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Blue light derived from multiple sources, including sunlight, generates reactive oxygen species (ROS) and negatively affects the skin in a manner similar to that of ultraviolet light. Cyanidin‑3‑O‑ glucoside (C3OG) and cyanidin‑3‑O‑ruti‑ noside (C3OR) are anthocyanin antioxidants that have protective effects on various tissues and cell types. However, the effects of anthocyanins on blue light ‑mediated changes remain unconfirmed. In this study, we determined the protective effects of C3OG and C3OR isolated and purified from waste cherry fruits (Prunus serrulata L. var. tomentella Nakai) against the blue light‑induced ROS formation and inflammatory responses in HaCaT cells. It is showed that the treatment of C3OG and C3OR significantly reduced the blue light ‑induced cytotoxicity and ROS production in a dose dependent manner. Furthermore, we found that focal adhesion kinase (FAK) is a major upstream of blue light‑ induced expression of inflammatory cytokines ( TNF‑α, IL ‑6 and IL ‑8), and these effects were attenuated by C3OG or C3OR treatment. In the initial reaction, blue lights increased the phosphorylation of inhibitory‑κB Kinase α (IKKα), c‑jun N‑terminal kinase (JNK), and p38. The phosphorylation of these intracellular proteins was reduced via FAK inhibitor, NAC (ROS scavenger), and anthocyanin treatments. After 24 h of blue light irradiation, C3OG or C3OR treatment mark‑ edly inhibited caspase‑3‑mediated apoptosis and cleaved‑FAK ‑mediated anoikis, which is cell detachment ‑induced apoptosis. Therefore, our results indicate that C3OG and C3OR effectively protected human keratinocytes from harm‑ ful blue light‑induced cytotoxicity and inflammation. Keywords Blue light, Anthocyanins, Cyanidin 3‑O‑ glucoside, Cyanidin 3‑O‑rutinoside, Reactive oxygen species, Focal adhesion kinase, Agri‑food waste exposure to blue light stimulating melanocytes and caus- Introduction ing pigmentation problems such as spots and age spots Similar to ultraviolet (UV) radiation, blue light can [2, 3]. Moreover, blue light has a longer wavelength and induce the generation of reactive oxygen species (ROS) lower energy than UV light. It can penetrate deeper into and thereby cause oxidative damage to the skin , with the skin  and promote photoaging and inflammatory changes in a wider range of skin layers than UV. *Correspondence: Jun‑Sub Kim Recently accumulated evidence has indicated that red firstname.lastname@example.org light promotes an increase in cell proliferation, whereas Department of Biotechnology, Korea National University blue light reduces the viability of different cell types by of Transportation, Jeungpyeong, Chungbuk 27909, Korea inducing changes in mitochondrial function, including © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Lee and Kim Applied Biological Chemistry (2023) 66:3 Page 2 of 12 aberrant ROS formation [5–8]. In particular, blue light oxidative stress . Among the diverse variety of antho- has been demonstrated to have a detrimental effect cyanins, cyanidin-3-O-glucoside (C3OG) has been iden- on the skin. At the molecular level, blue light (410 nm) tified as the most common and abundant anthocyanins has been established to cause a reduction in period cir- in fruits , and the findings of numerous studies have cadian regulator 1 (a clock gene involved in the cir- indicated that C3OG has beneficial effects on the treat - cadian rhythm) transcription and an increase in ROS ment of a range of inflammatory disorders, aging, cancer, production, DNA damage, and release of inflammatory and diabetes . To date, however, it has yet to be estab- cytokines (interleukin [IL]-1α, IL-6, IL-8, and tumor lished whether anthocyanins have any beneficial effects necrosis factor-alpha [TNF-α]) in keratinocytes . At with respect to blue light-induced cytotoxicity. longer wavelengths, blue light (470–480 nm) has been found to promote an increase in ROS release, which Results is dependent on transient receptor potential vanilloid Eec ff ts of cyanidin 3‑O‑glucoside (C3OG) and cyanidin 1 . Also, blue light at 460 nm was shown to induce 3‑O‑rutinoside (C3OR) on blue light‑induced cytotoxicity an increase in ROS production by mediating flavin exci - in HaCaT cells tation, which may, in turn, cause aging of skin . Fur- In our previous study, we demonstrated that high-inten- thermore, high-intensity blue light (412, 419, 426, and sity LED blue light has a cytotoxic effect on human skin 453 nm) is toxic to endothelial cells and keratinocytes keratinocytes (HaCaT cells) mediated via the generation , and blue-violet light (380–495 nm) has been dem- of large amounts of ROS . Further, C3OG and C3OR onstrated to have destructive effects on the carotenoids can contribute to inhibiting intracellular ROS produc- in the skin . Collectively, the findings of these stud - tion induced by diverse stimuli [26, 27]. Consequently, in ies thus indicate that ROS generation is the main factor the present study, we evaluated the effects of C3OG or underlying the detrimental effects attributable to blue C3OR isolated from cherry fruits on blue light-induced light. cytotoxicity. Focal adhesion kinase (FAK) is a protein tyrosine We found that, compared with the controls, whereas kinase activated via integrins and growth factor receptors blue light at lower light intensities (2,500 and 5,000 lx) . Having been activated, FAK promotes cell migra- gradually increased cell viability, exposure to higher tion and proliferation, including those associated with intensities (10,000 and 20,000 lx) significantly reduced tumor metastasis and angiogenesis [14, 15], and plays a viability (Fig. 1A). Based on these observations, we major role in inflammatory cytokine signaling [16, 17]. selected blue light of 20,000 lx, which had the highest Notably, ROS is linked to the activation of several protein cytotoxic effect, for further experiments. In contrast, tyrosine kinases, including FAK, proline-rich tyrosine compared with the controls, neither C3OG nor C3OR kinase 2, and Src [18–21]. Furthermore, we have previ- showed any clear evidence of cytotoxicity up to a con- ously shown that FAK is activated by ROS generated dur- centration of 200 µM (Fig. 1B). Having established these ing exposure to illumination from different light-emitting effects, we subsequently examined the effects of C3OG diodes (LEDs) and is involved in downstream signaling and C3OR on blue light-induced cytotoxicity. Accord- pathways, including those of mitogen-activated protein ingly, we observed that both C3OG and C3OR dose- kinase (MAPK) and inhibitory-κB Kinase α (IKKα) . dependently reversed the blue light-induced cytotoxicity The springtime cherry blossom festival is a particularly in HaCaT cells (Fig. 1C). This effect was also observed in important tourist event in countries such as Japan and crude extract of cherry fruit (Additional file 1: Fig S1). Korea . Consequently, cherry trees are extensively planted in many areas as a policy to enhance the added Eec ff ts of C3OG and C3OR on blue lightinduced ROS value of a region. However, the large numbers of fruits production produced by these trees (Prunus serrulata L. var. tomen- Given that the cytotoxic effects of blue light are depend - tella Nakai) tend to have an unpleasant taste and texture ent on excessive ROS generation [1, 8], we sought to and hence go to waste. In this study, we show the func- determine whether C3OG and C3OR can attenuate the tional utility of this wasted commodity, which can con- blue light-induced generation of cellular ROS. To this tribute to resource recycling and creating added value. end, we exposed HaCaT cells pre-treated with either Anthocyanins, which are produced in abundance in C3OG or C3OR to blue light for 1 h and subsequently colored fruits and vegetables, such as berries, red grapes, measured the cellular ROS production using H DCFDA purple sweet potatoes, and red cabbages, are among the staining. Compared with the controls, we observed most important and interesting classes of flavonoids, that exposure to 20,000 lx blue light promoted ROS and accumulating evidence is indicating their beneficial generation and that this response could be blocked by effects in preventing human diseases associated with NAC (a ROS scavenger) treatment (Fig. 2). In line with L ee and Kim Applied Biological Chemistry (2023) 66:3 Page 3 of 12 MAPKs in HaCaT cells , and numerous other stud- ies have observed crosstalk between ROS and FAK or MAPKs . Consequently, we evaluated the effects of C3OG and C3OR on FAK and MAPK activity in HaCaT keratinocytes by exposing cells to blue light for 30 min, and analyzing cell lysate using western blotting. We found that NAC and FAK inhibitor (FAK-i) treatments reduced the blue light-induced phosphorylation of FAK, MAPKs, or IKKα (Fig. 3). Treatment with C3OG or C3OR significantly reduced phosphorylation in a concen - tration-dependent manner (Fig. 3). Eec ff ts of C3OG and C3OR on blue light‑induced inflammation Although the activation of ROS-induced MAPKs is reportedly an important process in the development of inflammation , the mechanisms underlying the blue light induction of these events have yet to be suffi - ciently studied. However, although we previously found that ROS generated by blue light activates MAPKs via the activation of FAK, we did not confirm whether this mechanism is associated with an inflammatory response . In the present study, to investigate the effects of C3OG and C3OR on pro-inflammatory cytokine produc - tion in HaCaT cells, we pre-treated cells with C3OG or C3OR for 24 h, followed by exposure to blue light for 1 h, and then monitored the levels of TNF-α, IL-6, and IL-8 in cell culture supernatants. The results of the respective ELISA assays revealed that blue light induced the expression of all three of these pro-inflammatory cytokines in HaCaT cells (Fig. 4). Moreover, we demonstrated that the expression of these Fig. 1 Eec ff ts of C3OG and C3OR on the viability of HaCaT cells exposed to blue light. A HaCaT cells cultured in 96‑ well plates were cytokines was significantly inhibited by pretreatment exposed to blue light for 1 h. After 24 h, cell viability was quantified with NAC or FAK-I. However, when administered indi- using an MTT assay (n = 3, ±SD). ** p < 0.01 and *** p < 0.001 vs. vidually, the effect of FAK-i in this regard was less pro - the control. B Cells were treated with or without the indicated nounced than that of NAC. In addition, we found that concentrations of C3OG or C3OR for 24 h, and then cell viability was pre-treatment with a combination of NAC and FAK-i quantified using an MTT assay (n = 3, ±SD). C Cells were pre‑treated with or without the indicated concentrations of C3OG or C3OR for had no clear synergistic effect (Fig. 4). These findings thus 24 h prior to blue light exposure for 1 h. After 24 h, cell viability was tend to indicate that FAK-mediated signaling is one of quantified using an MTT assay (n = 3, ±SD). * p < 0.05, ** p < 0.01, and many ROS-regulated signaling pathways associated with *** p < 0.001 vs. blue light (20,000 lx) the activation of an inflammatory response. Similarly, we found that both C3OG and C3OR significantly inhib - ited the blue light-induced expression of TNF-α, IL-6, and IL-8. The inhibitory effect was dose-dependent and expectations, we found that pre-treatment with either the inhibitory effect of at the 200 µM concentration was C3OG or C3OR conferred a significant dose-dependent superior to that obtained using FAK-i (Fig. 4). protective effect against blue light-induced cellular ROS production (Fig. 2). Eec ff ts of C3OG and C3OR on blue light‑induced apoptosis Eec ff ts of C3OG and C3OR on FAK and MAPK activation In our previous study, we found that high-intensity blue in response to blue light exposure LED light induced apoptosis via the activation of cas- We have recently reported that red, green, and blue pase-3 activation and cleavage (inactivation) of FAK light-induced ROS stimulates the activation of FAK and . Consequently, in the present study, we examined Lee and Kim Applied Biological Chemistry (2023) 66:3 Page 4 of 12 Fig. 2 Eec ff ts of C3OG and C3OR on ROS production in HaCaT cells exposed to blue light. HaCaT cells cultured in 96‑ well plates were exposed to blue light (20,000 lx) for 1 h and then stained with H DCFDA (A, B). HaCaT cells were pre‑treated with the indicated concentrations of C3OG or C3OR for 24 h or with NAC (10 mM) for 1 h prior to exposure to blue light (A, B). The generation of ROS was visualized using a fluorescence microscope, Scale bar, 100 μm (A), or measured using a fluorescence plate reader (B). The bar graph presents the fold changes in ROS production in cells exposed to blue light relative to the unexposed control cells (n = 3, ±SD). ** p < 0.01 and *** p < 0.001 vs. blue light (20,000 lx) the effects of C3OG and C3OR on blue light-induced Having removed organic acids and polysaccharides from apoptosis. the crude juice by ion-exchange column chromatography, Based on a comparison of cell lysates prepared after we succeeded in achieving a more than three-fold enrich- 24 h following a 1-h exposure to blue light, we found that ment in the total content of anthocyanins in the eluate blue light increased the cleavage of PARP, caspase-3, and compared to the original juice and an overall 10% yield of FAK and that pre-treatment with NAC or z-DEVD-fmk purified C3OG and C3OR (Additional file 1: Fig. S2). (a caspase-3 inhibitor) blocked the cleavage of these pro- Blue light exposure can lead to cellular dysfunction via teins (Fig. 5). In line with expectations, we observed that the generation of ROS in the skin , with high-level blue 200 µM C3OG or C3OR also markedly reduced the cleav- light-induced ROS causing damage to the skin barrier, age of FAK and PARP via caspase-3 activation (Fig. 5). leading to aging [1, 31], hyperpigmentation , inflam - It has previously been shown that exposure to high- mation , and melasma . In this regard, although intensity blue light induces the activation of caspase-3, the antioxidant properties of anthocyanins are well estab- which in turn induces apoptosis and the cleavage of FAK lished, their effects with respect to the detrimental effects , and subsequently anoikis . In the present study, of blue light are yet to be confirmed. we observed that blue light could also reduce cell adhe- Especially, C3OG has already confirmed its effect sion and increase cell detachment from the extracellular in UVB-induced cytotoxicity studies. Cimino et al. matrix (Fig. 6). However, these detrimental effects could observed that UVB-exposed cells showed increase of be significantly reduced by treatment with C3OG, C3OR, the translocation of transcription factors NF-kB and z-DEVD-fmk, or NAC (Fig. 6). AP-1, overexpression of the proinflammatory cytokine Collectively, our findings indicate that C3OG or C3OR IL-8, cleavage of procaspase-3, and DNA fragmentation isolated from cherry fruits are effective suppressors of and these effects inhibited by C3OG treatment . Hu blue light-induced cytotoxic effects and inflammatory et al. reported that C3OG inhibit UVB-induced ROS responses. production, DNA damage, and apoptosis . He et al. demonstrated that C3OG could effectively prevent the UVB-induced apoptosis of HaCaT cells by the scaveng- Discussion ing of ROS and the suppression of COX-2 expression by The two major anthocyanins evaluated in this study, interaction with the MAPK and Akt signaling pathways C3OG and C3OR, were isolated by cation exchange col- . Consistently with this, we found that positive effects umn chromatography and semi-preparative HPLC to of C3OG and C3OR on blue light-induced inflammation yield 8 and 35 mg of pure compounds, respectively, from and apoptosis in HaCaT cells mediated via the generation 1 L of fresh cherry juice, with an estimated total antho- of ROS. Although DNA damage by blue light irradiation cyanins content of 428.6 ± 3.8 mg/L in fresh cherry juice. L ee and Kim Applied Biological Chemistry (2023) 66:3 Page 5 of 12 Fig. 3 Eec ff ts of C3OG and C3OR on the activation of FAK and MAPK induced by blue light. HaCaT cells in six ‑ well plates were exposed to blue light (20,000 lx) for 30 min. Cells were pre‑treated with or without NAC (10 mM) and FAK ‑i (1 µM) for 1 h or different concentrations (10, 50, 100, or 200 µM) of C3OG or C3OR for 24 h prior to blue light exposure (A). Immunoblots show pY397 FAK, p‑IKKα, p ‑ JNK, p‑ERK, and p ‑p38, the corresponding total proteins, and GAPDH used as a loading control. B–D Calculated fold changes in pY397 FAK, p‑IKKα, p ‑ JNK, p‑ERK, and p ‑p38 (n = 3, ±SD). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. blue light (20,000 lx) was not confirmed in this study, it is expected that the is a major upstream signal molecule implicated in several results will not be different from those of UVB. However, pathophysiological mechanisms, C3OG and C3OR may in order to clarify the difference between UVB and blue potentially be used to treat various diseases characterized light, confirmation is necessary. by FAK-associated mechanisms. Notably, 36 have reported We also established that FAK is an important upstream that black rice-derived anthocyanins can inhibit breast factor regulating the inflammatory responses induced cancer epithelial-mesenchymal transition-mediated metas- by blue light (Fig. 4) and that C3OG and C3OR are effec - tasis by suppressing FAK activation in human breast can- tive inhibitors of this regulation (Fig. 7). Given that FAK cer cells . Although these authors demonstrated that Lee and Kim Applied Biological Chemistry (2023) 66:3 Page 6 of 12 activity. In the present study, we showed that blue light- induced ROS production and FAK phosphorylation are reduced concentration-dependently by treatment with either C3OG or C3OR (Figs. 2 , 3). Moreover, we have previously established that ROS promotes the phospho- rylation of FAK in both intact cells and a cell-free system . Thus, it is conceivable that the antioxidant effects of anthocyanins may contribute to suppressing FAK activity in breast cancer cells. As mentioned in the Introduction section, blue light- induced cytotoxicity and inflammation have detrimen - tal effects on exposed skin. However, the effects of blue light tend to differ according to cell and tissue types. For example, in THP-1 cells, blue light has been demonstrated to inhibit the cytotoxic effects of low levels of lipopoly - saccharide (LPS) and reduce the release of inflamma - tory cytokines. However, these effects were not observed at high LPS levels . Blue light has also been shown to increase the expression of Nrf2 in A431 epidermoid carci- noma cells, thereby promoting an increase in the expres- sion of heme oxygenase 1, an antioxidative factor . However, whereas at high intensities, blue light (412– 426 nm) appears to have toxic effects, at 453 nm, blue light irradiation has been demonstrated to be non-toxic up to an intensity of 500 J/cm in primary human keratino- cytes and immortalized skin-derived HMEC-1 cells . Accordingly, these observations indicate that blue light may have beneficial effects at low intensities. However, these controversies due to the divergence of protocols used [39, 40] and the different mechanisms employed by cells to counter oxidative stress . Accumulated evi- dence suggests that low levels of ROS are considered to have a protective role in cells, and excessive amounts cause damage to cells . Concomitantly with this, our previ- Fig. 4 Eec ff ts of C3OG and C3OR on the expression of inflammatory ous study  and this study showed low intensity of blue cytokines induced by blue light. HaCaT cells in six‑ well plates were light make low level ROS and did not affect cell viability or exposed to blue light (20,000 lx) for 1 h. Cells were pretreated with or without NAC (10 mM), FAK‑i (1 µM), or NAC + FAK‑i for 1 h or different slightly increased (Figs. 1 , 2). concentrations (10, 50, 100, or 200 µM) of C3OG or C3OR for 24 h Since this study used only skin cell lines, there are many prior to blue light exposure. After 24 h, supernatants were collected, limitations in explaining the clear effect of C3OG or C3OR and the amounts of TNF‑α (A), IL ‑6 (B), and IL ‑8 (C) were determined on blue light in actual skin. Pure C3OG or C3OR is unsta- by ELISA (n = 3, ±SD). ** p < 0.01 and *** p < 0.001 vs. blue light ble even at room temperature and is easily oxidized or (20,000 lx) degradation by pH, light, oxygen, solvents, temperature and metal ions . When oral uptake, it is broken down phosphorylation of FAK is reduced by anthocyanin treat- more rapidly and converted into various metabolites . ment, they did not elucidate the mechanisms by which Therefore, even in animal experiments, the effect of C3OG these anthocyanins contribute to the regulation of FAK or C3OR is likely to vary depending on the method of (See figure on next page.) Fig. 5 Eec ff ts of C3OG and C3OR on blue light ‑induced FAK cleavage and caspase ‑3 activation. HaCaT cells in six ‑ well plates were exposed to blue light for 1 h and then treated with or without NAC (10 mM) or z‑DEVD ‑fmk (200 µM) for 24 h. Cells were pre ‑treated with 3OG (200 µM) or C3OR (200 µM) for 24 h prior to blue light exposure. (A) Immunoblots of total‑FAK, PARP, procaspase ‑3, active caspase ‑3 (cleaved), and the GAPDH loading control are shown. (B–D) Calculated fold changes of cleaved‑FAK, ‑PARP, and ‑ caspase‑3 calculated (n = 3, ±SD). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. blue light (20,000 lx) L ee and Kim Applied Biological Chemistry (2023) 66:3 Page 7 of 12 Fig. 5 (See legend on previous page.) Lee and Kim Applied Biological Chemistry (2023) 66:3 Page 8 of 12 Fig. 6 Eec ff ts of C3OG and C3OR on blue light ‑induced cell detachment. HaCaT cells in six ‑ well plates were exposed to blue light for 1 h and then treated with or without NAC (10 mM) or z‑DEVD ‑fmk (200 µM) for 24 h. Cells were pre ‑treated with C3OG (200 µM) or C3OR (200 µM) for 24 h prior to blue light exposure (A, B). Representative phase‑ contrast images are shown (A), and the percentages of cell adhesion and detachment relative to the control (B) were quantified (n = 3, ±SD). Scale bar, 100 μm. * p < 0.01 and *** p < 0.001 vs. blue light (20,000 lx) Consequently, our findings indicate that high-inten - sity blue light induces high levels of ROS production, inflammation, and apoptosis. The anthocyanins C3OG and C3OR are effective in suppressing the detrimental effects of blue light on HaCaT cells. Materials and methods Cells and reagents Human skin keratinocytes (HaCaT cells) were obtained from the American Type Culture Collection (USA) and maintained in Dulbecco’s modified essential medium containing 10% fetal bovine serum (Thermo Fisher Sci - entific, Waltham, MA, USA), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 units/mL peni- cillin, and 100 µg/mL streptomycin. N-acetyl-l-cysteine (NAC), z-DEVD-fmk, and cell proliferation kit I (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA); FAK inhibitor (FAKi; PF-562,271) was Fig. 7 C3OG and C3OR block blue light‑induced cytotoxicity. obtained from MedKoo (Chapel Hill, NC, USA); and Exposure to blue light initiates FAK‑related inflammation signaling 2′,7′-dichlorodihydrofluorescein diacetate (H DCFDA) events via ROS, triggering apoptotic signaling. Treatment with the 2 was purchased from Invitrogen (Carlsbad, CA, USA). anthocyanins C3OG and C3OR effectively suppress the detrimental effects of blue light via a reduction in the generation of ROS. Antibodies against FAK (Millipore, MA, USA), pY397- FAK (Invitrogen), extracellular signal-regulated kinase (ERK), p-ERK, c-Jun N-terminal kinase (JNK), p-JNK, direct application to the skin and oral feeding. For com- p38, p-p38, IKKα, p-IKKα, procaspase-3, cleaved cas- mercial use of C3OG or C3OR, new methods prepar- pase-3, PARP (Cell Signaling, Danvers, MA, USA), ing much more stable anthocyanins needs to be further and GAPDH (Millipore, MA, USA) were obtained as explored. indicated. L ee and Kim Applied Biological Chemistry (2023) 66:3 Page 9 of 12 Preparation of Cyanidin‑3‑O‑glucoside (C3OG) Cell viability assay and cyanidin 3‑O‑rutinoside from cherry fruits (Prunus Cell viability was determined using the thiazolyl blue serrulata L. var. tomentella Nakai) tetrazolium bromide (MTT) assay. HaCaT cells were Cyanidin-3-O-glucoside (C3OG) and cyanidin-3-O- cultured in 96-well plates (clear-bottomed, dark-sided rutinoside (C3OR) were extracted from cherry fruits 96-well microplate; Thermo Fisher Scientific, Rochester, collected in June 2019 from trees in Jeungpyeong NY, USA) at a density of 1 × 10 cells/well and pretreated Gun, Chungbuk, South Korea (Fig. 1 A). 1.0 L of 95% with or without NAC (10 mM) for 1 h, and C3OG (10– ethanol was added to 0.20 kg of the fresh cherry fruits, 200 µM) or C3OR (10–200 µM) for 24 h prior to expo- grinded and squeezed to produce a purple juice, which sure to 2,500–20,000 lx blue LED light for 1 h. After 24 h, was filtered through a vacuum filtration device to yield the cells were incubated with MTT (0.25 mg/mL) at 37 °C crude extract that was used for further preparation in a CO incubator for 4 h. The resulting MTT formazan of anthocyanin-enriched extracts. The crude extract products were dissolved in DMSO, and absorbances were was purified by column chromatography through a measured at 570 nm using a microplate reader (Bio-Tek Dowex ECR-S resin. The preparation thus obtained Instruments Inc., Santa Clara, CA, USA). was washed with acidified water (0.1% HCl) to remove naturally-occurring sugars and acids contained in the ROS measurement fruits. Thereafter, the resulting preparation was eluted HaCaT cells were cultured in 96-well plates (clear-bot- with acidified methanol (0.1% HCl), concentrated, and tomed, dark-sided) at a density of 1 × 10 cells/well and freeze-dried to yield an anthocyanin-rich powder . pretreated with or without NAC (10 mM) for 1 h and The two target anthocyanins, C3OG and C3OR, were C3OG (10–200 µM) or C3OR (10–200 µM) for 24 h isolated by semi-preparative HPLC using a YMC Tri- prior to exposure to 20,000 lx blue LED light for 1 h. art C18 column (250 × 10 mm, 5 μm, 12 nm), yielding Thereafter, the cells were stained with 10 µM H DCFDA 8 and 35 mg of pure anthocyanin compound, respec- for 30 min at 37 °C and subsequently washed with tively, from 1 L of cherry juice (200 g of fresh cherry phosphate-buffered saline prior to analysis using a flu - fruits). We included anthocyanins content of extracts in orescence microplate reader or visualized under a fluo - Additional file data (Additional file 1 : Fig. S2). rescence microscope. LED light exposure Direct sunlight can reach a light intensity of up to High performance liquid chromatography (HPLC‑DAD) 100,000 lx and 25,000 lx in full daylight. Comparatively, All HPLC analyses were performed using a Youngrin indoor light intensities are considerably lower, with YL9100 HPLC system equipped with a photodiode array standard office lighting typically not exceeding 500 lx. (PDA) detector. Samples were analyzed using a YMC During the day, light levels are determined by the pres- ODS-A C18 column (250 × 4.6 mm, 5 μm, 12 nm), for ence of clouds and haze. They can vary to differing which we used a gradient mobile phase comprising sol- degrees and durations, depending on factors such as the vent A (H O containing 0.1% trifluoroacetic acid) and prevailing cloud cover and atmospheric turbidity. Con- solvent B (acetonitrile). The gradient elution program sequently, in the present study, we simulated overcast used was as follows: an initial A to B ratio of 90:10, fol- (2500 lx) and full-daylight (20,000 lx) conditions for 1 h, lowed by an increase in B from 10 to 15% in 40 min at as was used in a previous study . a flow rate of 1.0 mL/min, and a subsequent increase in As a source of illumination, we used LED (GT-P25G6: B from 15 to 80% in 10 min. The retention time of com - blue, 460–470 nm, 9 W, GT-P25WB; Shenzhen Get- pound 1 was 27.4 min, whereas compound 2 was eluted ian Opto-Electronics Co., Ltd., Shenzhen, China), which at 30.0 min. Based on comparing the chromatogram were attached to a fan and a heat sink to reduce the peaks obtained for isolated compounds and those of transfer of heat to samples. To eliminate unwanted ther- standard compounds, compounds 1 and 2 were identified mal effects caused by the LED light, we monitored that as C3OG and C3OR, respectively (Fig. 8B and D). The the temperature of the incubator and cell culture dish results of full-wave scanning (Fig. 8 C and E) revealed was maintained at 37 °C in all experiments performed. that both C3OG and C3OR have two maximum absorp- HaCaT cells were seeded into 96-well cell culture plates tion peaks at approximately 281 and 518 nm and 279 and (for MTT assays or ROS measurement) or six-well cell 515 nm, respectively, which is consistent with the previ- culture plates (for western blotting or adhesion assays). ously reported characteristics of these anthocyanins. We After 24 h, the cells were exposed to blue (450 nm) LED also further confirmed the chemical structures of two light at a unified illuminance of 2,500–20,000 lx for 1 h in major anthocyanin compounds using NMR spectroscopy an incubator equipped with an LED box. Illuminance was (Additional file 1: Figs. S3, S4) [45, 46]. Lee and Kim Applied Biological Chemistry (2023) 66:3 Page 10 of 12 Fig. 8 Chemical structures of the anthocyanins cyanidin 3‑O‑ glucoside (C3OG) and cyanidin 3‑O‑rutinoside (C3OR) (A) . The 280 nm wavelength scan used to determine the purity of compounds 1 and 2 (B and D). A full‑ wave scan of C3OG and C3OR (C and E). An HPLC chromatogram of anthocyanin standard compounds (F): cyanidin 3‑O‑ glucoside (*) and cyanidin 3‑O‑rutinoside (**) L ee and Kim Applied Biological Chemistry (2023) 66:3 Page 11 of 12 measured and adjusted using a Hioki 3423 lx HiTester lux multiple comparisons test (Prism software, v7.0d; Graph- meter (Hioki E. E. Corporation, Japan) on the sample sur- Pad Software, La Jolla, CA, USA). face. As a control, a similar plate of cells was incubated in an unilluminated incubator. To avoid any potential bias, Supplementary Information the cells used for the control and light irradiation treat- The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13765‑ 023‑ 00767‑5. ments cells were derived from the same stock. Additional file 1: Figure S1. Eec ff ts of cherry fruitextract (CFE) on the Immunoblotting viability of HaCaT cells exposed to blue light. (A)HaCaT cells were treated with or without the indicated concentrations of CFEfor 24 h, and then cell To observe cellular canonical signaling, HaCaT cells in viability was quantified using an MTT assay (n =3, ±SD). (B) Cells were six-well plates were pre-treated with NAC (10 mM) and pre‑treated with or without the indicatedconcentrations of CFE for 24 h PF-271 (1 µM) for 1 h or C3OG (10–200 µM) or C3OR prior to blue light exposure for 1 h. After 24h, cell viability was quanti‑ fied using an MTT assay (n = 3, ±SD). * p< 0.05, ** p < 0.01, and *** p < (10–200 µM) for 24 h prior to exposure to blue LED illu- 0.001 vs. blue light(20,000 Lux). Figure S2. Total anthocyanine content of mination (20,000 lx) for 30 min, and thereafter immedi- the crude extract (1)and the anthocyanine enriched extract (2) from ion ately lysed (Fig. 4). Similarly, to determine the activation exchange chromatography. 1;428.6 ± 3.8mg/L, 2; 1185.6 ± 3.4mg/L. Fig‑ ure S3. NMR spectra of cyanidin 3‑O‑ glucoside (C3OG). A; H‑NMR, B; of caspase-3, HaCaT cells in six-well plates were pre- 13 1 C‑NMR. H‑NMR(400 MHz, D O) 8.08 (d, 1H, J=10.4 Hz), 7.28 (s, 1H), treated with NAC (10 mM) and DEVD-fmk (200 µM) for 6.94(d, 1H, J=15.6Hz), 6.29 (t, 1H, J=10.2Hz), 6.16 (t, 2H, J=8.6Hz), 5.0 (s, 1 h or C3OG (200 µM) or C3OR (200 µM) for 24 h prior 1H),3.93 (d, 1H, J=12.0Hz), 3.79 (m, 1H), 3.58 ~ 3.51 (m, 4H). C‑NMR(100 MHz, D O) 168.7, 158.8, 156.5, 154.6, 153.3, 144.6, 142.9,132.9, 126.3, 118.4, to exposure to blue LED illumination (20,000 lx) for 1 h, 116.1, 111.2, 102.4, 101.2, 100.3, 94.7, 75.7, 75.5, 72.5,71.8, 70.3, 69.8, 69.1, followed by cell lysis (Fig. 6). Clarified lysates were run 68.5, 66.3, 16.4. Figure S4. NMR spectra of cyanidin 3‑O‑rutinoside (C3OR). 1 13 1 on 4–12% NuPAGE Tris-Bis gels (Invitrogen). The sepa - A; H‑NMR, B; C‑NMR. H‑NMR(400 MHz, D O) 8.10 (s, 1H), 7.40 (d, 1H, J=8.4 Hz), 7.13 (s,1H), 6.45 (d, 1H, J=8.8Hz), 6.27 (d, 2H, J=8.0Hz), 4.95 (d, rated proteins were transferred to polyvinylidene fluoride 1H, J=7.2Hz),3.98 (d, 1H, , J=10.8Hz), 3.83 (s, 1H), 3.53 (m, 8H), 3.28 (t, 1H, membranes, blocked with 3% bovine serum albumin, and , J=9.6Hz),1.05 (d, 3H, , J=6.0Hz). C‑NMR (100 MHz, D O) 168.7,158.8, incubated overnight with primary AT antibodies at 4 °C. 156.5, 154.6, 153.3, 144.6, 142.9, 132.9, 126.3, 118.4, 116.1, 111.2,102.4, 101.2, 100.3, 94.7, 75.7, 75.5, 72.5, 71.8, 70.3, 69.8, 69.1, 68.5,66.3, 16.4. The following day, the membranes were washed with Tris-buffered saline containing 0.1% Tween 20 deter- gent and then incubated with horseradish peroxidase- Acknowledgements Not applicable. conjugated secondary antibodies. Bound antibodies were visualized using Enhanced chemiluminescence in con- Author contributions junction with a ChemiDoc MP Imaging System (Bio-rad, JS KIM and HY LEE designed the research, performed the experiments, ana‑ lyzed data, interpreted data, and wrote the manuscript. Hercules, CA, USA). Funding The research was supported by the Basic Science Research Program through Cell detachment assay the National Research Foundation of Korea (NRF) funded by the Ministry of HaCaT cells in six-well plates were pretreated with NAC Education (No. 2021R1A6A1A03046418) and partly supported by the technol‑ (10 mM) and DEVD-fmk (200 µM) for 1 h or C3OG (200 ogy development program of MSS (S2952823). µM) or C3OR (200 µM) for 24 h prior to being exposed Availability of data and materials to blue LED illumination (20,000 lx) for 1 h. 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Applied Biological Chemistry – Springer Journals
Published: Jan 13, 2023
Keywords: Blue light; Anthocyanins; Cyanidin 3-O-glucoside; Cyanidin 3-O-rutinoside; Reactive oxygen species; Focal adhesion kinase; Agri-food waste
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