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Abstract

Objectives: The present study explored the protective effects of Pruns persica leaf extract (AstraPL) on photoaging, and investigated the associated molecular mechanisms. Methods: HaCaT keratinocytes or SKH1 hairless mice that had been treated with AstraPL were exposed to ultraviolet (UV) rays. Results: Treatment with AstraPL significantly inhibited the decrease in cell viability of UV-irradiated HaCaT cells. In addition, AstraPL treatment decreased phosphorylation of mitogen-activated protein kinases and cleavage of caspase-3 and poly (ADP-ribose) polymerase. Moreover, UV-mediated lipid peroxidation and its protein adducts were reduced by AstraPL, which is along with accumulation of nuclear factor E2-related factor 2 (Nrf2) in the nucleus, transactivation of Nrf2, and expression of Nrf2-dependent antioxidant genes. AstraPL significantly increased phosphorylation of AMP-activated protein kinase α (AMPKα), but AMPK inhibition blocked AstraPL-mediated nuclear Nrf2 accumulation and antioxidant genes induction. Consistently, topical application of AstraPL to mice significantly prevented increases in epithelial thickness, apoptosis, dryness, wrinkle length and wrinkle depth in UV-irradiated dorsal skin tissue. Furthermore, AstraPL restored UV-mediated imbalance between oxidative stress and antioxidant defense system with an increase in phosphorylated AMPKα in the skin tissue. Conclusion: The present results suggest that AstraPL may be a valuable cosmetic and therapeutic candidate for managing various UV-related skin disorders.

Keywords

AMP-activated protein kinase (AMPK)HaCaT cell nuclear factor E2-related factor 2 (Nrf2)oxidative stress skin photoaging ultraviolet (UV) ray

Introduction

Skin photoaging is caused by repeated and chronic exposure of ultraviolet (UV) rays. According to the definition of the Commission internationale de l'éclairage, UV rays are subdivided into three types based on their wavelength range: UVA (315-400 nm), UVB (280-315 nm), and UVC (100-280 nm). UVA accounts for 95% of UV reaching the Earth's surface and can lead reactive oxygen species (ROS)-mediated skin wrinkles by penetrating to the dermis. UVB has stronger energy and shallower penetration depth than UVA, which can cause skin redness and sunburn.¹ In contrast, UVC can not reach the Earth's surface because it is absorbed by stratospheric ozone layer. Therefore, UVA and UVB are major causes of skin photoaging which includes deep wrinkles, dryness, irregular pigmentation, and increased fragility.^2,3 In addition, microscopic pathologic changes of photoaged skin reveal thickened epithelium, dystrophic elastin, disorganized collagen, and inflammatory cell infiltration.^2,4 Because skin is the primary defense organ to maintains the body's homeostasis from external toxic substances, uncontrolled skin damages due to UV irradiation can progress to more serious cutaneous diseases, including eczema, dermatitis, and even cancer.³

UV radiation excites various photosensitizers/chromophores in skin tissue. These photoactive and unstable chemical moieties transfer their energy to adjacent oxygen atoms to produce singlet oxygen, superoxide anion, hydrogen peroxide, hydroxy radical, and so on. Excess amounts of ROS and reactive nitrogen species generated by UV radiation can overwhelm the skin's intrinsic antioxidant defense system, causing oxidative/nitrosative stress to the skin. Then, oxidative stress directly attacks macromolecules to downregulate their functions (eg, peroxidation of membrane lipids) and also triggers activation of cellular signaling pathways associated with cell death, extracellular matrix remodeling, and inflammation.^3,5 Therefore, medicinal herbs with potent antioxidant properties have attracted great attention for managing various skin disorders caused by UV rays.^2,6

Of diverse valuable medicinal plants, Prunus (Rosaceae) is a representative plant genus rich in polyphenol antioxidants.^7,8 Peach, Prunus persica (L.) Batch, is a deciduous tree cultivated throughout temperate regions for obtaining its delicious fruits. Worldwide, about 26 million tons of peach fruit over 2000 cultivars were produced in 2022, and in Korea, it ranked fifth among the most popular consumed fruits. Albeit leaves of P. persica have been long considered a useless byproduct of fruit harvesting, traditional knowledge suggests that the leaves also exhibit diverse beneficial activities, including diuretic, laxatives, vermifuges, insecticides, sedatives, and febrifuges. In addition, the leaves have used for the treatment of whooping cough and leukoderma.⁹ Moreover, modern pharmacological study provides evidence that P. persica leaves have greater radical scavenging ability than green tea,¹⁰ can inhibit the growth of several pathogenic/commensal microorganisms,¹¹ decrease inflammatory response in lipopolysaccharide-stimulated glial cells,¹² reduce postprandial elevation of blood glucose,¹³ and enhance spasmogenic and spasmolytic activity of intestine.¹⁴

Despite the potent abilities of P. persica leaf for promoting human health, ingestion of the leaves as food ingredient has been prohibited in Korea under the regulations of the Korea Food and Drug Administration. However, their external use (eg, medicated patches, ointments, cosmetics, etc) is currently permitted. Interestingly, it has already been reported that topical application of the leaves ethanolic extract alleviates wrinkles and epidermal hyperplasia in the UV-exposed mouse skin through its potent antioxidant, anti-elastase, anti-collagenase, and anti-tyrosinase activities.^7,10 But, the detailed cellular and molecular mechanisms of how P. persica leaves protect the skin from UV radiation have not been extensively elucidated yet. Thus, present study investigated molecular mechanisms correlated with skin protection of P. persica leaves in UV-exposed keratinocytes, an outermost barrier of the skin, and confirmed our findings in the skin of hairless mice irradiated with UV light by comparing its efficacy with ascorbic acid, as a well-known representative antioxidant.^4,15

Materials and Methods

Materials

The reporting of this study confirms to ARRIVE 2.0 guidelines.¹⁶ P. persica (cultivar, Gyeongbong) leaves were collected from a peach farm located in Yeongcheon, Gyeongsangbuk-do, Republic of Korea. To prepare AstraPL, P. persica leaves were added 10 times (weight : volume ratio) of 30% prethanol A (Duksan Pure Chemical Co.; Ansan, Republic of Korea) and extracted twice for 8 h at 70 °C. The extract was pooled, filtrated using a 60 mesh filter, and then dried using a FD8508 freeze dryer (ilShin BioBase Co.; Yangju, Republic of Korea). The yield of AstraPL was 17.53%, and the powder was dissolved in sterile water for treatment of cells or animals.

Astragalin was purchased from Biopurity Phytochemicals Ltd (Chengdu, China), and Cell Counting Kit-8 (CCK-8) was from Dojindo (Tokyo, Japan). Horseradish peroxidase-conjugated secondary antibodies (Cat. No. 7074 and 7076) as well as primary antibodies for phosphorylated extracellular signal-regulated kinase (ERK) (Thr²⁰²/Tyr²⁰⁴)(Cat. No. 9101), ERK (Cat. No. 4695), phosphorylated p38 mitogen-activated protein kinase (MAPK) (Thr¹⁸⁰/Tyr¹⁸²)(Cat. No. 9211), p38 MAPK (Cat. No. 8690), phosphorylated c-jun N-terminal kinase (JNK) (Thr¹⁸³/Tyr¹⁸⁵)(Cat. No. 9251), JNK (Cat. No. 9252), caspase-3 (Cat. No. 9662), cleaved caspase-3 (Asp¹⁷⁵)(Cat. No. 9661), poly (ADP-ribose) polymerase (PARP)(Cat. No. 9542), and phosphorylated AMP-activated protein kinase α (AMPKα) (Thr¹⁷²)(Cat. No. 2535) were provided by Cell Signaling Technology (Beverly, MA, USA). Antibodies for nuclear factor E2-related factor 2 (Nrf2)(Cat. No. sc-722), NADPH quinone oxidoreductase 1 (NQO1)(Cat. No. sc-32793), AMPKα (Cat. No. sc-74461), and cleaved PARP (H215)(Cat. No. sc-23461) were supplied from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). An anti-Lamin A/C antibody (Cat. No. 612162) was purchased from BD Bioscience (San Jose, CA, USA). Anti-glutamate cysteine ligase catalytic subunit (GCLC)(Cat. No. ab41463) and anti-4-hydroxynonenal (4-HNE)(Cat. No. ab46545) antibodies were obtained from Abcam (Cambridge, UK), and an anti-heme oxygenase-1 (HO-1) antibody (Cat. No. ADI-SPA-896) was from Enzo Life Sciences (Farmingdale, NY, USA). Vectastain Elite ABC kit and peroxidase substrate kit were purchased from Vector Lab (Burlingame, CA, USA). Luciferase expressing reporter plasmids, pGL4.37[luc2P ARE Hygro] and pRL-SV40, were provided by Promega (Madison, WI, USA). BODIPY^® 581/591 undecanoic acid (C₁₁-BODIPY) was supplied from Thermo Fisher Scientific (Rockford, IL, USA). _L-Ascobic acid (AA), anti-β-actin antibody (Cat. No. A5316), anti-nitrotyrosine antibody (Cat. No. 06-284), 2,2-diphenyl-1-picrylhydrazyl (DPPH), hematoxylin & eosin staining solution, compound C, and other analytical graded reagents were obtained from Merck (Seoul, Republic of Korea).

Quantification of Astragalin

AstraPL powder in methanol (0.01 g/mL) was solubilized for 30 min under JAC-5020 ultrasonicator (KODO; Hwaseong, Gyeonggi-do, Republic of Korea), and filtrated using a 0.45 μm polyvinylidene fluoride syringe filter (PALL Corp.; Port Washington, NY, USA). AstraPL was loaded into Alliance e2695 high-performance liquid chromatography (HPLC) system (Waters; Milford, MA, USA) equipped with CAPCELL PAK C18 MGII column (column size, 4.6 × 250 mm; pore size, 5 μm)(Osaka Soda Ltd; Osaka, Japan) and 2998 photodiode array detector (Waters). Mobile phase comprising 0.05% trifluoroacetic acid and acetonitrile was used as follows: Ratio of trifluoroacetic acid : acetonitrile was maintained at 95 : 5% for 20 min, and the ratio was gradually changed to 85 : 15% by 40 min and 60 : 40% by 80 min. Flow rate was 1.0 mL/min, and eluants was detected at 210 nm. The concentration of astragalin contained in AstraPL was quantified by interpolating the peak area with the same retention time as astragalin in the AstraPL eluants into the standard curve of astragalin.

Cell Culture, UV Exposure and Treatment

HaCaT cells were generous gift of Prof. SH Ki (Chosun University, Kwangju, Republic of Korea) and maintained in Dulbecco's modified Eagles medium (Welgene; Gyeongsan, Gyeongsangbuk-do, Republic of Korea) with 10% fetal bovine serum (Welgene) and 1% penicillin-streptomycin (Welgene) at 37 °C under 5% CO₂. After HaCaT cells were pretreated with 0.1–1 mg/mL AstraPL for 1 h, the cells were washed with phosphate-buffered saline (PBS). For UV irradiation, cells were placed in CL-1000 M UV crosslinker (Analytik Jena; Upland, CA, USA). The spectra illuminated by CL-1000 M were directly measured using a spectral UV meter (UV100N, UPRtek Europe; Arachen, Germany). The UV crosslinker irradiated 59,931.5 mW/m² of energy, and peaked wavelength was 312.8 nm. UV rays consisted of 41.67% of UVA, 57.35% of UVB, and 0.97% of UVC. The cells were exposed to about 100 mJ/cm² UV (approximately 17 s). UV exposed cells were further incubated in culture medium with appropriate concentration of AstraPL for the indicated time periods. In the same way as the experimental group, the control cells were washed with PBS, left in the turned off UV chamber for the same of time, and then the medium was replaced. For some experiments, cells were was pretreated with compound C (5 μM) 1 h before AstraPL treatment.

Cell Viability Assay

AstraPL-treated HaCaT cells with or without UV exposure were additionally incubated with CCK-8 reagent for 1 h, and the resulting plate were measured optical intensity at 450 nm using an Enspire^TM microplate reader (PerkinElmer; Waltham, MA, USA). Relative cell viability was calculated as a percentage of the absorbance of vehicle-treated control cells.

Immunoblot Analysis

Whole cell lysates using a radioimmunoprecipitation buffer and nuclear subfraction using hypo- and hypertonic lysis buffers were prepared as previously reported.¹⁷ Equal amounts of protein samples were separated on a sodium dodecyl sulfate polyacrylamide gel, electrotransferred to a nitrocellulose membrane, and verified by Ponceau S staining. After the membrane was then sequentially incubated with a primary antibody of the protein interest and a horseradish-conjugated secondary antibody, specific protein on the membrane was visualized by SuperSignal^TM West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific; Rockford, IL, USA) and Amersham ImageQuant 800 biomolecular imager (Cytiva; Marlborough, MA, USA). Uncropped original images of the immunoblots were presented in Supplementary Figure 1. Densitometric quantification of gel image was carried out using an ImageJ software (https://imagej.nih.gov/ij; accessed on 12 June 2020). The expression level of phosphorylated protein was normalized to the expression level of the unphosphorylated form, and other proteins were normalized to the expression level of β-actin (for whole cell lysate) or Lamin A/C (for nuclear protein). Moreover, intensity of HNE-conjugated proteins was normalized by band intensity in the same molecular weight range on Ponceau S-stained membrane.

Radical Scavenging Assay

Radical scavenging activity of AstraPL was analyzed using DPPH. Briefly, 0.03–1 mg/mL of AstraPL were incubated with 150 μM DPPH for 0.5 h, and then optical intensity of the mixture was measured at the wavelength of 517 nm. Scavenging of DPPH radicals by AstraPL was expressed as a percentage of the absorbance of vehicle (control), and IC₅₀ was calculated from linear regression analysis of the relative DPPH absorbance (%) with respect to AstraPL concentration.

Flow Cytometry

After cells were additionally incubated with 3 μM C₁₁-BODIPY for 1 h, the cells were collected by trypsinization and resuspended in PBS with 1% fetal bovine serum. The mean intensity of green fluorescence from 10,000 cells was analyzed by using an Accuri^TM C6 Plus flow cytometer (BD Biosciences; San Jose, CA, USA).

Reporter Gene Assay

pGL4.37 reporter plasmid which expresses firefly luciferase gene under control of 4 copies of antioxidant response element (ARE)(500 ng) and pRL-SV40 which expresses constitutively Renilla luciferase (50 ng) were cotransfected into HaCaT cells for 6 h by using a FuGENE^® HD transfection reagent (Promega), and the cells were further treated with 0.3 or 1 mg/mL AstraPL for 18 h. Firefly and Renilla luciferase activities were determined using Dual-Luciferase^® Reporter Assay System (Promega) and Lumi^TM single tube luminometer (Micro Digital; Seongnam, Republic of Korea). Firefly luciferase activity was normalized by Renilla luciferase activity.

Animal Husbandry and Drug Treatment

Experimental design and research ethics using animals was approved from Institutional Animal Care and Use Committee of Daegu Haany University (Approval No., DHU2021-055; Approval date, July 23 2021). Thirty two female CrlOri:SKH1-h hairless mice were obtained from OrientBio (Seungnam, Republic of Korea) and maintained in standard conditions of animal facility of College of Korean Medicine, Daegu Haany University, as previously.⁴ After 1 week of acclimatization, mice were randomly divided into four experimental groups (n = 8/group); Control, UV, UV + AA, and UV + AstraPL. To induce skin photoaging, mice in UV, UV + AA, UV + AstraPL were exposed 330 mJ/cm² of UV (CL-1000 M UV crosslinker, Analytik Jena) three times per week for 15 weeks. Mice in control group stayed under UV crosslinker turned off for the same amount of time (approximately 55 s). Using an art brush (No. 4, HWAHONG; Hwasung, Republic of Korea), mice in UV + AstraPL was topically applied 200 μL of 2% (w/v) AstraPL on the dorsal skin once daily for 15 weeks. Instead of AstraPL, distilled water was used for mice in Control and UV groups. In addition, mice in UV + AA was orally administered 200 mg/kg of AA, as a reference drug.^4,15 On the day of UV irradiation, each drug was treated 1 h after UV exposure. All mice were euthanized 24 h after the last drug application, and then general images of dorsal skin were captured using a FinePix S700 digital camera (Fujifilm; Tokyo, Japan). For the subsequent experiments, skin tissue was collected after obtaining skin replica.

Measurement of Body Weight and Skin Weight

The body weight of individual mice was measured at days −1, 0, 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 104, and 105 (ie, day 0 = the first day of drug application) using an electric balance (XB320 M, Precisa instrument; Zürich, Switzerland). Skin weight was measured by the weight of skin tissue obtained by punching with a diameter of 6 mm.

Histopathology and Immunohistochemistry

Generation of paraffin embedded skin tissue, general hematoxylin & eosin staining, and immunohistochemical staining using specific antibody were conducted, as previously described.^4,17 Thickness, number of microfolds, and number of immunoreactive cells against specific antibody in the skin epithelium were quantified under Eclipse 80i light microscope (Nikon; Tokyo, Japan) equipped with ProgRes^TM C5 digital camera (Jenoptik Optical System GmbH; Jena, Germany) and iSolution FL 9.1 image analyzer (IMT i-solution Inc.; Bernaby, BC, Canada).

Measurement of Skin Moisture Content

The moisture content of punched skin with a diameter of 6 mm was measured using an MB23 moisture balance (Ohaus; Pine Brook, NJ, USA).

Analysis of Skin Replica

After dorsal skin replicas of euthanized mice were generated using Repliflo Cartridge Kit (CuDerm Co.; Dallas, TX, USA), wrinkle length and depth of skin replicas were analyzed using a SV600 Skin-Visiometer System (Courage & Khazaka; Cologne, Germany), as previously described.⁴

Measurement of Superoxide Anion, Lipid Peroxidation, and Glutathione

Skin tissue was homogenized in 1.15% of KCl (w/v) using TacoPre^TM bead beater (GeneReach Biotechnology; Taichung, Taiwan) and KS-750 ultrasonic cell disruptor (Madell Technology Co.; Ontario, CA, USA). After the homogenate was clarified by centrifugation at 10,000× g for 10 min, the resulting supernatant was collected. For detecting the level of superoxide anion, skin homogenates were reacted with nitroblue tetrazolium, and optical intensity from dissolved formazan crystals was measured at a wavelength of 600 nm. In addition, skin homogenates deproteinated by trichloroacetic acid-mediated precipitation were further incubated with 0.67% of thiobarbiturate or 1 mg/mL of o-phthalaldehyde for measuring lipid peroxidation or glutathione levels, respectively. Optical intensity at a wavelength of 535 nm (for lipid peroxidation) or fluorescence intensity at excitation wavelength of 350 nm and emission wavelength of 420 nm (for glutathione) was monitored, and each value was normalized to protein concentration.

Quantitative Polymerase Chain Reaction (qPCR)

Isolation of total RNA from skin tissue, reverse transcription using dT₁₆ oligonucleotide, and PCR incorporated with SyBr Green were conducted, as previously described.^4,17 Expression levels of specific gene were quantified through normalization to β-actin expression. Following primer pairs were used for the amplification of specific genes: NADPH oxidase 2 (nox2), 5′-AGCTATGAGGTGGTGATGTTAGTGG-3′ (forward) and 5′-CACAATATTTGTACCAGACAGACTTGAG-3′ (backward); glutathione reductase (gsr), 5′-TGCGTGAATGTTGGATGTGTACCC-3′ (forward) and 5′-CCGGCATTCTCCAGTTCCTCG-3′ (backward); β-actin, 5′-AGCTGCGTTTTACACCCTTT-3′ (forward) and 5′-AAGCCATGCCAATGTTGTCT-3′ (backward).

Statistical Analyses

Numerical values are expressed as means ± standard deviations, and statistical analyses were conducted using a SPSS software (version 18, SPSS Inc.; Chicago, IL, USA), as previously described.^4,17 If one way analysis of variance (ANOVA) or Welch's ANOVA is significant (eg, P < .05), Tukey's honestly significant difference test for homoscedasticity samples or Dunnett's T3 test for heteroscedasticity samples was carried out to compare means within experimental groups.

Results

AstraPL Prevents Cell Death Induced by UV Irradiation in HaCaT Keratinocytes

Before investigating the beneficial effects of AstraPL on skin exposed to UV light, we quantified the concentration of astragalin, a representative active compound found in AstraPL.¹⁸ By interpolating the peak area showing the same retention time as the chemical standard in HPLC chromatogram, we found that AstraPL used in this study contained 12.47 ± 0.23 mg/g of astragalin (Figure 1a).

Figure 1.

AstraPL Protects HaCaT Cells from UV Irradiation. (a) HPLC chromatogram of astragalin (Left) and AstraPL (Right). Eluants are monitored at the wavelength of 210 nm. The numbers in the parentheses of chromatograms are retention time (min) of astragalin. (b) Cell viability assay. After HaCaT cells were treated with 0.1–1 mg/mL AstraPL for 24 h, Cell viability was determined by CCK-8 assay. (c) Effect of AstraPL on UV mediated cell damage. HaCaT cells that had been pretreated with 0.3 or 1 mg/mL of AstraPL for 1 h were exposed to 100 mJ/cm² of UV. Cell viability was determined at 6 (left) or 15 h (right) after UV irradiation. (d and e) Immunoblot analysis. Cells were harvested 1 h after UV Irradiation. Expression of specific proteins was visualized by chemiluminescence (left), and protein expression was normalized by expression level of unphosphorylated form of MAPKs (for d) or β-actin (for e) immunoblotting (right). Dashed Lines in immunoblot images indicate cropped images of the same membrane. ^** p < .01, vVersus vehicle treated control cells; ^# p < .05, ^## p < .01, versus UV exposed cells; AstraPL, ethanol extract of P. persica leaves; Cas-3, caspase-3; Cleav., cleaved; N.s., not significant; MAPKs, mitogen-activated protein kinases; PARP, poly (ADP-ribose) polymerase; p-ERk, phosphorylated extracellular signal-regulated kinase; p-JNK, phosphorylated c-Jun N-terminal kinase; p-p38, phosphorylated p38; Pre., precursor; UV, ultraviolet.

Next, we determined the effect of AstraPL on the viability of HaCaT cells, human immortalized keratinocytes. Because AstraPL at 1 mg/mL was the maximum concentration that did not cause precipitation in the culture medium, HaCaT cells were exposed to 0.1–1 mg/mL of AstraPL for 24 h. There was no difference in viability compared to vehicle-treated control cells (Figure 1b), suggesting AstraPL at concentrations up to 1 mg/mL is not cytotoxic to HaCaT cells, at least under our experimental conditions. Preliminary study was conducted to determine the effective irradiation intensity to damage HaCaT cells, and 100 mJ/cm² of UV irradiation was chosen, expecting that cell damage of around 50% would be optimal to observe changes in response to drug treatment (data not shown). Pretreatment with 0.3 and 1 mg/mL of AstraPL significantly inhibited the decrease in HaCaT cell viability caused by UV exposure (Figure 1c).

At the molecular level, it has been well established that UV irradiation causes damage to keratinocytes, accompanied by the activation of MAPKs and caspase-3-dependent cleavage of PARP.^19,20 Thus, we further tested whether AstraPL can protect HaCaT cells from UV via regulating essential signaling molecules correlated with UV-mediated cell damage. As expected, phosphorylation of three types of MAPKs (Figure 1d) and cleavage of caspase-3 and PARP (Figure 1e) were significantly increased 1 h after UV irradiation in HaCaT cells. However, pretreatment with 1 mg/mL of AstraPL significantly suppressed UV-mediated increases in MAPKs phosphorylation, cleaved caspase-3, and cleaved PARP. Among the observed signaling molecules, only phosphorylation of ERK was reduced by pretreatment with 0.3 mg/mL of AstraPL. In addition, except for ERK, AstraPL treatment in the absence of UV exposure did not alter the phosphorylation status of MAPKs and the cleavage of caspase-3 and PARP (Figure 1d and 1e).

AstraPL Activates Antioxidant Genes in HaCaT Cells

Because oxidative stress is an essential process to trigger UV-mediated skin damage,^3,5 we further investigate whether AstraPL can protect UV-irradiated cell damage via upregulating antioxidant defense system. When AstraPL (0.03-1 mg/mL) was incubated with DPPH, AstraPL significantly and concentration-dependently scavenged DPPH radicals. IC₅₀ of AstraPL against DPPH radical was 227.03 ± 7.04 μg/mL (Figure 2a).

Figure 2.

AstraPL Activates Antioxidant Genes in HaCaTt Cells. (a) DPPH radical scavenging assay. After 0.03–1 mg/mL AstraPL were incubated with 150 μM DPPH for 0.5 h, Optical intensity was measured at the wavelength of 517 nm (optical intensity of control = 100%). (b) 4-HNE-conjuated protein adducts. After 6 h of UV irradiation (100 mJ/cm²), Protein adducts in whole cell lysates were visualized by immunoblotting against 4-HNE antibody (left). Band intensities of the protein sdducts ranging from 35 to 100 kDa were quantified using Image J and normalized to the band intensities of proteins stained with Ponceau S (right). (c) Flow cytometry. After 5 h of UV Irradiation, the cells were additionally incubated with 3 μM of C₁₁-BODIPY for 1 h. the Mean green fluorescence intensity obtained from 10,000 cells was analyzed using flow cytometer. (d) Nrf2 activation. Nuclear fractions obtained from 0.3–1 mg/mL AstraPL tTreated HaCaT cells for 15 h were subjected to Nrf2 immunoblotting. Expression level of nuclear Nrf2 was normalized by Lamin A/C expression (left). Cells that had been cotransfected with pGL4.37 and pRL-SV40 were treated with 0.3 and 1 mg/mL AstraPL for 18 h. Firefly luciferase activity was normalized by Renilla luciferase activity (right). (e) Antioxidant gene expression. Whole cell lysates obtained from 0.3–1 mg/mL AstraPL treated cells for 15 h were used for immunoblotting of antioxidant genes (left). Expression levels of specific proteins were normalized by β-actin expression (right). * p < .05, ** p < .01, versus vehicle treated control; ^# p < .05, ^## p < .01, versus UV exposed cells; DPPH, 2,2-diphenyl-1-picrylhydrazyl; GCLC, glutamate cysteine ligase catalytic subunit; 4-HNE, 4-hydroxynonenal; HO-1, heme oxygenase-1; Nucl., nuclear; Nrf2, nuclear factor E2-related factor 2; NQO1, NADPH quinone oxidoreductase 1.

We further explored antioxidant potential of AstraPL in UV-irradiated HaCaT cells. As expected, UV exposure in HaCaT cells significantly increased protein adducts conjugated with 4-HNE, a representative marker of lipid peroxidation. However, AstraPL pretreatment concentration dependently reduced the level of 4-HNE-conjugated protein adducts (Figure 2b). Consistently, results from flow cytometry also indicated that pretreatment with AstraPL significantly decreased the mean green fluorescence intensity emitted from C₁₁-BODIPY (Figure 2c), another fluorescence sensor of lipid peroxidation.

Next, immunoblot analysis using subcellular fractionated protein extracts showed that AstraPL (0.3 and 1 mg/mL) significantly accumulated Nrf2 in the nucleus (Figure 2d_left). In addition, HaCaT cells, which had been transiently cotransfected with pGL4.37 and pRL-SV40 plasmids, were treated with 0.3 and 1 mg/mL of AstraPL. Treatment with AstraPL significantly increased ARE-driven luciferase activity (Figure 2d_right). Moreover, treatment with 0.3 and 1 mg/mL AstraPL for 15 h significantly increased the expression of NQO1, GCLC, and HO-1, which are representative antioxidant genes induced by Nrf2 transactivation,²¹ in HaCaT cells (Figure 2e).

AstraPL Activates AMPK for Induction of Antioxidant Genes in HaCaT Cells

Because AMPK has been regarded as an upstream kinase to regulate Nrf2 activity via direct phosphorylation,²² we investigated whether AstraPL can phosphorylate AMPK. Consistent with Nrf2 activation, immunoblot analysis showed AstraPL significantly phosphorylated AMPKα in the presence or absence of UV exposure (Figure 3a). In addition, AstraPL-dependent nuclear accumulation of Nrf2 was significantly blocked by pretreatment with 5 μM of compound C (a chemical inhibitor of AMPK)(Figure 3b), suggesting AMPK is an upstream activator of Nrf2 in HaCaT cells. Moreover, pretreatment with compound C significantly reduced the expression of antioxidant genes by AstraPL (Figure 3c).

Figure 3.

AstraPL Activates AMPK for the Induction of Antioxidant Genes. Whole cell lysates prepared 1 (For a) or 15 h (for b And c) after UV irradiation (100 mJ/cm²) were subject to monitor expression level of phosphorylated AMPKk (a), nuclear Nrf2 (b) and antioxidant genes (c). 5 μM of compound C was pretreated 1 h prior to AstraPL treatment (for bB And c). Dashed lines in immunoblot images indicate cropped Images of the sSame membrane. ** p < .01, versus vehicle treated control; ^## p < .01, versus UV exposed cells; AMPK, AMP-activated protein kinase; Comp. C, compound C; Nucl., Nuclear.

AstraPL Ameliorates Skin Damages in UV-Irradiated Mouse

We previously established in vivo photoaging model using hairless mice irradiated by 330 mJ/cm² of UV (equivalent to 180 mJ/cm² of UVB) three times per week for 15 weeks.^4,23 And we and others have already reported that 200 μL is sufficient amount to apply dorsal skin to explore efficacy of skin protective candidates.^23,24 Thus, to expand the keratinocyte protective effect of AstraPL against UV irradiation, we adopted a UV-mediated photoaging model in mice. 2% AstraPL (200 μL; topical application once daily) was selected as the maximum dose that did not cause precipitation in solvent (eg, distilled water), and AA was used as positive control.⁴ Although there was no difference in body weight change between the experimental groups throughout the experimental period (data not shown), repetitive exposure of UV significantly increased skin weight. However, topical application of AstraPL significantly blocked the skin weight gain (Figure 4a). In addition, histopathological analysis of hematoxylin & eosin-stained skin sections showed that UV irradiation resulted in significant thickening of the skin epithelium due to hyperplasia of epidermal keratinocytes (Figure 4b). However, UV-mediated abnormal histopathological change was decreased by topical application of AstraPL (Figure 4b and 4c). In addition, AstraPL treatment significantly inhibited moisture loss from the skin due to UV irradiation (Figure 4d). Furthermore, immunohistological analysis of skin tissue showed that UV irradiation increased the number of immunoreactive cells for specific antibodies to cleaved caspase-3 as well as cleaved PARP in the skin epithelium (Figure 4e), suggesting repetitive UV exposure provokes apoptosis of skin epithelial cells. However, AstraPL treatment significantly reduced the number of these immunoreactive cells (Figure 4e and 4f). The effects of AstraPL on epithelial thickness and epithelial apoptosis were more potent than those of AA.

Figure 4.

AstraPL Ameliorates Skin Damages in UV-Irradiated Mouse. (a) Skin weight. The dorsal skin of euthanized mice was punched to a diameter of 6 mm and weighed. (b and c) Representative histological image from paraffin-embedded skin section was obtained after staining with hematoxylin & eosin. Scale bars indicate 200 μm, and black arrows are microfolds formed on the surface of epidermis (b). Length from stratum corneum to basale layer (white dDouble arrow) was measured using an automated image analyzer (c). (d) Moisture content with a diameter of 6 mm skin was measured using a moisture balance. (e and f) Representative immunohistological image was obtained after staining skin section with cleaved caspase-3 (e-left) or cleaved PARP antibody (e-right). Scale brs Indicate 100 μmM (e). Number of immunoreactive cells per 100 epithelial cells was quantified using an automated image analyzer (f). ** p < .01, versus mice in control group; ^## p < .01, versus mice in UV group; AA, _L-ascorbic acid; Ab, antibody; Cleav., cleaved; CM, cutaneous muscle; DE, dermis; EP, epithelium; SE, sebaceous gland.

AstraPL Suppresses UV-Mediated Wrinkle Formation in Mouse Skin

Besides epidermal thickening and skin dryness, wrinkle formation is another representative phenotypic change of photoaged skin.² Thus, we generated replica of dorsal skin to further explore the effect of AstraPL on wrinkle formation in UV-irradiated mice. Analysis of skin replica using skin visiometer system showed that repeated UV exposure increased the length (Figure 5a and 5b) and depth (Figure 5a and 5c) of wrinkles. However, topical application of AstraPL significantly reduced wrinkle length and depth increased by UV irradiation (Figure 5a–5c). In addition, histopathological observations showed that AstraPL treatment significantly suppressed the formation of UV-induced microfolds on the skin epidermis (Figure 4b and 5d). AstraPL's effects on macroscopic as well as microscopic wrinkles were similar to those of AA.

Figure 5.

AstraPL Suppresses UV-Mediated Wrinkle Formation in Mouse Skin. (a) Representative image of dorsal skins (upper) and their replicas (lower). Scale bars indicate 10 mm. (b and c) Wrinkle length (for b) and depth (for c) in skin replica were measured using a visiometer. (d) Number of microfolds on epidermal surface were quantified in hematoxylin & eosin-stained skin section. ** p < .01, versus mice in control group; ^## p < .01, versus mice in UV group.

AstraPL Relieves Oxidative Stress in UV-Irradiated Mouse Skin

To determine whether AstraPL inhibits skin photoaging via reducing UV-mediated oxidative stress, we monitored oxidative stress and endogenous antioxidant levels in mouse skin. UV irradiation increased levels of lipid peroxidation (eg, malondialdehyde and 4-HNE), superoxide anion, protein nitration, and nox2 mRNA (a subunit of NADPH oxidase complex)(Figure 6a and 6b). On the contrary, glutathione, a representative endogenous antioxidant, and the level of gsr mRNA, a glutathione regenerating enzyme, were suppressed in mouse skin irradiated with UV light (Figure 6c), which imply that UV irradiation provokes oxidative stress via depleting antioxidant in mouse skin. However, AstraPL treatment significantly attenuated the aforementioned changes related to oxidative stress and endogenous antioxidant (Figure 6a–6c). Similar to the result of AMPKα phosphorylation in HaCaT cells, topical application of AstraPL significantly inhibited the UV-mediated reduction of phosphorylated AMPK in the epidermis (Figure 6d). AstraPL's effects on relieving oxidative stress and restoring endogenous antioxidant were similar to those of AA, except for the levels of malondialdehyde, nitrotyrosine-immunoreactive cells, and phosphorylated AMPKα-immunoreactive cells.

Figure 6.

AstraPL Relieves Oxidative Stress in UV-Irradiated Mouse Skin. (a) Oxidative stress level. Malondialdehyde (upper), superoxide anion (middle) and nox2 mRNA (lower) were quantified in mouse skin, as described in Materials and Methods section. (b) Immunohistochemistry. skin section was immunochemically stained with specific antibodies recognizing 4-HNE (upper left) or nitrotyrosine (upper right). Scale bars Indicate 100 μm (upper). Immunoreactive cells in 100 epithelial cells were counted using an automated image analyzer (lower). (c) Endogenous antioxidant level. Glutathione (uUpper) and gsr mRNA (lower) were quantified in mouse sSkin, as described in Materials and Methods section. (d) AMPKα phosphorylation. Mouse skin was immunochemically stained with a phosphorylated AMPKα (p-AMPKα) antibody (left), and immunoreactive cells in 100 epithelial cells were counted (right). Scale bars indicate 160 μm. ** p < .01, versus mice in control group; ^## p < .01, versus mice in UV group; Ab, antibody.

Discussion

In the present study, we showed that AstraPL used in the present study contained 12.47 ± 0.23 mg/g of astragalin, a kaempferol-_O-glycoside. It has been reported that Astragalin prevents UV-induced actinic keratosis by inhibiting p38-dependent mitogen and stress activated protein kinase and γ-histone H2A.X phosphorylation.²⁵ To elucidate skin protective candidates, phytochemicals in AstraPL were further identified by ultraperformance liquid chromatography-quadrupole time-of-flight/mass spectrometry. When selecting eluants within 50 min based on peak area (≥1.0×e⁶ arbitrary unit) and library score (≥ 90), we found at least 49 major phytochemicals (15 compounds in positive ion mode, 22 compounds in negative ion mode, and 12 compounds in both ion mode) in AstraPL (Supplementary Table 1). In addition to astragalin, trigonelline, cordycepin, neochlorogenic acid, epicatechin, chlorogenic acid, caffeic acid, p-coumaric acid, rutin, ferulic acid, hyperoside, isoquercitrin, cinnamic acid, 3,5-dicaffeoyl quinic acid, afzelin, luteolin, naringenin, isorhamnetin, madecassic acid, asiatic acid, and ursolic acid have known to be effective in alleviating skin damages caused by UV.^6,26–39 Therefore, aforementioned phytochemicals present in AstraPL may collaboratively contribute to mitigating UV-induced skin photoaging.

Although various types of programmed cell death (eg, pyroptosis, necroptosis, autophagy and ferroptosis) are closely linked to UV-mediated skin damages,^1,40 it has been reported that pretreatment with caspase inhibitor suppresses UV-mediated keratinocyte's death,⁴¹ supporting the concept that apoptosis is one of the major types of cell death during UV-mediated skin photoaging. Excessive ROS caused by UV radiation react with membrane components in mitochondria, disrupting the permeability of the mitochondrial membrane. Translocation of mitochondrial proteins (eg, cytochrome c) from leaky mitochondria into the cytosol initiates the intrinsic pathway through the formation of apoptosome complex composed of cytochrome c, apoptotic protease-activating factor 1, and caspase-9. Then this complex transduces cellular signal by cleaving executioner caspases (eg, caspase-3), which in turn cleave various substrates (eg, PARP). In addition, UV radiation also directly increases expression of Fas and its downstream effector, Fas-associated death domain,^42,43 for initiating extrinsic pathway. In vitro and in vivo results from this study also showed that UV radiation increased the cleavages of caspase-3 and PARP, common final step of intrinsic as well as extrinsic apoptosis. Therefore, present results demonstrating AstraPL could inhibit UV-induced caspase-3 and PARP cleavages suggest that AstraPL may protect skin from UV radiation by reducing epidermal cell apoptosis. The effects of AstraPL on other types of programmed cell death other than apoptosis remain to be studied in the future.

In the present study, we also showed that AstraPL decreased the phosphorylation of three MAPKs in UV-irradiated HaCaT cells. MAPKs are large protein family of a serine/threonine protein kinase that transduce cellular signaling primarily through ERK, p38 and JNK pathways. Especially, UV-mediated ROS production has been known to be a major trigger for activating three MAPK signaling pathways in skin.^19,20 In addition, UV ray also causes phosphorylation of cytokine receptor (eg, epidermal growth factor receptor), which turns on signaling cascades for MAPKs activation.^6,20 MAPK-dependent phosphorylation cascades regulate the cell cycle, induce cytokine expression for inflammatory response, and increase matrix metalloproteases transcription for extracellular matrix remodeling, ultimately contributing to UV-induced photoaging of the skin.^2,5 Therefore, inhibition of MAPKs phosphorylation by AstraPL may be one of the plausible molecular mechanisms by which AstraPL alleviate UV-induced photoaging.

Oxidative stress is a critical factor in causing UV-mediated skin photoaging.⁵ Present results from in vitro and in vivo study clearly showed that AstraPL could efficaciously reduce lipid peroxidation (eg, 4-HNE protein adducts, C₁₁-BODIPY green fluorescence intensity, 4-HNE, and malondialdehyde) and superoxide anion production in response to UV irradiation. In addition, AstaPL suppressed UV-mediated decreases in glutathione and gsr mRNA. More importantly, present results indicated that AstraPL could induce antioxidant genes through accumulating nuclear Nrf2 and enhancing Nrf2 transactivation. Nrf2 has been regarded as a guardian to protect the organism from oxidative stress.^21,44 In resting state, Nrf2 resides in the cytoplasm as a protein complex with Kelch-like ECH-associated protein 1 and is rapidly degraded by the proteasome system. External stimuli like electrophilic antioxidants can disrupt the interaction between Nrf2 and Kelch-like ECH-associated protein 1, and the stabilized Nrf2 then translocates into the nucleus, where it binds to ARE for transactivating a series of antioxidant genes, including HO-1, GCLC, NQO1, and GSR.²¹ Especially, it has been also reported that mouse deficient with nrf2 gene worsens UV-induced wrinkle formation, epidermal thickening, and loss of skin elasticity. In addition, nrf2 deficiency results in greater accumulation of UV-induced cutaneous lipid peroxidation as well as greater depletion of skin glutathione compared to UV-exposed to wild type mouse.⁴⁵ Moreover, many herbal extracts and those derived natural compounds have been suggested to protect the skin from UV-induced photoaging via activating Nrf2 signaling pathway,⁴⁶ implying that Nrf2 is a critical transcription factor to ameliorate UV-induced skin photoaging by AstraPL.

The activity of Nrf2 has been reported to be orchestrated by several mechanisms, such as ubiquitin proteasome system, microRNA, autoregulation, and posttranslational modifications.⁴⁷ Of diverse regulatory mechanisms, many serine, threonine, and tyrosine residues existed in Nrf2 serve potential sites for phosphorylation as posttranslational modification. Protein kinase C, glycogen synthase kinase 3, casein kinase 2, protein kinase RNA-like endoplasmic reticulum kinase, cyclin dependent kinase, MAPKs, and AMPK have been identified as direct upstream kinases to regulate Nrf2 activity via ubiquitin-dependent degradation, acceleration of nuclear translocation, inhibition of nuclear export, and recruitment of coactivator/corepressor.^22,47 Especially, AMPK can phosphorylate Ser⁵⁵⁰ residue within Nrf2-ECH homology 1 (Neh1) domain of mouse Nrf2 (Ser⁵⁵⁸ in case of human Nrf2).⁴⁸ Because Neh1 contains nuclear export signal motif, Ser⁵⁵⁰ phosphorylation of Nrf2 in the cytoplasm has been reported to lead nuclear accumulation by masking interaction between nuclear export signal motif and nuclear export protein complex (eg, exportin, Ran).^47,48 Although there were differences in basal and UV-exposed AMPKα phosphorylation between in vitro and in vivo studies, present results from two models clearly showed that AstraPL treatment was capable to increase the level of phosphorylated AMPKα. In addition, AstraPL-mediated antioxidant gene induction in HaCaT cells was completely blocked by pretreatment with an AMPK inhibitor, suggesting that AMPK probably is an upstream signaling molecule for AstraPL-mediated Nrf2 activation.

Throughout in vitro as well as in vivo experiments, present results provide concrete evidence that AstraPL can efficaciously alleviate skin photoaging, and relieving oxidative stress via AMPK-dependent Nrf2 activation is involved in the process. In addition, present results show that most of the skin protective effects by AstraPL are largely similar to those of AA, but certain biomarkers (eg, epithelial thickness and AMPK phosphorylation) are more potent with AstraPL. Nevertheless, present study has several limitations. 1) In addition to quantitative analysis of phytochemicals contained in AstraPL, it is necessary to further identify the key compounds as well as relevant mechanisms associated with skin protection of AstraPL. 2) The in vivo efficacy of AstraPL has been confirmed at a single dose only. Thus, additional studies are needed to determine the effective dose range of AstraPL, with appropriate positive control showing a similar molecular mechanism to AstraPL. 3) To overcome safety concerns of AstraPL, more purified or standardized forms of AstraPL need to be developed for human application.

Conclusion

If the remaining issues are successfully resolved, AstraPL will be a valuable natural extract for topical application that helps protect the skin from various tissue damage caused by repeated and chronic UV exposure.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251330952 - Supplemental material for Prunus persica Leaf Extract Mitigates Ultraviolet-Induced Skin Damage Via Activation of AMP-Activated Protein Kinase

Supplemental material, sj-docx-1-npx-10.1177_1934578X251330952 for Prunus persica Leaf Extract Mitigates Ultraviolet-Induced Skin Damage Via Activation of AMP-Activated Protein Kinase by Eun Ok Kim, Dae Geon Lee, Cheol Jong Jung, Yeong Eun Yu, Museok Hong, Il Je Cho and Sae Kwang Ku in Natural Product Communications

Footnotes

Acknowledgements

The authors express thanks to Dr SH Ki (College of Pharmacy, Chosun University, Republic of Korea) for kind donation of HaCaT cells and Dr SM Park (High-Tech Material Analysis Core Facility, Gyeongsang National University, Republic of Korea) for technical assistance in identifying phytochemicals of AstraPL using an UPLC-QTOF/MS analysis.

Statement of Human and Animal Rights

The study was conducted in accordance with guidelines stipulated by the Ministry of Food and Drug Safety for the Care and Use of Laboratory Animals and was approved by Institutional Animal Care and Use Committee of Daegu Haany University (Approval no. DHU2021-055; Approval date, July 23. 2021).

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iDs

Eun Ok Kim

Dae Geon Lee

Cheol Jong Jung

Yeong Eun Yu

Museok Hong

Il Je Cho

Sae Kwang Ku

Ethical Considerations

This study was approved by Institutional Animal Care and Use Committee of Daegu Haany University (Approval no. DHU2021-055; Approval date, July 23. 2021).

Author Contributions/CRediT

Conceptualization, CJ Jung, M Hong, IJ Cho and SK Ku; methodology, EO Kim, DG Lee, IJ Cho and SK Ku; validation, EO Kim, DG Lee, IJ Cho and SK Ku; formal analysis, EO Kim, YE Yu, CJ Jung and IJ Cho; investigation, EO Kim, DG Lee, YE Yu, IJ Cho and SK Ku; data curation, IJ Cho; writing—original draft, EO Kim, IJ Cho and SK Ku; writing—review and editing, EO Kim, DG Lee, IJ Cho and SK Ku; supervision, CJ Jung, M Hong and SK Ku; All authors have read and agreed to the published version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Conflicting Interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Authors (EO Kim, DG Lee, CJ Jung, YE Yu, and IJ Cho) were employed by the Okchundang Inc. The remaining authors declare that the research was conducted in the absence of commercial or financial relationship that could be constructed as a conflict of interest.

Data Availability

All datasets are included within the article and its supplementary information file.

Supplemental Material

Supplemental material for this article is available online.

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