Table of Contents    
ORIGINAL ARTICLE
Year : 2020  |  Volume : 11  |  Issue : 1  |  Page : 45-54  

Anthocyanins isolated from Oryza Sativa L. protect dermal fibroblasts from hydrogen peroxide-induced cell death


1 Department of Dermatology School of Anti-Aging and Regenerative Medicine, Mae Fah Luang University, Chiangrai, Thailand
2 Department of Pharmacology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand

Date of Submission10-Aug-2019
Date of Decision26-Sep-2019
Date of Acceptance06-Nov-2019
Date of Web Publication11-Mar-2020

Correspondence Address:
Dr. Salunya Tancharoen
Department of Pharmacology, Faculty of Dentistry, Mahidol University, No. 6, Yothi Road, Ratchathewi, Bangkok 10400
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_171_19

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   Abstract 


Background: Oxidative stress, cellular toxicity, and inflammation lead to skin damage, which results in premature skin aging. Recently, anthocyanins (ANT) have received much attention as dietary anti-oxidants involved in the prevention of oxidative damage. Materials and Methods: This study investigated the effects of ANT extracted from black rice (Oryza sativa L.) on the survival of rat dermal fibroblasts (RDFs) after oxidative stress-induced cellular damage by hydrogen peroxide (H2O2) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We further investigated the apoptosis-inducing effects of ANT using 4′,6-diamidino-2-phenylindole and Annexin V staining. The effect of ANT extract on autophagy was confirmed by reverse transcription polymerase chain reaction of the autophagy-related microtubule-associated protein 1B light chain 3 (LC3-II) and ffluorescence microscopy of the LC3-II protein. Results: The high-performance liquid chromatography results indicated the presence of cyanidin-3-O-glucoside in both extracts. The study demonstrated that the addition of crude or purified ANT extract before H2O2 treatment increased RDF cell viability. Pretreatment with ANT decreased the number of cells exhibiting dense chromatin fragments and DNA condensation, which are characteristics of apoptotic cell death. ANT decreased the number of late apoptotic/necrotic (Annexin + and propidium iodide (PI) +) cells and early apoptotic (Annexin V + and PI-) cells. Furthermore, ANT inhibited the H2O2-mediated induction of LC3-II gene expression in RDFs. Conclusion: The contribution of autophagy induction to the protective effects of ANT was verified by the observed decrease in the mRNA and protein expression of LC3-II. These results suggest the therapeutic potential of polyphenolic compounds extracted from O. sativa L. in oxidative damage-induced skin aging.

Keywords: Anthocyanin, autophagy, cell death, microtubule-associated protein 1B light chain 3 (LC3-II), Oryza sativa L., oxidative stress


How to cite this article:
Palungwachira P, Tancharoen S, Dararat P, Nararatwanchai T. Anthocyanins isolated from Oryza Sativa L. protect dermal fibroblasts from hydrogen peroxide-induced cell death. J Nat Sc Biol Med 2020;11:45-54

How to cite this URL:
Palungwachira P, Tancharoen S, Dararat P, Nararatwanchai T. Anthocyanins isolated from Oryza Sativa L. protect dermal fibroblasts from hydrogen peroxide-induced cell death. J Nat Sc Biol Med [serial online] 2020 [cited 2020 Sep 19];11:45-54. Available from: http://www.jnsbm.org/text.asp?2020/11/1/45/280129




   Introduction Top


Reactive oxygen species (ROS) contribute to skin aging by producing oxidative damage in cells, and their roles in survival, life span, and age-related pathology are being revealed.[1] Aging is caused by cumulative damage resulting from interactions among biological, physical, and biochemical processes that lead to cell damage and disturb cellular functions.[2] Stress responses are known to induce autophagic cell death,[3] and novel signaling molecules in the oxidative stress-induced survival pathway of autophagy have been identified.[4]

Plants have multiplexes of anti-oxidants to cope with strong damage from external stimuli. Recently, many extracts from raw plant materials, such as flavonoids and other phenolic compounds, have been discovered to have anti-oxidant effects. Carotenoids and polyphenols (bioflavonoids) are the two main classes of plant-derived antioxidants.[5] Anthocyanins (ANT) are natural phenolic compounds that have been shown to scavenge free radicals and protect against lipid peroxidation.[6] We previously found that black rice (Oryza sativa L.), a widely distributed crop in South-Eastern Asia, is rich in ANT.[7] The major ANT in O. sativa L. extracts is cyanidin-3-O-glucoside (C3G),[8] which is considered a phytotherapeutic agent due to its antioxidant properties.[9]

The potential mechanisms underlying the beneficial effects of antioxidants on skin diseases are complex. Recently, several studies have shown that anti-oxidant compounds can delay the senescence of fibroblasts and can counteract oxidative stress.[10] Vitamins and polyphenols can improve skin damage by quenching free radicals and delaying aging. Consequently, many topical anti-oxidant products have been developed in recent years to protect and delay skin aging; however, the role of diet in preventing oxidative skin damage remains unclear due to insufficient data.[11] Our recent findings suggested that ANT from O. sativa L. has anti-oxidant and anti-inflammatory properties, such as modulating type I collagen gene expression and suppressing H2O2-induced nuclear factor-kappa B activation in skin fibroblasts.[12]

Autophagy is a cell-autonomous innate defense mechanism that functions to degrade cellular organelles and abnormal proteins[13] and to maintain cellular homeostasis by recycling toxic protein aggregates or damaged organelles in the cell.[14] In addition to enhancing cell survival, autophagy can lead to cell death. Treatment with chemical agents, such as arsenic trioxide,[15] or the overexpression of tumor-suppressor proteins, such as the short mitochondrial form of p19ARF, initiates an autophagic response that leads to cell death. Many cellular stresses can induce autophagy, such as starvation, growth factor deprivation, endoplasmic reticulum stress, the accumulation of unfolded proteins, and infection.[16] Accumulating evidence suggests that a balance between autophagy and oxidative stress is essential for maintaining cellular function. Oxidative stress has been shown to induce autophagy under starvation and ischemia/reperfusion conditions in cardiac myocytes.[17] He et al. found that short exposure to oxidative stress could induce autophagy in myocardial cells, whereas long-term oxidative stress inhibited autophagy and led to cardiac muscle cell death.[18] Therefore, the stability of autophagy plays an important role in protecting different types of cells from oxidative stress. Epidermal keratinocytes activate autophagy in response to ultraviolet A (UVA)- and UV-oxidized phospholipids.[19] Under stress conditions, the excessive generation of ROS results in substantial oxidative damage to cell structures and in the degradation of damaged organelles.[4] H2O2 treatment increases microtubule-associated protein 1B light chain 3 (LC3-II) levels and the numbers of double-membraned autophagosomes and autophagic vacuoles.[20] Damaged proteins and organelles are known to accumulate in aged cells. These changes result in a decrease in energy supply and an increase in intracellular damage and oxidative stress, all of which accelerate aging.

Anti-oxidant agents abolish autophagosome formation and proteolysis.[21] Whereas excessive autophagy can lead to cell death, the moderate enhancement of autophagy during dietary restriction slows aging and increases the life span of cells and organisms.[22] A diverse range of stimuli that induce both ROS and autophagy have been described, and autophagy induction by these agents is antagonized by anti-oxidants.[23] Autophagy signaling is known to be involved in the enzymatic anti-oxidant system through the nuclear-factor (erythroid-derived-2)-2-mediated anti-oxidant system.[24] The loss of anti-oxidant activity and an increase in oxidized phospholipids in autophagy-related gene 7-deficient keratinocytes have been reported.[19] Despite the identified central roles of autophagy in the aging process and the anti-oxidant system, there has been little research on the potential roles of autophagy in skin aging.

The supplementation with natural anti-oxidant components in skincare is gaining increasing recognition among dermatologists and other medical professionals. Considering the important role of dermal fibroblasts in skin aging, the aim of the present study was to investigate thein vitro impact of H2O2-related changes in the viability of rat dermal fibroblasts (RDFs), in which autophagic death had been induced by oxidative stress. Crude and purified extracts from black rice were used to identify an isolated compound. The crude extract that contains high levels of ANT compounds and has favorable results in reversing the effects of H2O2 on cell viability was further studied in autophagy and apoptosis analyses. We also investigated whether ANT has protective effects against H2O2-induced cell death, possibly in association with the inhibition of LC3-II, a marker of autophagy.


   Materials and Methods Top


Chemicals, reagents, and antibodies

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was obtained from Promega (Madison, WI, USA). The Annexin V-ffluorescein isothiocyanate (FITC) with propidium iodide (PI) kit and 4′,6-diamidino-2-phenylindole (DAPI) staining solution were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). An isotype control immunoglobulin G (IgG) antibody (Ab), a rabbit polyclonal anti-LC3-II Ab, and an anti-rabbit FITC secondary Ab were obtained from Abcam (Cambridge, MA, USA). Unless otherwise stated, all other reagents were supplied by Sigma-Aldrich, Inc. (St. Louis, MO, USA).

Plant material and extraction

Black rice was obtained from Chachoengsao Province, Thailand, and was subjected to ANT extraction following a previously reported method.[7] ANT was extracted in ethanol (60/40, v/v%), concentrated using a Büchi Labortechnik B-490 rotary evaporator (Büchi Labortechnik AG, Flawil, Switzerland), and lyophilized with a freeze-dryer (Labconco Corp., Kansas City, MO, USA). The crude extract was stored at room temperature (RT). The purified extracts were prepared following previously reported methods.[25] Briefly, a C18 Sep-Pak cartridge (Waters Corp., Milford, MO, US) was activated for 30 min. The ANT extract was then loaded onto the column and eluted with methanol-containing 0.01% HCl. The ANT solution was then collected and condensed at 40°C using a Büchi Labortechnik AG B-490 rotary evaporator with a vacuum.

Determination of cyanidin-3-O-glucoside in crude and purified extracts of Oryza sativa L. by high-performance liquid chromatography

The composition of the ANT extract was identified using high-performance liquid chromatography (HPLC) (Prominence, Shimadzu, Japan) equipped with a photodiode array (PDA) detector and a C18 column (TSK® gel ODS-100 V, 150 mm length × 4.6 mm id, 5 μm, Tosoh, Tokyo, Japan). The solvents were aqueous 5% formic acid (A) and a formic acid solution in methanol (B). The gradient was 10% B for 5 min, 10%–60% B for 15 min, and 60%–10% B for 18 min at a flow rate of 1.0 ml/min. The separated C3G components were measured by the PDA detector at a wavelength of 520 nm and identified based on their retention times. The ANT was quantified by UV-Vis spectroscopy, as previously described.[26] The model reaction solution was diluted with 0.01% HCl in distilled water. The absorbance of the samples at 510 nm was analyzed in parallel with known standard solutions using a Genesys 10 UV spectrophotometer (Thermo Scientific, Grand Island, NY, USA).

Cell culture and treatment

Primary RDFs, growth media, and passaging solutions were purchased from Cell Applications (San Diego, CA, USA). Cells were maintained in growth media in a humidified incubator with 5% CO2 at 37°C. Cells were cultured in serum-free medium before treatment with ANT extract to eliminate any possible side effects of growth factors. For this study, the cells used were from passages 6–14.

Cell viability test

Cell viability was measured by MTT assay in accordance with a previously described method.[27] Briefly, after the cells were cultured at a density of 1 × 105 cells/mL in 96-well plates, they were preincubated with the selected doses of ANT from crude or purified black rice extract (BRE; 5, 10, 25, 50, 100, 200, 500, 1000, 2000, and 5000 μg/mL). RDF viability was evaluated after exposure to different concentrations of H2O2(0.6, 1.2, and 1.8 mM) for 24 h to evaluate the effects of ANT extracted from BRE. The absorbance of the solution was measured at 570 nm with an automatic microplate reader (ImmunoMini NJ-2300; InterMed, Tokyo, Japan). The relative percentage of viable cells was evaluated.

4′,6-diamidino-2-phenylindole staining

The apoptotic morphology of RDFs was investigated by staining with the fluorescent DNA-binding dye DAPI. The morphological alterations in RDF nuclei following treatment with either H2O2 and ANT or H2O2 alone were investigated. The cells were seeded in four-well chamber slides at a density of 2 × 104 cells/well. RDFs were pretreated with 10 or 25 μg/mL ANT and then treated with 0.6 mM or 1.2 mM H2O2. Following 24 h of incubation, the cells were washed with PBS + 0.1% Tween 20 and then fixed in methanol. After washing with PBS, the cells were stained with DAPI (0.1 μg/mL) at RT for 2 min. The cells on the glass coverslips were embedded in mounting medium (Aquamount, Pittsburgh, PA, USA). Images of the stained cells were acquired using a fluorescence microscope (BX53 Digital Upright Microscope; Olympus, Tokyo, Japan) to examine the degree of apoptotic nuclear and chromatin condensation. For quantification, at least 100 cells from each sample were counted manually in four randomly chosen fields to calculate the percentage of apoptotic cells.

Annexin V-fluorescein isothiocyanate and PI staining

RDFs were seeded onto a chamber slide at a density of 2 × 104 cells/well. The RDFs were pretreated with 10 or 25 μg/mL ANT for 2 h and then treated with 0.6 mM or 1.2 mM H2O2 for 24 h. Next, the cells were washed with 100 μL of 1 × binding buffer and incubated for 15 min in the dark at RT with 10 μL of Annexin V-FITC and 5 μL of PI according to the manufacturer's protocol. The slide was subsequently observed under an inverted light microscope (BX53 Digital Upright Microscope; Olympus, Tokyo, Japan).

RNA extraction and quantitative reverse transcription-polymerase chain reaction

Time-course experiments were performed to identify changes in LC3-II gene expression levels, which were evident at the 10 h time point. RDFs were pretreated with 10 or 25 μg/mL ANT for 2 h and then treated with 0.6 mM H2O2 for another 10 h to induce autophagy. We used only 0.6 mM H2O2 due to the previous result showed that 1.2 mM H2O2 markedly increased the number of late apoptotic and necrotic cells, which might not be suitable for our objectives that focus on autophagic activity and early apoptosis. After removing the supernatant and collecting the cell pellets, total RNA was isolated from the cells using the FavorPrep Tissue Total RNA Mini Kit (Favorgen Biotech, Pingtung, Taiwan). One microgram of RNA was subjected to DNase I digestion followed by reverse transcription using a DNase I RNase-free kit (Thermo Fisher Scientific, MA, USA). Total RNA (1 μg) was reverse-transcribed into cDNA using the iScript™ Select cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) in a total volume of 20 μL using oligo-dT primers. The PCR primers were as follows: LC3-II (Accession number NM_199500.2) forward: 5′-CATGCCGTCCGAGAAGACCT-3′, reverse: 5′-CTCTGAGCAGTGGTGCATGT-3′; GAPDH (Accession number NM_017008.4) forward: 5′-CCCCCAATGTATCCGTTGTG-3′, reverse: 5′-TAGCCCAGGATGCCCTTTAGT-3′. PCR amplification was conducted using template cDNA, primers, and 2× KAPA SYBR® FAST qPCR Universal Master Mix (KAPA Biosystems, Selangor, Malaysia). The following cycling parameters were used: enzyme deactivation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 3 s and annealing/extension at 60°C for 30 s. Melting curve analysis was then performed, with stepwise (0.5°C) increases from 65°C to 95°C at 10-s intervals. All qPCRs were performed in triplicate. Relative expression was quantified using CFX Manager Software (version 2.0; Bio-Rad) by measuring SYBR green fluorescence.

Immunofluorescence staining for LC3-II in H2O2-treated rat dermal fibroblasts

RDFs were seeded onto a four-well chamber slide (Nunc™ Lab-Tek™ II Chamber Slide™ System; Thermo Fisher Scientific) at a density of 2 × 104 cells per well. The cells were pre-incubated with ANT at a concentration of 10 or 25 μg/mL for 2 h, and apoptosis was induced by 0.6 mM H2O2 for 24 h. A prolonged period of H2O2 incubation (24 h) was used to observe post-transcriptional modifications. Then, the cells were washed with PBS, fixed with methanol, blocked with 1% BSA + 0.1% Tween 20 to eliminate nonspecific binding. After washing with PBS, the cells were incubated for 1 h at RT with an anti-LC3-II Ab (1:2000 dilution). Other cells were incubated with an isotype control rabbit monoclonal Ab as a negative control. Then, the cells were incubated for 1 h with a FITC-conjugated goat anti-rabbit IgG H&L Ab (1:2500 dilution). The nuclei were counterstained with 0.1 μg/mL DAPI. After washing with PBS, the cells on the glass coverslips were covered with mounting medium (Aquamount, Pittsburgh, PA, USA) and then photographed with a ffluorescence microscope (BX53 Digital Upright Microscope; Olympus, Tokyo, Japan).

Statistical analysis

The results are expressed as the mean ± standard deviation SPSS software (version 20.0; SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Significant differences among groups were identified by one-way ANOVA, followed by Tukey-Kramer multiple comparison tests. The level of statistical significance was set at P < 0.05.


   Results Top


Separation and characterization of anthocyanins in Oryza sativa L. by high-performance liquid chromatography

The ANT composition in O. sativa L. was determined by HPLC/PDA. The resulting chromatograms at 520 nm are shown. The chromatogram with the retention time of 14.245 min indicated the presence of C3G in the crude extract [Figure 1]a, and the chromatogram with the retention time of 14.453 min identified C3G in the purified extract [Figure 1]b. In addition, the crude and purified extracts contained C3G at ~755.6 mg/kg and ~430.8 mg/kg total extract, respectively (calculated as C3G equivalents).
Figure 1: High-performance liquid chromatography analysis of anthocyanins from (a) crude extract and (b) purified extract of Oryza sativa L. representative high-performance liquid chromatography/photo diode array chromatograms of cyanidin-3-O-glucoside in the extracts at 520 nm. Peaks were detected with retention times of 14.245- and 14.453- min. Duplicate experiments were performed

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Cell viability after treatment with anthocyanins from crude and purified black rice extracts

The viability of RDFs was evaluated after exposure to different concentrations of crude and purified BREs by the MTT method. We assessed the viability of RDFs after treatment with ANT from crude and purified BREs at concentrations up to 5000 μg/mL to determine the LC50 values. As shown in [Figure 2], ANT from crude and purified BREs had no cytotoxic effect at 24 h at concentrations up to 25 μg/mL. Both extracts were found to have LC50 values above 5000 μg/mL, the highest concentration tested. The LC50 for crude extract was 5,378.44 μg/mL, and the LC50 for purified extract was 5,828.90 μg/mL. To minimize confounding effects due to reduced cell viability, the ANT concentration used for both crude and purified extracts in subsequent experiments was 25 μg/mL, which is the maximal noncytotoxic concentration, along with 10 μg/mL to establish dose-dependent effects.
Figure 2: Effects of different concentrations of ANTs on rat dermal fibroblast viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. rat dermal fibroblasts were treated with crude or purified ANTs extracted from black rice at 5–5000 μg/mL for 24 h. The percent viability of rat dermal fibroblasts relative to the percent viability of control cells is expressed as the mean ± standard deviation of three independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001 versus control

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Cell viability after exposure to H2O2 and the protective effect of pretreatment with anthocyanins from crude and purified black rice extracts

To examine the consequences of prolonged free radical stimulation, RDF viability was evaluated after exposure to different concentrations of H2O2. The H2O2-induced cytotoxicity was dose-dependent, and H2O2 concentrations >0.6 mM resulted in significant decreases in cell viability (data not shown). H2O2 at concentrations of 0.6 mM, 1.2 mM, and 1.8 mM decreased RDF viability to 90.03 ± 1.97%, 83.35 ± 2.73%, and 70.37 ± 3.53%, respectively, of the control, and the addition of crude ANT extract [Figure 3]a or purified ANT extract [Figure 3]b before H2O2 treatment resulted in increased cell viability. In contrast, pretreatment with either crude or purified ANT extract at 10 μg/mL had no significant impact on cell viability under any of the H2O2 treatment conditions. Pretreatment with 25 μg/mL ANT before the addition of 0.6 mM H2O2 increased cell viability to 97.04 ± 1.69% of the control for the crude extract and to 98.03 ± 1.00% for the purified extract (P< 0.01). These increases represented cell viability increases of 70.33 ± 2.40% [Figure 3]c and 80.22 ± 0.82% [Figure 3]d for the crude and purified extracts, respectively, relative to viability on treatment with 0.6 mM H2O2 alone.
Figure 3: Rat dermal fibroblast viability was investigated by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cells were incubated with various concentrations of H2O2for 24 h following a 2-h pretreatment with (a) crude anthocyanins extract at 10 or 25 μg/mL or (b) purified anthocyanins extract at 10 or 25 μg/mL. The percent increase in cell viability after a 2-h pretreatment with anthocyanins from (c) crude ANT extract or (d) purified ANT extract is shown. Data are expressed as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001

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Similar results were observed for cells incubated in 1.2 mM H2O2, where crude and purified ANT extracts significantly increased the viability to 92.11 ± 3.92% [Figure 3]a and 94.19 ± 3.96% [Figure 3]b, respectively, compared to the controls (P< 0.05). However, the percent increase in cell viability by ANT extracts was lower in the presence of 1.2 mM H2O2 than in the presence of 0.6 mM H2O2(52.63 ± 3.13% [Figure 3]c and 65.13 ± 2.74% [Figure 3]d for crude and purified extracts, respectively). For the subsequent experiments, crude ANT extract was used due to the favorable result in reversing the effects of H2O2 on cell viability.

The effects of H2O2 and anthocyanins treatment on rat dermal fibroblast chromatin condensation as determined by 4′,6-diamidino-2-phenylindole staining

DAPI staining was conducted to detect the extent of densely stained chromatin fragments and the distribution of stained DNA, indicating DNA condensation characteristic of apoptotic cell death. As shown in [Figure 4]a, the cells in the control group, with intact DNA, were only weakly visible in the fluorescence microscope image. H2O2 treatment caused increases in the fluorescence intensity, the number of densely stained chromatin fragments and DNA condensation. Compared with the control, pretreatment with 25 μg/mL ANT significantly decreased the number of cells exhibiting H2O2-induced dense chromatin fragments and DNA condensation at both 0.6 mM H2O2(21.75 ± 4.62 vs. 9.75 ± 1.70, P < 0.01) and 1.2 mM H2O2(28.25 ± 3.09 vs. 15.00 ± 2.58, P < 0.001, [Figure 4]b. These results suggest that ANT might have cytoprotective effects against H2O2-induced apoptotic cell death.
Figure 4: Protective effects of ANTs on H2O2-induced apoptosis. rat dermal fibroblasts were treated with 10 or 25 μg/mL crude ANT and then exposed to H2O2. (a) DNA condensation was detected by 4′,6-diamidino-2-phenylindole staining. Regions of condensed chromatin appear as dense blue areas (white arrows). Scale bar: 100 μm (low magnification images) and 50 μm (high magnification images). (b) The graph presents the percentage of apoptotic cells out of the total cells. The results are expressed as the mean ± standard deviation of three independent experiments. * P < 0.05, ** P < 0.01 and ***P < 0.001

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Protective effects of anthocyanins on the H2O2-induced apoptotic cell death of rat dermal fibroblasts investigated by Annexin V and propidium iodide staining

To investigate whether ANT has a cytoprotective effect against H2O2-induced cell damage, a double-staining method using FITC-labeled Annexin V and PI was used to detect apoptotic cells. Early apoptotic cells, with only Annexin V-positive staining, were recognized by a green-stained plasma membrane, whereas late-stage apoptotic cells, with both Annexin V-and PI-positive staining, were recognized by a green-stained plasma membrane and a red-stained nucleus. Viable cells are negative for both Annexin V-FITC and PI. Twenty-four hours of treatment with H2O2 induced death in many cells, which displayed the characteristics of late apoptosis/necrosis (that is, Annexin + and PI+) or early apoptosis (Annexin V+ and PI−). ANT pretreatment at concentrations of 10 and 25 μg/mL suppressed the induction of apoptosis by H2O2[Figure 5]. The Annexin and PI staining of cells treated with only ANT were not different from that of control cells. These results demonstrate the ability of ANT to inhibit apoptosis.
Figure 5: Rat dermal fibroblasts were incubated in 0.6 mM H2O2for 24 h with or without crude anthocyanins and stained with Annexin V/propidium iodide. Red fluorescence represents propidium iodide, and green fluorescence represents Annexin V. Scale bar: 100 μm for the lower magnification images and 50 μm for the higher magnification images

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Anthocyanins inhibit LC3-II expression in H2O2-treated rat dermal fibroblasts

It is believed that under oxidative stress conditions, intracellular components are targeted for degradation by autophagy. One of the most commonly used approaches for studying autophagy is to evaluate the appearance and disappearance of a specific intracellular protein, LC3-II, which can be used as a marker of autophagic flux.[28] As shown in [Figure 6]a, an increased mRNA level of LC3-II, a key component of the autophagosome, was observed in the H2O2 group relative to the control group, and pretreating the fibroblasts for 2 h with ANT promoted a decrease in LC3-II mRNA levels. To visualize the process of autophagy, we introduced fluorescently-tagged LC3-II Abs into fibroblasts [Figure 6]b. The accumulation of LC3-II in intracellular puncta is thought to represent the formation of autophagosomes.[28] Fluorescent puncta were visualized in fibroblasts exposed to H2O2, and an increase in puncta-positive cells was observed in the H2O2 condition relative to the control condition (1.75 ± 0.96% vs. 10.5 ± 1.29%, P < 0.001) [Figure 6]c. The percentage of puncta-positive cells significantly decreased after pretreatment with 10 and 25 μg/mL ANT (8.0 ± 0.82% and 5.75 ± 1.25%, respectively, P < 0.01). Treatment with 25 μg/mL ANT alone did not cause a difference in the number of puncta-positive cells in the treated group compared to the control group. No positive staining was detected in the cells incubated with isotype-matched control IgG Abs [Supplementary Figure 1].
Figure 6: The protective effects of ANTs on the H2O2-induced expression of the autophagy-related LC3-II gene. (a) reverse transcription-polymerase chain reaction of LC3-II in H2O2-stimulated fibroblasts without or with ANT pretreatment. Scale bar: 50 μm (lower magnification images) and 25 μm (higher magnification images). (b) Fluorescence microscopy of LC3-II protein in H2O2-stimulated fibroblasts with or without anthocyanins. (c) The percentages of puncta-positive cells (>10 puncta/cell) out of the total number of fluorescently stained cells. The data are from four independent experiments. *P < 0.05, and **P < 0.01

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   Discussion Top


In recent years, interest in research on herbal medicine has been increasing worldwide. Cultivated throughout Asia, black rice is now considered a superfood with highly beneficial attributes. The content of phenolic compounds in BRE is higher than that in white rice extract.[29] Moreover, C3G,[30] a compound found in BRE, has been shown to exhibit various pharmacological activities, such as anti-oxidant, anti-inflammatory, and anti-aging activities, and to reduce lipid peroxidation and the deleterious effects of ROS.[31] Despite these findings, the beneficial effects of black rice-derived ANT in wound healing or other skin-related protective mechanisms of black rice-derived ANT have not been fully investigated.

Oxidative stress is defined as an imbalance between ROS and anti-oxidant levels at the cellular and tissue levels. ROS-induced oxidative stress plays important role in lifestyle-related diseases and in the aging process. Dermal fibroblasts are located within the dermis layer of the skin and are responsible for generating the connective tissues that comprise the extracellular matrix.[10] Although ANT has been reported to have antioxidant activities, the protective roles of these polyphenols against oxidant-induced cell death are poorly understood. With the MTT viability assay in the present study, it was possible to assess whether ANT from BREs can recover cell viability after prolonged oxidative stress at different H2O2 concentrations. We found that RDFs exposed to an oxidative stressor had reduced cell viability. Pretreatment with ANT at concentrations as low as 10 μg/mL rescued the cells from H2O2-induced cell death, suggesting that ANT from BREs may be useful for attenuating oxidative stress-induced skin damagein vitro study. Our results are consistent with those reported by Ghosh et al.,[32] who found that the ANT and phenolic fractions of blackcurrants effectively protected the DNA of HL-60 human promyelocytic cells from the oxidative stress induced by dopamine and amyloid β25–35.

Since we found that H2O2 reduced cell viability, we then examined the effects of H2O2 on cell death and apoptosis to clarify the mechanisms by which H2O2 influences cell viability. DAPI staining revealed the typical characteristics of chromatin fragments and DNA condensation in apoptotic cells. Compared to the negative control, H2O2 treatment significantly induced RDF apoptosis. However, when the RDFs were co-treated with ANT and H2O2, the effect of H2O2 on cell viability was significantly reversed, similar to the findings of previous reports describing the effects of a polyphenol-rich strawberry extract on human dermal fibroblasts exposed to H2O2.[33] To validate the apoptotic potential of H2O2, we performed Annexin V-PI staining of H2O2-stimulated RDFs after treatment with ANT. The H2O2-treated cells showed increases in the numbers of early and late apoptotic cells, and pretreatment with ANT decreased the numbers of these apoptotic cells. This finding suggested that apoptosis may be the mechanism by which H2O2 induces cell death in RDFs and that anti-oxidants may reverse this effect of H2O2.

Autophagy is a natural process for clearing damaged cells from the body, and it helps the body regenerate new, healthy cells. Thus, autophagy is undoubtedly involved in the aging process.[14] Oxidative stress participates in the pathological process of aging by producing excessive amounts of oxygen free radicals, which alter mitochondrial function, trigger cell senescence, and activate apoptotic signals.[34] The assessment of autophagy signaling in cells requires measuring the expression of autophagy marker proteins, such as LC3-II, and observing the recruitment to autophagosome membranes using electron microscopy.[28] Studies of antioxidants in larval zebrafish models of polyglutamine disease found that co-treatment with N-acetylcysteine (NAC) or vitamin E and ammonium chloride, an autophagy-inducing stimulus, caused significant decreases in LC3-II levels in the co-treated groups relative to the group treated with ammonium chloride alone, suggesting that these antioxidants can inhibit autophagic flux.[35] A study of tenofibroblasts demonstrated that oxidative stress-induced autophagy can be reduced by antioxidants: after exposure to H2O2, more autolysosomes were observed, whereas fewer were observed following pretreatment with cyanidin.[36] In addition, a relationship between the anti-aging effect of resveratrol (RESV) and autophagy was confirmed in human umbilical vein endothelial cells (HUVECs) treated with the autophagy inhibitor 3-methyladenine (3-MA) along with RESV. After pretreatment with 3-MA, the levels of phosphorylated Rb and LC3 decreased in the control group relative to the RESV treatment group, suggesting that RESV may delay the senescence of HUVECs through the autophagy pathway.[37] In our study, we observed autophagic vacuoles in cultured dermal fibroblasts using immunofluorescence staining. We found that the number of these vacuoles increased on exposure to H2O2 alone but decreased in the context of H2O2 with ANT pretreatment. In addition, ANT extract dose-dependently downregulated the expression of the autophagic cell death-related protein LC3-II. These results revealed that ANT can regulate cell apoptosis and may have beneficial anti-aging effects. However, scanning electron microscopy should be employed to directly investigate autophagosome and autolysosome recruitment to determine the protective effects of ANT against the cellular damage induced by H2O2.

The role of autophagy in the skin is poorly understood, especially in the dermis. Many recent studies have implicated ROS in the induction of autophagy in many cell types,[38] and autophagy is known to be activated to reduce oxidative damage.[19] A study by Cavinato et al. demonstrated that blocking ROS accumulation by treatment with the anti-oxidant NAC prevents autophagy activation on UVB exposure, suggesting the important roles of autophagy-mediated ROS signaling-induced senescence in fibroblasts.[39] In addition to these observations, our findings imply a role for the autophagy response in oxidative stress, which was enhanced in skin fibroblasts after H2O2 activation and suppressed by the ANT antioxidants. Our findings highlight the complex relationship between oxidative stress and autophagy. As reported in previous studies,[3] anti-oxidants play a protective role against autophagic cell death by inhibiting intracellular ROS production. Underwood et al.[35] verified that NAC inhibits both the basal and inducible autophagy of proteins associated with neurodegenerative disease, which is consistent with our findings. Using the potential autophagy-targeting nutrient (-)-epigallocatechin-3-gallate, excessive muscle autophagy in type 2 diabetic rats was ameliorated through the downregulation of ROS signaling, which led to reduce oxidative stress and the inhibition of mitochondrial loss and dysfunction.[40] Similarly, ANT inhibits oxidative stress-induced apoptosis by elevating cellular anti-oxidant capacity[41] and contribute to anti-proliferative activity by inducing apoptosis in cancer cells.[42] Despite their antioxidant action, ANT can paradoxically exhibit pro-oxidant activity, resulting in the selective death of leukemia cells.[43] Autophagy has dual functions, as it is a pathway involved in both cell death and cell survival.[44] Based on the results of the current study, further investigations are necessary to explore the effects of ANT treatment on apoptosis-related proteins and autophagy signaling.


   Conclusion Top


We observed that LC3-II is induced by ROS to promote autophagy, which can lead to RDF cell death. ANT protects against H2O2-induced apoptosis and inhibits LC3-II expression. Understanding the apparent close relationships among ANT, ROS and autophagy may lead to the development of a new protective regimen for aging skin. The effects of ANT on the progression of age-related diseases should be monitored with clinical trials.

Financial support and sponsorship

This work was supported in part by Mahidol University Faculty of Dentistry Grant (2018).

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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