Farrerol Enhances Nrf2-Mediated Defense Mechanisms against Hydrogen Peroxide-Induced Oxidative Damage in Human Retinal Pigment Epithelial Cells by Activating Akt and MAPK

Oxidative stress of the retinal pigment epithelium (RPE) is an essential element contributing to the progression of age-related macular degeneration (AMD). Notably, the activation of Nrf2 is regarded as an effective strategy for controlling oxidation. The novel 2,3-dihydroflavonoid compound farrerol, which is extracted from Rhododendron, possesses antioxidant properties. In this study, we investigated the mechanism by which farrerol protects against oxidative damage mediated by hydrogen peroxide (H2O2) in adult retinal pigment epithelial cell line 19 (ARPE-19) cells. Farrerol supplementation conspicuously reversed H2O2-related cell damage through declining the generation of intracellular reactive oxygen species (ROS) and MDA and increasing the concentrations of GSH and SOD. According to the results of the apoptosis assay, a farrerol pretreatment decreased the protein expression of the Bax/Bcl-2, cleaved caspase-3, PARP, caspase-8, and caspase-9 proteins. Furthermore, farrerol markedly activated Nrf2, thereby increasing the levels of antioxidant enzymes downstream of Nrf2, such as HO-1, NQO1, and GCLM. Knockdown of Nrf2 with a specific siRNA successfully suppressed farrerol-mediated HO-1 transcription and partially abolished the cytoprotective effect on ARPE-19 cells. Meanwhile, farrerol induced Akt and MAPK phosphorylation in a dose-related way. However, inhibiting Akt and MAPK substantially blocked the cytoprotective functions of farrerol. Therefore, farrerol enhanced Nrf2-mediated cytoprotection of oxidative damage caused by H2O2, which may be inseparable from the activation of Akt and MAPK.


Introduction
Age-related macular (AMD) degeneration is an acquired disorder which substantially stimulates the macular area of the retina and causes the patient to gradually lose central vision [1,2]. Early-stage AMD is generally asymptomatic, although RPE abnormalities, including extracellular drusen deposits, which are located between RPE cells and Bruch's membrane, are clinically observed in the eye's central posterior pole [3]. In addition, late-stage AMD also involves geographic atrophy of RPE or the choroidal neovascular complex. This stage of AMD will make loss of central visual acuity and consider-able visual impairment, which may decrease the patient's quality of life [4,5]. Currently, AMD is still the third main cause of severe irreversible vision loss worldwide [6]. Meanwhile, an estimate of the global prevalence rate also indicated that the number of AMD cases worldwide will reach nearly 300 million by 2040, which constitutes a major public health problem that imposes a substantial burden on society and the economy [7].
Although the specific mechanisms of the development of AMD remain unclear, many studies have shown that oxidative stress and apoptosis acted an essential part in these processes [8]. RPE cells possess strong metabolism and survive in the presence of a large amount of endogenous ROS, consisting of superoxide anions (O2 •-), hydroxyl radicals ( • OH), and H 2 O 2 [9]. Meanwhile, the phagocytosis of photoreceptors and accumulation of lipofuscin may result in further ROS production [10,11]. In addition, detrimental elements like aging, smoking, and additional UV exposure also increase ROS production. Oxidative damage caused by the long-term accumulation of ROS may lead to RPE cell dysfunction [5]. However, supplementation with antioxidants containing ascorbic acid (vitamin C), provitamin A, and lutein alleviates retinal damage and modulates AMD progression. Therefore, treatments that reduce the oxidative damage to RPE cells are considered an advantageous method to prevent the occurrence and progression of AMD [12,13]. Nuclear factor erythroid-related factor 2 (Nrf2) is effectively activated to trigger the endogenous antioxidant defense system under stress conditions [14]. Moreover, Nrf2 regulates ROS production and biological metabolism by regulating multiple antioxidants and phase II detoxification [15]. Under oxidative stress conditions, Kelch-like ECH-related protein 1 (Keap1) undergoes modifications that cause a conforma-tional change, thereby restraining the ubiquitination of Nrf2 [9]. Subsequently, Nrf2 translocated to the nucleus and can bind to small tendon fibrosarcoma (sMaf) protein to form a heterodimer. This heterodimer recognizes and binds to ARE, thereby activating the transcription of downstream genes like heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase-1 (NQO1), glutamatecysteine ligase catalytic subunit (GCLC), and glutamatecysteine ligase modifier subunit (GCLM) [16,17]. Additionally, Nrf2 is also regulated via the phosphorylation of Keap1 by several kinases, such as phosphatidylinositol 3 kinase (PI3K)/Akt [18] and mitogen-activated protein kinases (MAPK), including JNK, ERK, and P38 [19,20]. Therefore, the inhibition of oxidative damage through an approach targeting Nrf2 molecules represents a novel therapeutic strategy for AMD.
Farrerol is a major Nrf2 activator and novel 2,3-dihydroflavonoid compound extracted from Rhododendron. As shown in our previous experiments, farrerol possesses biological activities, including antibacterial, anti-inflammatory, and antioxidant functions [21,22]. According to our   [23]. In addition, farrerol also protects against acetaminophen-induced liver damage by regulating Nrf2 and autophagy signaling pathways [24]. Here, we determined the cytoprotection of farrerol on H 2 O 2 -associated oxidation and apoptosis in vitro and further explored the underlying interaction between the Nrf2 regulatory pathway and potential mechanisms.       2.6. Flow Cytometry. ARPE-19 cells were grown and handled as described previously. Annexin V and propidium iodide (PI) were utilized to quantitate apoptosis according to the manufacturer's instructions. Afterwards, the proportion of apoptotic cells was measured using a flow cytometer.  Oxidative Medicine and Cellular Longevity and lysed according to related reagent instruction. We used the BCA protein assay to measure the protein concentration.
After that, proteins were transferred to a PVDF membrane.
The membrane was sealed with 5% skim milk. After incubations with the corresponding primary and secondary antibodies, the bands were developed utilizing ECL and quantified using scanning densitometry.  (Figure 1(b)). Hence, we utilized H 2 O 2 (at a dose of 300 μM) in subsequent experiments to appraise the cytoprotection of farrerol against H 2 O 2 -mediated damage. Furthermore, as shown in Figure 1(c), a remarkable difference in viability was not observed in cells pretreated with farrerol at concentrations ranging from 5 to 10 mg/L; however, at a concentration of 20 mg/L, this may result in statistical difference of cell viability. Based on statistical analyses described above, the cells were pretreated with farrerol (0-20 mg/L) and then incubated with 300 μM H 2 O 2 . The farrerol supplement, particularly at a dose of 20 mg/L, attenuated the cytotoxicity of H 2 O 2 (Figure 1(d)).  (Figures 2(a)-2(c)). In addition, farrerol distinctly decreased intracellular ROS levels and cell death, as evidenced by the results of the DCFH-DA staining (Figures 2(d) and 2(e)).

Discussion
As a retinal disorder, AMD mainly causes irreversible blindness among the aged population in the developed world [26]. Approximately 11 million of Americans suffer from AMD, and this figure may continuously increase and will probably become a global medical burden [27]. A feasible therapy for AMD is not available, and thus, the demand for new treatments has become increasingly urgent. Notably, AMD is a complex disease caused by genetic and environmental factors  Oxidative Medicine and Cellular Longevity [28]. Although the precise mechanism of its pathogenesis is unknown, the progressive degeneration of the macular RPE cells in the retina may cause AMD. The degeneration of the RPE involves crosstalk between oxidation and apoptosis pathways and is a well-known essential factor contributing to the pathogenesis of AMD [29]. The retina is a tissue with a high oxygen consumption rate. Its photoreceptor cells are continuously exposed to oxygen and light, and thus, they are more vulnerable to oxidative stress [30]. Excessive ROS production induced by chronic oxidative damage is the main factor leading to AMD, and its pathophysiology may cause oxidative damage to cellular components and severely destroy a proportion of RPE cells. Consistent with these findings, H 2 O 2 significantly increased ROS production in the present study (Figures 2(d) and 2(e)). Moreover, antioxidants also remarkably decrease the rate of AMD progression in the clinic [29,31]. Thus, a new method for inhibiting oxidative stress would be a potential treatment for AMD. The production of MDA and the consumption of GSH and SOD have been frequently used as indicators of oxidative damage. In our present study, H 2 O 2induced oxidation resulted in higher levels of MDA and lower levels of SOD and GSH (Figures 2(a)-2(c)).
Farrerol, a new 2,3-dihydroflavonoid compound extracted from Rhododendron, possesses antibacterial, antiinflammatory, antioxidant, and other biological activities [21,22]. As shown in Figures 2(a)-2(c), farrerol effectively reversed the changes in the indicators described above. In addition, farrerol significantly reduced the H 2 O 2 -induced increase in ROS levels in cells (Figures 2(d) and 2(e)). Consistent with these findings, farrerol visibly attenuated oxidative damage and potentially represents a treatment for H 2 O 2induced cytotoxicity (Figure 1(d)). In addition, a large amount of accumulated ROS may cause mitochondrial dysfunction in RPE cells and induce apoptosis [32]. In our study, farrerol significantly decreased apoptosis compared with the H 2 O 2 treatment alone, as determined using flow cytometry (Figures 3(a) and 3(b)). In addition, we also observed the levels of the Bax/Bcl-2, cleaved caspase-3, and cleaved PARP protein by performing western blot analyses. The levels of these apoptosis-related proteins were distinctly increased in the H 2 O 2 treatment group, and the farrerol pretreatment substantially reduced their levels (Figures 3(c)-3(f)). As we all know, caspase-3, as an important effector molecule in the apoptosis pathway, can trigger mitochondrial and the death ligand pathways by interacting with caspase-9 and caspase-8, respectively [33]. In the mitochondrial activation pathway, mitochondrial cytochrome c can be released into the cytoplasm and cause the cleaved caspase-9 to activate the expression of downstream caspase-3 [34]. In the death ligand activation pathway, death receptors (such as FasL and FasR) can cause apoptosis to activate downstream cleaved caspase-8, thereby activating the expression of caspase-3 [35]. In the following study, western blot analysis showed that farrerol downregulated the levels of cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 in ARPE-19 cells induced by H 2 O 2 . However, the protective effects of farrerol on cleaved caspase-8 and cleaved caspase-9 were not observed when Nrf2 expression was suppressed using siNrf2 when compared with the siNrf2 alone group (Figures 5(d)-5(i)). These results indicated that farrerol improves H 2 O 2induced ARPE-19 cell damage by restraining death receptors and mitochondrial apoptotic pathways.
As shown in our previous study, farrerol ameliorates renal toxicity caused by cisplatin and acetaminopheninduced liver damage by activating the Nrf2 signaling to improve oxidative damage [23,24]. With the aim of further studying the pharmacological effects of farrerol and based on the aforementioned results, we investigated antioxidant molecules to explore the mechanism underlying the interaction between Nrf2 and antioxidants. The Nrf2 signaling pathway is required to regulate the expression of antioxidant and antiapoptosis-related enzymes, and this pathway plays a considerable role in maintaining antioxidant homeostasis [36,37]. Under stress conditions, the newly synthesized Nrf2 translocates to the nucleus and subsequently activates downstream antioxidant genes to inhibit ROS production [38]. As shown in the present study, farrerol protected cells from H 2 O 2 -mediated oxidative stress by inducing the nuclear translocation of Nrf2 (Figures 4(a)-4(c)) and increasing its downstream like HO-1, NQO-1, and GCLM (Figures 4(d)-4(g) and 4(i)-4(k)). Additionally, the silencing of Nrf2 partially abolished the cytoprotective effects of farrerol ( Figure 5(c)) and decreased the HO-1 level (Figures 5(a) and 5(b)). A potential explanation for this finding is that farrerol-associated cytoprotective activities are carried out through the Nrf2/HO-1 pathway to some extent. Notably, the expression of ARE-dependent genes is also induced by activated kinase pathways (such as MAPK and PI3K/Akt) in cells [18][19][20]. In our present study, the farrerol treatment effectively induced the necessary phosphorylation of Akt and MAPK (Figures 6(a)-6(e)), which is crucial for subsequent Nrf2 activation in RPE cells. However, the Akt inhibitor (LY294002), JNK inhibitor (SP600125), ERK inhibitor (UO126), and P38 inhibitor (SB203580) nearly completely abolished the cytoprotective impact of farrerol in vitro stimulated with H 2 O 2 ( Figure 6(f)). Based on these results, Akt and MAPK activation is related to the cytoprotective effect of farrerol on RPE cells subjected to H 2 O 2 -induced oxidative damage and subsequent apoptosis.
Taken together, the results of this study indicated that farrerol has novel functions that protect RPE cells from H 2 O 2 -associated oxidation and apoptosis by inhibiting ROS generation. Farrerol ameliorates H 2 O 2 -induced cell death by increasing Nrf2/HO-1 generation via activating Akt and MAPK in ARPE-19 cells. Thus, farrerol shows promise in the treatment or prevention of AMD.

Data Availability
All datasets analyzed for this study are included in the article material.

Conflicts of Interest
The authors have no conflicts of interest to declare.