Oxidative stress in the retina plays a major role in the pathogenesis of dry AMD. While antioxidant defence systems in the retinal cells are appropriate in 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, are possible protections for curtailing the progression of AMD and vision loss . In the present study, we demonstrated the improved protective effects of TPP-Niacin for the first time; this is a mitochondrial targeting compound against oxidative damage in human RPE cells. In the mitochondrial level, the TPP-Niacin exerts improved protective effects via the 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 supplying the prevention against oxidative damage by increment the expression level of antioxidant enzymes, mainly HO-1 and NQO-1, via the 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 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 composition in the retina. H2O2 is a critical factor in producing oxidative damage and cell death 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 viability and LDH assays, pre-treatment with TPP-Niacin in the ARPE-19 cells significantly increased the cell viability against H2O2-induced cell death, whereas TPP-Niacin reduced the cell death from oxidative damage. Intriguingly, TPP-Niacin treatment alone was able to slightly increase the growth of ARPE-19 cells compared to the parent compound (Supplementary Figure 1A).
Intracellular accumulation of ROS is interconnected with oxidative stress and dysfunction of RPE cells . Diminishments of intracellular ROS may shield the RPEs from oxidative damage [5, 29]. The results in this study confirmed that TPP-Niacin markedly diminished H2O2-induced intracellular ROS levels in RPE cells, as observed via DCFDA and DHE staining. There are major antioxidant enzymes, including Cu/Zn-superoxide dismutase (cytosolic SOD, SOD1), manganese superoxide dismutase (mitochondrial SOD, SOD2), catalase, and glutathione peroxidase (GPx). The SODs dismutase 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 may combat oxidative stress. Additionally, as a result of catalase and GPx, the TPP-Niacin significantly increased catalase and GPx activities in the decreased by H2O2 in 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 falling the intracellular ROS status and by eliminating oxygen free radicals. In addition, we observed that ARPE-19 cells pre-treated with niacin and TPP-Niacin markedly reduced 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 the 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 TPP-Niacin is an effective derivative of the parent compound.
The pathological changes of mitochondrial-related dysfunction, including accumulation of ROS and superoxide in mitochondria and MMP (△Ψm) reduction, were discovered in AMD. . In other mitochondrial targeting compounds [10, 14, 31], we observed that pre-treatment 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 gene 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 and 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 function in various tissues , including the retina [27, 33, 34]. To intermediate their functions, the PGC-1α isoforms cooperate with transcription factors, such as estrogen-related receptor alpha (ESRRA), peroxisome proliferator-activated receptor α, γ (PPARα, γ), forkhead foxO1 and 3 (FOXO1), 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 necessitated 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 an excessive accumulation of mitochondrial ROS, was turned on by PGC-1α in RPE cells [27, 38]. Therefore, to determine the possible pathway of the protective effect of TPP-Niacin, we examined the gene expressions of PGC-1α related genes. We 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 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 mechanism associated with the protective ability of TPP-Niacin, it has appeared that the HO-1 and NQO-1 of downstream target genes of NRF2 signalling play major roles in the prevention of the cells from oxidative damage [39, 40]. Recently, many studies have reported that the activation of NRF2/HO-1 signalling is required for the alleviation of oxidative damage to RPE cells [40–45]. In this study, it was speculated that the anti-oxidative effects of TPP-Niacin might be combined with PGC-1α and NRF2 signalling. The results of the present study reveal that TPP-Niacin protects the ARPE-19 cells from H2O2-induced oxidative damage by activating the NRF2 signalling through upregulation of the expression of NRF2, NQO-1, and HO-1.
Initially, we thought that TPP-Niacin had an effect on nano-concentration state, as well as 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 was more effective than its original chemical against oxidative damage in RPE cells. These results underscore TPP-Niacin as a more 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. As many reported other 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, 48–50]. Our study so far has only been applied to focus on the improved antioxidant effect 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.
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 of antioxidant molecules. These results supply the first experimental evidence for TPP-Niacin as a possible therapeutic agent in the prevention of AMD.