Inhibitory Effects of Parachlorella Beijerinckii Extracts on the Formation of Advanced Glycation End Products and Glycative Stress-Induced Inflammation in an In Vitro Skin Dermis-Like Model

Advanced glycation end products (AGEs) are formed via a nonenzymatic glycosylation reaction called glycation. The formation and accumulation of AGEs increases in skin with age, contributing to the appearance of facial wrinkles and loss of skin elasticity. Therefore, inhibition of AGEs may delay skin aging. The microalgae Parachlorella beijerinckii has been used as a health food supplement for many years and contains carotenoids and vitamins that have antioxidant and anti-inflammatory effects. The aim of this study was to investigate whether Chlorella extract also has antiglycation activity. Antiglycation activity was measured using fluorescent AGEs, Nε-(carboxymethyl) lysine (CML), and Nε-(carboxymethyl) arginine (CMA) from glycated bovine serum albumin and type I collagen in vitro. A gel with a dermis-like structure consisting of collagen and a live fibroblast cell line was glycated with glyoxal. The content of fluorescent AGE, CML, and CMA, and the gel contraction activity were measured. In addition, to investigate the level of inflammation induced by the glycation of the collagen gel, the expression level of the receptor for AGEs and interleukin-8 were examined. Fat-solubleChlorella extract suppressed the formation of fluorescent AGEs, CML, and CMA in both models. These results indicated that Chlorella extract directly inhibited AGE formation. The collagen gel contracted over time during culturing, whereas contraction was inhibited in the glyoxal-treated collagen gel. Chlorella extract remarkably attenuated the glyoxal-induced gel contraction. Moreover, Chlorella extract substantially decreased the fluorescent AGEs, CML, and CMA in the collagen gels with glyoxal. Glyoxal exposure increased the expression levels of interleukin-8 and receptor for AGE proteins in collagen gels, while Chlorella extract inhibited this increase. This study showed that fat-solubleChlorella extract has a direct inhibitory effect on AGEs and decreases receptor expression for AGE-mediated inflammation by reducing AGEs. Chlorella may delay skin aging by inhibiting the formation and accumulation of AGEs.


Introduction
Protein glycation is a nonenzymatic reaction between reducing sugars and free amino groups in proteins that forms a reversible Schif base. Te Schif base spontaneously rearranges into an Amadori product, which induces further oxidation, giving rise to advanced glycation end products (AGEs). In the skin, an increase in autofuorescence with age refects the accumulation of fuorescent AGEs. Nε-(carboxymethyl) lysine (CML), a typical nonfuorescent AGE, as well as fuorescent AGEs, have been shown to be positively correlated with chronological age [1,2]. Moreover, the accumulation of these AGEs is linked to an increase in facial wrinkles and loss of skin elasticity [3,4].
Dermal extracellular proteins are considered major targets for glycation. Collagen is the main structural protein in the dermis, and its glycation induces the cross-linking of proteins. Accumulation of glycated collagen results in a long half-life and slow renewal of proteins in vivo [5]. Moreover, AGEs adversely afect dermal homeostasis [6]. For example, accumulation of AGEs into the dermis layer inhibits the proliferation [7], accelerates apoptosis and senescence of fbroblasts [7][8][9], and impairs extracellular matrix (ECM) synthesis [10]. Tese biological responses interfere with the normal maintenance of the dermal structure. Inhibition of the maintenance of skin homeostasis by AGEs is considered to be dependent on enhanced receptor for AGEs (RAGE) signaling, which is a proinfammatory process. Ligands binding to RAGE results in the upregulation of RAGE expression, thereby perpetuating cellular infammatory responses [11]. Terefore, inhibiting the formation and accumulation of AGEs and attenuating RAGE expression may be efective for maintaining skin homeostasis and suppressing skin aging.
Synthetic inhibitors, such as aminoguanidine (AG), have been used to inhibit the formation of AGEs and to strongly inhibit glycation. However, prolonged use of AG has been reported to cause some side efects [12]. Glycation increases with age and glycation products are considered to accumulate over several years [1,2]. Terefore, prolonged glycation inhibition is vital to suppress their accumulation. Natural plant-derived glycation inhibitors are considered to have fewer side efects and are safer than synthetic products, allowing their prolonged use. Terefore, many plant-derived components have been studied for their ability to inhibit AGE formation worldwide [13][14][15][16][17][18]. Tese components have antioxidant and antiinfammatory efects, which also exhibit inhibitory activity on the formation and accumulation of AGEs. Chlorella, a unicellular green alga, has been used as a health food supplement for many years and has been considered to be safe. It contains a wide variety of vitamins and carotenoids, which have recently been reported to have antioxidant, anti-infammatory, and antidiabetic efects [19][20][21]. We have already reported that Chlorella-a Parachlorella beijerinckii strain-has inhibitory efects against obesity, dementia, muscle atrophy, and infammasome activation in rodent models [22][23][24][25]. Tese inhibitory efects have been shown to be the synergistic efects of multiple carotenoids contained in P. beijerinckii. Tese fndings suggest that P. beijerinckii has the potential to suppress the formation and accumulation of AGEs and AGEs-induced infammation. However, no studies have evaluated the antiglycation activity of P. beijerinckii.
Te purpose of this study is to examine whether the components of P. beijerinckii exhibit direct antiglycation activity by inhibiting the glycation of bovine serum albumin (BSA) and collagen by glucose and glyoxal using an in vitro model. We also determined the inhibitory efects of glyoxalinduced glycative stress in a dermis-like model constructed from collagen gel and live fbroblasts and evaluated its potential contribution to the suppression of skin aging.

Chemicals and Reagents
. D-glucose, aminoguanidine hydrochloride (AGH), 40% glyoxal solution, chloroform, 2propanol, and 70% ethanol were purchased from Wako Pure Chemical Corporation (Osaka, Japan). BSA was purchased from Sigma-Aldrich (St. Louis, MO, USA). Acid-soluble bovine skin collagen type I was purchased from Nippi (Tokyo, Japan). (CE). Chlorella extract was prepared according to a previous method [25]. Briefy, Chlorella (Parachlorella beijerinckii) was cultured, dried, and powdered by Chlorella Industry Co. Ltd. Chlorella powder was extracted with methanol and chloroform (1 : 2) at 25°C overnight in the dark. Te extract was fltered through a flter paper, evaporated under reduced pressure, and dissolved in ethanol. For saponifcation, 50% KOH (w/v) was added to the extracted solution for 2 h. After saponifcation, 3% NaCl (w/v), distilled water, and diethyl ether were added. Te residue was extracted with diethyl ether three times. Te upper layers were evaporated and dissolved in tetrahydrofuran, dimethyl sulfoxide, and ethanol (1 : 1 : 2) to prepare a stock solution. Te stock solution was stored in the dark at −80°C. Carotenoids and tocopherol in Chlorella extracts were measured using high performance liquid chromatography (HPLC) as described in a previous method [25]. Te HPLC analysis was performed by the Japan Food Research Laboratories. Te extracts had the highest concentration of lutein (429.3 mg/g extracts), followed by β-carotene (97.7 mg/g extracts), zeaxanthin (40.3 mg/g extracts), α-tocopherol (27.8 mg/g extracts), and α-carotene (12.5 mg/g extracts). However, approximately 40% of the extract weight was unknown.

In Vitro Glycation
Assay. BSA was dissolved in phosphate bufer (0.1 M, pH 7.4) with glucose or glyoxal. Glyoxal is a physiological metabolite formed by lipid peroxidation, ascorbate autoxidation, oxidative degradation of glucose, and degradation of glycated proteins. It has also been identifed as a reactive dicarbonyl formed during AGE generation and is far more reactive than D-glucose and most other reducing sugars [26]. Te glycation of BSA and collagen was carried out in accordance with a literature method [27][28][29][30][31][32] with some modifcation. For the glucose glycation test, 10 mg/mL BSA was incubated with glucose (0.5 M) at 37°C for four weeks [27,28]. For the glyoxal glycation test, 2 mg/mL BSA was incubated with 20 mM glyoxal at 37°C for one week [29,30]. CE treatment concentrations in the all experiments were chosen based on lutein in CE, the most abundant carotenoid in the extract. Concentrations of 1.65 and 16.5 μg/mL CE (each contained 1 and 10 μM of Lutein) were used for experimentation. AGH solution (1 mM) was used as a positive control [28]. Collagen was dissolved in phosphate bufer (0.1 M, pH 7.4) with glucose or glyoxal. For the glucose glycation test [31], collagen (2 mg/mL) was incubated with 100 mM glucose in phosphate bufer with or without CE (1.65 and 16.5 μg/mL) at 37°C for four weeks. For the glyoxal glycation test, collagen (1 mg/mL) was incubated with 1 mM glyoxal in phosphate bufer with or without CE (1.65 and 16.5 μg/mL) at 37°C for one week [32]. Glucose-BSA and glucose-collagen samples were collected at zero, two, and four weeks. Glyoxal-BSA and glyoxal-collagen samples were collected at zero and one week. Samples were kept at −80°C until analysis.

Determination of Fluorescent AGE Formation.
Fluorescent AGEs, the irreversible products at the end stage of nonenzymatic glycation, were determined using

Contraction Capacity Study and CE Treatment Using an In
Vitro Dermis-Like Skin Model. Dermis-like structure collagen gels were prepared by modifying previously described methods [33][34][35] using skin normal diploid fbroblasts (TIG-118 (JCRB0535)) and collagen. In brief, the gelation collagen solution was prepared using 1080 μL of type I collagen (3 mg/mL) dissolved in 5 mM acetic acid, 198 μL of 10× Hanks' solution, and 522 μL of 5 mM acetic acid. Ten, NaOH was added dropwise to neutralize the solution. Te gelation collagen solution (1800 μL in total) was mixed with fbroblasts (3.0 × 10 5 ), resuspended in 200 μL of fetal bovine serum and seeded into a 6-well culture plate, and the gels were detached from the rim of the wells and incubated at 37°C and 5% CO 2 further for three days. Te gels were photographed using a SONY DSC-WX350 camera (Sony Corporation, Tokyo, Japan). Gel areas were calculated using ImageJ software (National Institutes of Health, MD, USA). We previously reported that the ingestion of Chlorella increased plasma lutein concentration to about 1.2 μM [36]. Terefore, in experiments using a collagen gel dermis-like model, we considered the maximum concentration of the extract to be applied to the culture medium as 1.65 μg/mL CE (1 μM lutein). To examine the efect of CE on contraction capacity, fbroblasts used for collagen gelation were pretreated with CE (0, 0.165, and 1.65 μg/mL) or AGH (50, 2000 μM) for three days. Following this, they were treated again with the same concentration of CE or AGH. Ten, to induce glycation, glyoxal (fnal concentration 400 μM) was added and the cells were cultured for three days. In this experiment, AGH was examined at two concentrations, with the lowest concentration (50 μM) being the blood concentration when 0.3 g of AG was ingested [37]. Tus, we designed this study using both low AG and high CE concentrations based on their actual blood concentration levels after oral administration.

AGE Extraction from Glycated Collagen Gels.
Collagen gels were cut into small pieces with scissors. HCl (0.01 M) was added to the fragmented collagen gel to make a 100 mg/mL collagen solution (w/v). Te solution was incubated overnight at 37°C and centrifuged at 15000 rpm for 5 min. AGEs were recovered from the supernatant. CML, CMA, and RAGE in the supernatant samples were analyzed using CML ELISA and western blotting. Samples collected from incubation were loaded onto gels after heating with 4× SDS sample bufer at 96°C for 5 min. Electrophoresis was conducted using 10% gels.

RNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) of Glycated Collagen
Gels. To examine cell-mediated contraction, the gels were detached from the rim of the wells and incubated for up to 24 h. Te gels were recovered and 1 mL ISOGEN (Nippongene, Tokyo, Japan) was added to glycated collagen gels. Ten, the gel was cut with scissors and homogenized using a BioMasherII (Nippi, Tokyo, Japan). Total RNA was extracted from cells in the glycated collagen gels using ISOGEN in accordance with the manufacturer's instructions. RNA samples were reverse transcribed using the PrimeScript TM RT Reagent Kit (TAKARA, Shiga, Japan), in accordance with the manufacturer's instructions. Additionally, qRT-PCR analysis was performed using specifc primers (Table S1) and TB Green Premix Ex Taq II (Tli RnaseH Plus) (TAKARA). β-Actin was used as the reference gene. Gene expression was determined by qPCR using a thermal cycler (StepOne Plus; Applied Biosystems, Waltham, MA, USA). Relative quantifcation was performed using the ΔΔ C t method in combination with reference genes for data normalization.

Statistical Analysis.
All experiments were performed in triplicate or greater. All graphs display the mean ± standard deviation (SD). Statistical analysis was performed using the Bell Curve for Excel (Social Survey Research Information Co., Ltd. Tokyo, Japan). Values of p were calculated using unpaired two-tailed Student's t tests. Diferences between the groups were considered statistically signifcant at p < 0.05.

Efects of CE on Fluorescent AGE Formation from an In
Vitro Glycation Assay. We prepared fat-soluble and watersoluble crude extracts of Chlorella and investigated their inhibitory efects on fuorescent AGE formation in an in vitro system. Te fat-soluble extract greatly inhibited fuorescent AGE formation, whereas the water-soluble extract resulted in slight inhibition (Figures 1(a) and 1(b) and Figures S1(a)-S1(b)). Te results showed that the antiglycation activity of Chlorella was contained in the fatsoluble component; therefore, the fat-soluble CE was used in all subsequent experiments. Te formation of fuorescent AGEs was monitored every two weeks to determine the fuorescence intensity of the glucose-BSA and glucosecollagen solutions. Te fuorescence intensity increased over time in both solutions. Solutions containing CE also showed an increase in fuorescence intensity over time. However, this was substantially suppressed when compared with that in the control solution (Figures 1(a) and 2(a)). Next, glucose was replaced with glyoxal and incubated with BSA and collagen solutions to measure the fuorescence intensity. As with the results for glucose, the fuorescence intensity of these solutions increased, and CE suppressed the increase in fuorescence of both solutions (Figures 1(b) and  2(b)).

Efects of CE on Nonfuorescent AGE Formation from an In
Vitro Glycation Assay. CML and CMA are typically used as biomarkers for AGE formation. CML and CMA levels increased after glycation in the glucose-BSA and glucosecollagen solutions. Te solutions containing CE also showed an increase in CML and CMA levels when compared with that in the control solutions. However, the increase was considerably suppressed when compared with that in the control solutions (Figures 1(c), 1(e), 2(c) and 2(e)). Glucose was replaced with glyoxal and incubated with BSA and collagen solutions to measure CML and CMA. As with the glucose results, the CML and CMA of these solutions increased after glycation; however, CE suppressed the increase in CML and CMA in both solutions (Figures 1(d), 1(f ), 2(d), and 2(f )). Tese results indicated that CE directly inhibited both fuorescent and nonfuorescent AGEs in the in vitro glycation assay.

Attenuating Efect of CE on the Inhibition of Glyoxal-Induced Collagen Gel Contraction.
We examined cytotoxicity against fbroblasts used for in vitrodermis-like skin model. No cytotoxic efects were observed at the concentration of CE used in the experiment ( Figure S2). We cocultured fbroblasts and type I collagen to create a dermislike collagen gel and investigated the efect of CE on gel contraction. Te contraction of the collagen gel was induced by culturing for three days. When glyoxal-treated collagen gel was used, the contraction was inhibited, and the area expanded by about two times ( Figure 3). However, when CE was added to the glyoxal-treated collagen gel, inhibition of contraction by glyoxal was remarkably attenuated in a dose-dependent manner when compared with that in the control (Figure 3).

Inhibitory Efect of CE on the Formation of AGEs in
Glyoxal-Treated Collagen Gels. We measured the fuorescence of the collagen gel as well as the levels of extracted CML and CMA. At the concentration of glyoxal that inhibited contraction activity (400 μM) (Figure 3), the fuorescence of the gel itself increased only slightly (data not shown). Terefore, 10 mM glyoxal was used in the experiments on fuorescent AGEs in the collagen gel. Te addition of CE to the collagen gel did not suppress the increased fuorescence on the frst and second day after treatment with glyoxal, but considerably suppressed the increase in fuorescence on the third day (Figure 4(a)). In addition, formation and accumulation of CML and CMA in the gel were observed after treatment with 400 μM glyoxal but were remarkably inhibited by treatment with CE (Figures 4(b) and 4(c)). Tese results indicated that CE inhibited both fuorescent and nonfuorescent AGE accumulation in collagen gels induced by glyoxal glycation. Figure 4, the accumulation of AGEs was confrmed after glyoxal treatment of the collagen gel. Terefore, to investigate the enhancement of infammatory signals in collagen gels by AGEs, we measured the mRNA levels of proinfammatory chemokines and cytokines (interleukin-8 (IL-8), IL-6, and tumor necrosis factor alpha (TNF-α)). One day after glyoxal treatment of the collagen gel, the expression level of IL-8 mRNA was substantially increased in a dose-dependent manner (Figure 5(a)). However, no increase in IL-6 or TNF-α mRNA expression levels was observed ( Figure S3). Te increase in the IL-8 mRNA expression level by glyoxal treatment was suppressed by the addition of CE ( Figure 5(a)), which was consistent with the decrease in AGEs by CE (Figure 4).

CE Exerts an Anti-Infammatory Efect by Suppressing RAGE Expression. As shown in
We investigated the efect of CE on RAGE protein expression levels in collagen gels. RAGE expression increased by approximately 2.5-fold after treatment with glyoxal, but the addition of a high concentration of CE (16.5 μg/mL) suppressed RAGE expression to the same level as that of the control group ( Figure 5(b)).

Discussion
Our fndings provide evidence that Chlorella is a new antiglycation agent that may be useful against skin aging. It acts by two independent mechanisms: direct inhibition of the formation of AGEs and suppression of RAGE-mediated infammation.
In many studies, in vitroprotein-glucose or proteinglyoxal antiglycation assay models have been used as the frst step in testing the antiglycation activity of various ingredients [13][14][15][16][17][18]. Using the same model, we prepared water-and fat-soluble crude extracts of Chlorella powder and examined their antiglycation efects. Consequently, we found that the fat-soluble extracts had higher antiglycation 4 Evidence-Based Complementary and Alternative Medicine     activity than the water-soluble extracts (Figures 1 and S1). Zheng et al. compared the antiglycation activities of several Chlorella strains and found antiglycation activity in the fatsoluble portions [38], which is consistent with our results. In addition, astaxanthin, a carotenoid from Chlorella, has been found to strongly inhibit protein glycation [39]. Because we used the crude, fat-soluble extract, we could not identify the ingredients responsible for the antiglycation activity in this study. Moreover, the fat-soluble extract used in our experiments did not contain astaxanthin but contained several other carotenoids (lutein, zeaxanthin, α-carotene, β-carotene). Tese carotenoids may have synergistic efects that exhibited antiglycation activity in this study. However, given that approximately 40% of the components of fat-solubleChlorella extracts are unknown, further research is required to identify the ingredients of Chlorella that exert antiglycation activity.
Te formation of AGEs involves molecular processes based on simple or complex multistep reactions, including an oxidative step [40]. In this study, Chlorella suppressed the formation of CML and CMA, which passed the oxidation step during production. Furthermore, the Parachlorella beijerinckii strain that we used in this study has been shown to have antioxidant efects [23][24][25], suggesting that the antioxidant activity of this Chlorella extract might contribute to the inhibition of AGE formation. In addition, collagen type I is the major structural component of the dermal extracellular matrix and accounts for >70% of skin dry weight, providing the dermis with tensile strength and stability [41]. AGEs have been reported to accumulate in human and mouse skin tissues with age [1,2,42]. As CE also inhibited the formation and accumulation of collagen protein glycation (Figure 2), if fat-soluble components of Chlorella are able to reach the skin tissue, glycation and aging in the skin may be reduced.
In this study, we used aminoguanidine (AG) as a positive control for assessing antiglycation activity. AG is known to have strong antiglycation activity [43] and showed a higher inhibitory efect on glycation than the high concentration of CE in all experiments (Figures 1 and 2). Although AG has high antiglycation activity, it is not approved as a drug owing to its side efects [12]. Chlorella, more specifcally Parachlorella beijerinckii, has been used as a dietary supplement in Japan for about ffty years and no side efects have been observed. Its continuous ingestion for 6−16 months in animal experiments or for 1−5 months in clinical trials was also not associated with adverse efects [23,24,36,[44][45][46][47]. Because the accumulation of AGEs in the skin occurs over many years, long-term ingestion is desirable for prevention. Tus, Chlorella may be used as a long-term ingestible antiglycation supplement as it has no reports about side efects. As a high CE concentration was shown to be as efective as or better than a low AG concentration in all experiments using collagen gel, it may show the same inhibitory efect on glycation as AG in vivo. However, because these experiments were conducted in vitro, further in vivo studies are warranted.
AGEs not only alter the physicochemical properties of biomolecules, such as proteins, lipids, and nucleic acids, but also bind to RAGE, initiating a cascade of signals that infuence the cell cycle and proliferation, gene expression, infammation, and ECM synthesis [11]. In this study, we  Figure 5: Efect of Chlorella extract on IL-8 and RAGE expression in the glycated collagen gels. Te collagen gel was incubated with 0, 400, or 1000 μM glyoxal for 24 h and IL-8 expression ((a) n � 3) was measured using real-time polymerase chain reaction (RT-PCR). CE was administered at 1.65 μg/mL. Te collagen gel was incubated with 400 μM glyoxal for three days, and RAGE expression ((b) n � 3) was determined using western blotting. CE was administered at low (0.165 μg/mL) and high (1.65 μg/mL) concentrations. AGH was used as a positive control and administered at low (50 μM) and high (2000 μM) concentrations. Each value represents the mean ± SD. * p < 0.05, * * p < 0.01, * * p < 0.001 compared to the glyoxal-treated control gel.
Evidence-Based Complementary and Alternative Medicine found that Chlorella attenuated AGE-induced dysfunction in gels composed of skin fbroblasts and collagen (Figure 3). Tese results may be attributed to the direct inhibition of AGE formation and accumulation by Chlorella, as shown in Figures 1 and 2. Although the direct efect of Chlorella on glycation inhibition was not stronger than that of AG, Chlorella showed the same efect as AG in the experiments using collagen gel. Tis suggests that Chlorella has a mechanism to suppress the adverse efects induced by AGEs other than the direct inhibition of glycation. Owing to the fact that the lipophilic extract of Chlorella exhibits anti-infammatory efects [25], Chlorella may suppress infammatory signals in the fbroblasts contained in the collagen gels. Additionally, IL-8 expression was reported to be induced via signaling pathways activated by RAGE ligand recognition in normal skin fbroblasts [48]. We found that Chlorella reduced the mRNA expression level of IL-8. Tese results suggest that Chlorella has at least two mechanisms for alleviating the adverse efects of AGEs. Te frst is direct AGE inhibition, and the second is suppression of the infammatory pathway initiated by RAGE activation. RAGE gene and protein expression are upregulated by AGEs [48,49], and their increased expression leads to chronic infammation, which correlates with skin aging [50]. Terefore, regulation of RAGE expression is an important factor in delaying the process of skin aging. In this study, we found that Chlorella not only suppresses infammation but also suppresses the expression of RAGE. Tese results indicate that Chlorella may delay skin aging by suppressing chronic infammation. However, our study did not clearly indicate whether the antiglycation and anti-infammatory efects of Chlorella inhibit skin wrinkles and loss of elasticity; therefore, further research is required regarding the inhibitory efects of Chlorella on these processes.

Conclusions
In this study, we demonstrated that carotenoids-the major fatsoluble extract of Parachlorella beijerinckii, which has been used as a safe supplement for many years-directly inhibit the glycation of BSA and collagen, as well as maintain the normal contraction of derma-like collagen gel by suppressing the accumulation of AGEs and RAGE-mediated infammation. Tese results suggest that Parachlorella beijerinckii inhibits glycation stress and may slow age-related skin wrinkles and elasticity loss that occurs with an increase in age.

Data Availability
All relevant data are available and can be provided upon request by the corresponding author.

Conflicts of Interest
Te authors declare that YI, YN, and TK are employees of Chlorella Industry Co. Ltd.