4-(Phenylsulfanyl) Butan-2-One Attenuates the Inflammatory Response Induced by Amyloid-β Oligomers in Retinal Pigment Epithelium Cells

Age-related macular degeneration (AMD) is a progressive eye disease that causes irreversible impairment of central vision, and effective treatment is not yet available. Extracellular accumulation of amyloid-beta (Aβ) in drusen that lie under the retinal pigment epithelium (RPE) has been reported as one of the early signs of AMD and was found in more than 60% of Alzheimer’s disease (AD) patients. Extracellular deposition of Aβ can induce the expression of inflammatory cytokines such as IL-1β, TNF-α, COX-2, and iNOS in RPE cells. Thus, finding a compound that can effectively reduce the inflammatory response may help the treatment of AMD. In this research, we investigated the anti-inflammatory effect of the coral-derived compound 4-(phenylsulfanyl) butan-2-one (4-PSB-2) on Aβ1-42 oligomer (oAβ1-42) added to the human adult retinal pigment epithelial cell line (ARPE-19). Our results demonstrated that 4-PSB-2 can decrease the elevated expressions of TNF-α, COX-2, and iNOS via NF-κB signaling in ARPE-19 cells treated with oAβ1-42 without causing any cytotoxicity or notable side effects. This study suggests that 4-PSB-2 is a promising drug candidate for attenuation of AMD.


Introduction
Age-related macular degeneration (AMD) is a degenerative macular disease that causes vision loss in the aged population [1]. It can be categorized into wet (neovascular AMD) and dry (atrophic AMD) AMD. The wet type is found in around 10-20% of AMD patients and results from abnormal growth of blood vessels. The breaking and leakage of blood vessels leads to irreversible damage to the macula and photoreceptors, in turn leading to vision loss. Blocking the activity of vascular endothelial growth factor (VEGF) is used as a treatment for wet-type AMD [2,3], whereas there is no approach yet for treating dry-type AMD. The dry-type AMD shows slower progression than wet-type AMD, and it affects approximately 80-90% of AMD patients. It is also associated with the formation of drusen [4]. Drusen are small yellow deposits in the macular area and are a common pathological hallmark of early AMD. Increased size and number of drusen contribute to a higher risk of AMD development and are related to retinal pigment epithelium (RPE) dysfunction, RPE atrophy, and photoreceptor degeneration [5,6]. The RPE is a monolayer of pigmented cells derived from the neuroectoderm and is located in between the neurosensory retina and the vascular choroid. The important functions of RPE cells are to maintain homeostasis of the outer retina, including helping the absorption of light, phagocytosis of old rod outer segments, transportation of nutrients and ions, protection from oxidative stress, immune privilege, and secretion of cytokines [7,8]. The amyloidbeta (Aβ) aggregates existing in drusen are related to increased secretion of inflammatory cytokines from RPE cells [9].
Aβ peptide contains 39-43 amino acids and presents mainly in senile amyloid plaques in the brain of Alzheimer's disease (AD) patients and in the drusen of AMD patients [6,10,11]. In eyes, Aβ aggregation is primarily located among the outer segments of photoreceptors and between the RPE and Bruch's membranes. oAβ  secretion is elevated in human RPE cells by aging and is associated with oxidative stress [12], autophagy [13], and expressions of inflammatory molecules such as interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) [14,15]. These inflammatory-related cytokines and molecules were also increased in AMD patients [16][17][18]. Therefore, finding and developing a novel compound that can effectively reduce the inflammatory response in retinal cells is urgent for treating AMD.
In the past decade, more than 20% of novel marine compounds have been discovered from soft corals [19]. The natural products isolated from soft corals have been demonstrated to exhibit various biological activities such as anti-tumor, anti-viral, and anti-inflammatory functions with minimum adverse effects [19][20][21][22][23]. The compound 4-(phenylsulfanyl) butan-2-one (4-PSB-2), modified from dihydroaustrasulfone alcohol, is synthetic precursor of soft coral (Cladiella australis)-derived natural compound, austrasulfone. It has an antimelanogenic effect via the suppression of tyrosine kinase activity in zebrafish embryos [24]. It can also reduce expression levels of iNOS, and COX-2 increased after the optic nerves of rats were crushed [25]. Based on these lines of evidence, we investigated whether 4-PSB-2 can suppress inflammatory responses in human adult retinal pigment epithelial cell line (ARPE-19 cells) treated with oAβ  . Here, we report that 4-PSB-2 can effectively reduce the expressions of TNF-α, COX-2, and iNOS via NF-κB signaling in oAβ 1-42 -treated ARPE-19 cells without notable cytotoxicity. Our results suggest that 4-PSB-2 is a promising therapeutic compound for treating AMD.

The Addition of oAβ 1-42 Caused Morphological Changes and Mild Cell Death in ARPE-19 Cells
RPE cells are the major cell type affected by oAβ  in AMD [26]. To investigate the effect of adding oAβ  in ARPE-19 cells, the Aβ peptide was incubated at 37 • C for 24 h, and the presence of the oligomeric form was further confirmed by immunocytochemistry staining of A11 or Aβ oligomer markers ( Figure 1A). Three different concentrations of oAβ 1-42 (0.1, 1, and 10 µM) were administered to ARPE-19 cells for 48 h. Then, the thiazolyl blue tetrazolium blue (MTT) assay was performed and showed no significant cell death (F( 3,30 ) = 0.792, p = 0.513; Figure 1B). However, the morphological changes were observed in the 10 µM of oAβ 1-42 group. Cell bodies and nuclei of ARPE-19 cells with added oAβ 1-42 became smaller, and the cytoplasm contained many small vesicles ( Figure 1C), which were suspected to be autophagosomes. Autophagy is a self-clearance mechanism that leads to the transport of cytoplasmic materials to vesicles for degradation and recycling. Autophagic dysfunction has been observed in AMD and is linked to the progression of disease [27,28]. To determine whether the small vesicles observed in the oAβ 1-42 -treated ARPE-19 cells were autophagosomes, we investigated the expression of autophagy-related molecules in oAβ 1-42 -treated ARPE-19 cells. Expressions of several autophagy-related molecules including LC3B I ( Figure 2B), LC3B II ( Figure 2C), BECLIN 1 ( Figure 2D), and p62 ( Figure 2E) were detected, indicating the existence of autophagasomes. p62 expression was decreased, suggesting an increase in autophagic activity. cultures for 24 h, and we used the MTT assay to measure cell viability. Results of the MTT analysis indicated that 4-PSB-2 did not cause toxicity in ARPE-19 cells; on the other hand, 25 µ M of 4-PBS-2 significantly enhanced cell viability (F(5,28) = 6.818, p < 0.001; Figure 1E).

4-PSB-2 Repressed Elevated Expression of Inflammation Markers in oAβ 1-42 -Treated ARPE-19 Cells
4-PSB-2 has been reported to have anti-inflammatory and neuroprotective effects via inhibiting iNOS and COX-2 expression in a rat optic nerve crush model [25]. We thus wanted to investigate whether it had the same effects on oAβ 1-42 -treated ARPE-19 cells. MTT results are demonstrated in Figure 1E. ARPE-19 cells were administered with 10 µM of oAβ  , and 24 h later 4-PSB-2 was added, and cells were allowed to rest for an additional 24 h ( Figure 3A). Then, the expression levels of inflammatory cytokines were measured. We detected higher expression levels of inflammatory cytokines including TNF-α, COX-2, and iNOS after 10 µM of oAβ
Previous studies have reported that exposure of ARPE-19 cells to oAβ resulted in a reduction in cell viability in a dose-and time-dependent pattern [14,31]. Our results are in line with an earlier study showing that 10 µM of oAβ can alter the structure and function of RPE cells [5], but not cause cell death. It is noted that different concentrations of oAβ added to ARPE-19 cells cause distinct degrees of change. Five micromolars of oAβ
Previous studies have reported that exposure of ARPE-19 cells to oAβ resulted in a reduction in cell viability in a dose-and time-dependent pattern [14,31]. Our results are in line with an earlier study showing that 10 µM of oAβ can alter the structure and function of RPE cells [5], but not cause cell death. It is noted that different concentrations of oAβ added to ARPE-19 cells cause distinct degrees of change. Five micromolars of oAβ increased ARPE-19 cell proliferation and inhibited apoptosis, whereas significant ARPE-19 cell death was observed after treatment with 25 µM of oAβ for 48 h [32]. Here, we found that the morphology of ARPE-19 cells was changed, with shrinking cell bodies and nuclei, and filling small vesicles in the cytoplasm. Transmission electron microscopy identified that the small vesicles were autolysosomes and autophagosomes [13]. Our results revealed that the p62-autophagy marker was decreased in ARPE-19 cells treated with oAβ peptides, which supports the findings from the aforementioned study. p62 directly binds to microtubule-associated protein 1A/1B-light chain 3 (LC3) to negatively modulate autophagic activity [13,33]. In addition, we found that expressions of inflammatory markers including TNF-α, COX-2, and iNOS were increased via NF-κB signaling in ARPE-19 cells induced with oAβ 1-42 , which were consistent with studies on human RPE cells exposed to oAβ   [14,15] and AMD patients [16][17][18]. Interestingly, a study in the Russian population also reported a significant association between AMD and single-nucleotide polymorphisms of TNF-α [34]. TNF-α, a proinflammatory cytokine, is synthesized and secreted by ac-tivated macrophages and T-cells, and it regulates the biological activities of cells [35]. Chronic activation of TNF-α in RPE cells can change cell morphology, alter tight-junction organization, and decrease the immunosuppressive capacities by inducing transforming growth factor β (TGF-β) expression [36]. The activation of TNF-α is mediated by NF-κB, which is the upstream mediator of COX-2 and iNOS [29]. Inhibition of NF-κB signaling can reduce the inflammatory expressions and angiogenic factors in RPE cells induced by oAβ 1-42 [37]. COX-2 is an enzyme inducible by pathologic stimuli such as lipopolysaccharides, IL-1β, TNF-α, and NF-κB [38]. Previous studies have shown that the expression of COX-2 in human choroidal neovascular membranes was related to AMD pathology by increasing the secretion of VEGF and TGF-β [38,39]. iNOS is an inducible isoform of nitric oxide and is induced by inflammatory cytokines. The increase in iNOS expression in choroidal neovascular membranes from patients with AMD directly links with VEGF [40]. Taken together, the overexpressions of inflammatory cytokines presented in ARPE-19 cells induced by oAβ 1-42 may be one of the causal factors of AMD; thus, reduction in the inflammatory response may become an effective therapeutic approach for this disease.

Cell Culture
A human retinal pigment epithelial cell line (ARPE-19) was provided by Rong-Kun Tsai at Institute of Medical Sciences, Tzu Chi University (Taiwan), and cultured in Du becco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12), containing 10% f fone and dihydroaustrasulfone alcohol are anti-inflammatory compounds that can inhibit the expressions of iNOS and COX-2 proteins in LPS-stimulated RAW264.7 macrophage cells [22]. Besides, the chemical structure of 4-PSB-2 is similar to BAY 11-7082, which also shows anti-inflammatory effects via NF-κB signaling [41]. Thus, the two compounds probably repress the inflammatory response via a similar mechanism. However, 25 µM of 4-PSB-2 did not modulate autophagic activity. Either 25 µM is not an effective dosage to rescue autophagic dysfunction, or this compound does not affect the autophagy pathway. Our results suggest that 4-PSB-2 may be a promising drug candidate for treating AMD. Further time-course, pharmacokinetic, and pharmacological metabolism studies are necessary to explore the possibility of further clinical application.

Preparation of Oligomeric Aβ 1-42 Solution
The Aβ 1-42 peptide (Bacham, Merseyside, UK) was dissolved in 0.1% NH 4 OH to a final concentration of 1 mg/mL. The Aβ 1-42 peptide was incubated at 37 • C for 24 h for aggregation, which was verified by Western blotting and immunocytochemical staining ( Figure 1A). The Aβ 1-42 peptide was dissolved in medium to final concentrations of 0.1, 1, and 10 µM for cytotoxicity examination.

Preparation of 4-PSB-2 Solution and Treatment
In this study, 4-PSB-2 was provided by the Research Center of National Research Program for Biopharmaceuticals, Taiwan, and its structure is shown in Figure 1D. To verify the cytotoxicity of 4-PSB-2, it was dissolved in DMSO and medium to final concentrations of 1, 25, 50, 100, and 200 µM.

Statistical Analysis
The mean ± standard error of the mean (mean ± SEM) were calculated and plotted. The data from the cell viability assay were analyzed by a two-tailed Mann-Whitney test. Western blotting and immunocytochemical staining data were analyzed by one-way ANOVA. Statistical significance for the differences among the groups was established at a p-value < 0.05. All graphs were plotted with GraphPad Prism 8.0 software (San Diego, CA, USA).

Conclusions
In this study, we identified that 4-PSB-2 exhibits anti-inflammatory effects via NF-κB signaling in oAβ 1-42 -treated ARPE-19 cells without notable side effects. Our results suggest a novel therapeutic approach to AMD.