisome proliferator-activated receptor α, γ (PPARα, γ), FOXO1, FOXO3, and nuclear respiratory factors 1 (NRF1) and Nfe212 (nuclear factor erythroid 2-related factor 2, NRF2) to control respiration, mitochondrial biogenesis, and expression of antioxidants [27, 35]. PGC-1α is necessary for the generation of ROS scavenging enzymes, including SOD1, SOD2, GPx, and CAT [36, 37]. Recently, several studies have shown that superoxide dismutase 2 (SOD2), an enzyme detoxifying the excessive accumulation of mitochondrial ROS, was turned on by PGC-1α in the RPE cells [27, 38]. Therefore, to determine the possible pathways of protective effects of the TPP-Niacin, we examined the gene expressions of PGC-1α related genes and observed that PGC-1α and PGC-1β were robustly upregulated by TPP-Niacin compared to the H2O2-induced oxidative damage group. In addition, when examining the potential downstream transcription factors responsible for these changes, ESRRA, FOXO1 and 3, and NRF1 and 2 were found to be upregulated by TPP-Niacin treatment.
On further investigating the possible mechanisms associated with the protective ability of TPP-Niacin, it appears that HO-1 and NQO-1, which are downstream targets of NRF2 signaling, play major roles in the prevention of oxidative damage in the cells [39, 40]. Recently, many studies have reported that the activation of NRF2/HO-1 signaling is required to alleviate oxidative damages in RPE cells [41, 42] [43–46]. In this study, it was speculated that the antioxidative effects of TPP-Niacin could be combined with PGC-1α and NRF2 signaling. The results of the present study show that TPP-Niacin protects the ARPE-19 cells from H2O2-induced oxidative damage by activating NRF2 signaling through upregulation of the expression of NRF2, NQO-1, and HO-1.
Initially, we thought that TPP-Niacin had an effect on the nanoconcentration state, as well as those of other mitochondrial targeting compounds, but TPP-Niacin showed antioxidant effects in the range of 10–200 μM. However, as shown in the comparison data with niacin, TPP-Niacin is more effective than its original chemical against oxidative damage in RPE cells. These results support TPP-Niacin as a potent antioxidant against oxidative stress compared to niacin and suggest that its improved protective effects are exerted via regulation of mitochondrial dynamics and antioxidant mechanisms. Contrary to expectations, the increment of antioxidant enzymes after induction of oxidative stress was in agreement with the study where the expression levels of HO-1 increased in ARPE-19 cells after H2O2 treatment [44, 45, 47–49]. However, some studies have presented that H2O2 treatment significantly reduces the expression levels of PGC-1α, HO-1, and NQO-1 in ARPE-19 cells [38,41]. The variances between these studies may be owed to the use of other concentrations of stimuli as well as the treatment time, which can control cellular response. As in reported research, it is well established that niacin exerts significant antioxidant, anti-inflammatory and anti-apoptotic activities in a variety of cells and tissues [19, 20, 50–53]. Our study thus far has only been applied to focus upon the improved antioxidant effects of TPP-Niacin, in terms of mitochondrial and ROS regulation. Further data collection would be needed to determine exactly how TPP-Niacin affects with antioxidant effect via mitochondrial biogenesis and dynamics. Additionally, when we exam the TPP-Niacin's own effects on normal ARPE-19 cells, as shown in Supplementary Figure 4, TPP-Niacin was not significantly changed of ARPE-19 cells compared with the control group in LDH, ROS, and MMP assay (S4 A~D). However, according to gene expression results, TPP-Niacin has significantly enhanced the SOD2 expression level assessed by RT-qPCR (S4 E). These results indicated that TPP-Niacin mediated cytoprotective activities that could be linked to the mitochondrial function on not only the normal state but also the oxidative stress.
In conclusion, this study shows that TPP-Niacin is an improved protective antioxidant than niacin against oxidative damage to ARPE-19, cells via the reduction of ROS levels and protection against oxidative-stress-induced cell death. The signal mechanisms by which TPP-Niacin presented such effects involve regulation of the mitochondrial quality control and transcriptional factors such as PGC-1α and NRF2, as well as a boost in the antioxidant molecules. These results provide the first experimental evidence for TPP-Niacin as a possible therapeutic agent in the prevention of AMD. Further studies are needed to determine its physiological functions and biological efficacies in both primary human RPE cells (at least fully differentiated ARPE-19 cell models) and in vivo models, as well as target identification in the near future.