Hawthorn polyphenol extract inhibits UVB-induced skin photoaging by

Jul 22, 2018 - Hawthorn polyphenol extract inhibits UVB-induced skin photoaging by regulating MMP expression and type I procollagen production in mice...
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Bioactive Constituents, Metabolites, and Functions

Hawthorn polyphenol extract inhibits UVB-induced skin photoaging by regulating MMP expression and type I procollagen production in mice Suwen Liu, Lu You, Yanxue Zhao, and Xuedong Chang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02785 • Publication Date (Web): 22 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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Journal of Agricultural and Food Chemistry

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Hawthorn polyphenol extract inhibits UVB-induced

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skin photoaging by regulating MMP expression and

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type I procollagen production in mice

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Suwen Liu†, Lu You†, Yanxue Zhao†, Xuedong Chang†,‡,§*

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Technology, Qinhuangdao, Hebei, 066004, China

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College of Food Science & Technology, Hebei Normal University of Science and

Hebei Yanshan Special Industrial Technology Research Institute, Qinhuangdao,

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Hebei, 066004, China

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§

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Hebei, 067000, China

Hebei (Chengde) Hawthorn Industrial Technology Research Institute, Chengde,

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*Corresponding author:

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Xuedong Chang, College of Food Science & Technology, Hebei Normal University of

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Science

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[email protected]

and

Technology,

Qinhuangdao,

Hebei,

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066004,

China.

E-mail:

Journal of Agricultural and Food Chemistry

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ABSTRACT: Ultraviolet (UV) B radiation can cause skin aging by increasing matrix

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metalloproteinase (MMP) production and collagen degradation, leading to the

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formation of wrinkles. This study investigated whether hawthorn polyphenol extract

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(HPE) protects against UVB-induced skin photoaging using HaCaT human

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keratinocytes, normal human dermal fibroblasts (HDFs), and mice. Analysis of the

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phenol composition of HPE by high-performance liquid chromatography-mass

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spectrometry showed that chlorogenic acid (13.5%), procyanidin B2 (19.2%), and

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epicatechin (18.8%) collectively accounted for 51.4% of total phenol content and

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represent the active ingredients of hawthorn fruit. A cell viability assay revealed that

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HPE treatment promoted cell proliferation in HaCaT cells and HDFs. On the other

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hand, MMP-1 and type I procollagen production was decreased and increased,

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respectively, in UVB-exposed cells treated with HPE as compared with those without

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treatment, as determined by enzyme-linked immunosorbent assay. Hematoxylin and

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eosin and Weigert staining of dermal tissue specimens from mice demonstrated that

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HPE also reversed UVB-induced epidermal thickening and dermal damage. The

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increase in production of reactive oxygen species and decrease in antioxidant

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enzyme activity as well as the increase in nuclear factor-κB activation and

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mitogen-activated protein kinase phosphorylation induced by UVB irradiation were

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reversed by HPE (100 or 300 mg/kg body weight), which also suppressed MMP

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expression and stimulated the production of type I procollagen in the dorsal skin of

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UVB-irradiated mice. These results suggest that HPE is a natural product that can

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prevent UVB radiation-induced skin photoaging.

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KEYWORDS: hawthorn polyphenol, HPLC-MSI-MS/MS, UVB radiation, antioxidant,

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NF-κB/MAPK

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INTRODUCTION

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Skin aging is caused by both internal and external factors that induce natural

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skin aging and photoaging, respectively. A major contributor to skin photoaging is

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exposure to ultraviolet (UV) radiation (wavelength of 290–320 nm) in sunlight,1,2 which

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can damage skin by altering its cellular composition and organization3 and causing

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loss of the extracellular matrix (ECM).4 The dermal ECM includes all intercellular

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materials (besides water) such as elastic and collagen fibers, amino polysaccharides,

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and proteoglycans. Exposure to UVB radiation increases the expression of matrix

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metalloproteinases (MMPs)—whose substrates are ECM proteins. It also leads to the

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degradation of fibrous connective tissue and decreases type I and III collagen fibers,

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resulting in the occurrence of wrinkles.5

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UVB-induced MMP expression leads to activation of multiple signaling

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pathways such as those of mitogen-activated protein kinase (MAPK) and nuclear

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factor kappa B (NF-κB).6-8 MAPKs are a family of serine and tyrosine kinases that

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includes extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase

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(JNK), and p38 MAPK in eukaryotic cells. MAPK transmits extracellular signals to

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regulate gene expression.9 The transcription factor NF-κB in human skin is a

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heterodimer composed of P50/P65 subunits that combines with inhibitor of κB protein

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to form an inactive complex in the cytoplasm under physiological conditions; the

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promoter of the MMP-1, -3, and -9 genes has NF-κB-binding sites, implying that they

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are regulated by this signaling pathway.10

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The production of reactive oxygen species (ROS) induced by UVB radiation is

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considered the starting point for activation of the NF-κB and MAPK signaling

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pathways.11 In addition, activator protein (AP)-1 stimulates the expression of c-Jun

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and c-Fos as well as MMPs and cytokines.8 Recent studies have shown that natural

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substances can block these signals—e.g., Pinus densiflora, grape seed, Rhus

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javanica, and Urtica thunbergiana extracts; youngiasides A and C isolated from

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Youngia denticulatum; and cod skin gelatin hydrolysates.12–17 Additionally, various

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phenolic compounds from plants are thought to inhibit UVB-induced skin aging.

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Hawthorn (Crataegus L.) is a member of the Rosaceae family of plants

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comprising about 1000 species that are mainly distributed in eastern and northern

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temperate regions of Asia, Europe, and North America; Chinese hawthorn was first

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identified 1700 years ago. The fruit is rich in polyphenols18 and has various

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physiological benefits including antioxidant and blood lipid- and glucose-lowering

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effects.19,20 However, it is not known if Hawthorn polyphenol extract (HPE) inhibits

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UVB-induced skin photoaging.

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To address this issue, the present study investigated the anti-photoaging

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effects of HPE by evaluating type I procollagen production in UVB-irradiated normal

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human dermal fibroblasts (HDFs) and MMP-1 expression in HaCaT human immortal

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skin keratinocytes and HDFs. We also assessed the effects of HPE treatment on UVB

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irradiation-induced skin photoaging in mice.

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MATERIALS AND METHODS

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Plant material and extraction

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“Yanranghong” hawthorn (Crataegus pinnatifida Bge. var. major N. E. Br.)

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fruits were collected at hawthorn cultivation demonstration park of Hebei (Chengde)

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Hawthorn Industrial Technology Research Institute in October 2016 from Hebei,

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Xinglong, China (latitude, 41°11′3″N and longitude, 117°12′35″E) according to a

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previously reported method,21,22 with slight modifications. In brief, fresh fruit samples

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were accurately weighed after removing the seeds, and 300 g of material (deseeded

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whole fruit) was homogenized using a food processor (JYL C022E; Joyoung, Jinan,

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China). After adding 70% acidic ethanol (hydrochloric acid 0.1%) at a ratio of 1:10 and

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mixing for 1 h at 30°C, the mixture was filtered in the dark. The filtrate was

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concentrated using a rotary evaporator (EYELA N1100; Tokyo Rikakikai, Tokyo, Japan)

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at 40°C. The filtrate was purified with AB-8 macroporous resin (Sigma-Aldrich, St.

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Louis, MO, USA) while protected from light and then washed for 4–6 h with distilled

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water. The samples were eluted with 70% acidic ethanol, concentrated in a rotating

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vacuum at 40°C, and refrigerated overnight for freeze drying (LGJ-15D freeze dryer,

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Beijing, Sihuan science instrument co., LTD. China). The powder was stored at −20°C

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in a brown glass tube.

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The polyphenol content of HPE was determined by high-performance liquid

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chromatography-electrospray

ionization-tandem

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(HPLC-ESI-MS/MS). The optimized mobile phases were 5% (v/v) formic acid in water

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(A) and acetonitrile (B). The elution program was as follows: 0–8 min, 5%–10% B;

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mass

spectrometry

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8–20 min, 10%–20% B; 20–28 min, 20%–25% B; and 28–30 min, 25%–5% B. An

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Eclipse XDB-C18 column (250 × 4.6 mm inner diameter, 5 µm; Agilent Technologies,

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Santa Clara, CA, USA) was used and operated at 30°C. The injected amount was 10

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µl, and the flow rate was maintained at 1.0 ml/min. Data were recorded at 280 nm. A

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mass spectrometer equipped with an ESI system was used to acquire MS/MS data

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(Ultimate 3000 LTQ XL; Thermo Fisher Scientific, Waltham, MA, USA).21

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Cell culture and UVB irradiation

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UVB irradiation and sample treatment were performed as previously

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described.14,23 HaCaT cells and HDFs (Huaao, Nanjing, China) were cultured at 37°C

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with 5% CO2 (HF-90; Heal Force BioMeditech, Shanghai, China) for 24 h. The HaCaT

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cells and HDFs were divided into the following eight groups. 1) In the normal control

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group, cells received no UVB radiation or HPE treatment. 2–4) Cells were treated with

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various concentrations of HPE (0, 5, or 10 µg/ml) for 1 h. Then, cells were irradiated

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with UVB at 30 mJ/cm2 for 40 s (wavelength range: 290–320 nm; peak: 312 nm;

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irradiation distance: 15 cm; UVB radiation intensity: 0.75 mW/cm2) with a UVB lamp

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(JY-T58; Zhongshan OURI Optoelectronic Technology, Zhongshan, China). Irradiance

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was measured by UV illumination (UV-340A; Luchang Electronic Enterprise, Taipei,

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Taiwan). After irradiation, the medium was replaced with fresh medium containing

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various concentrations of HPE (0, 5, or 10 µg/ml). 5–7) Cells in these groups were

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subjected to the same treatment as those in groups 2–4, using procyanidin B2 (PRB),

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epicatechin (EP), or chlorogenic acid (CA) (purity > 98%; Yuanye, Shanghai, China)

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instead of HPE at a concentration of 20 µmol/l. 8) Procyanidin (PR) derived from

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grape seeds was used as a positive control (purity > 95%; Yuanye). Dose selection

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was based on results of the cell viability experiment and previous reports.14,23,24

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Cell viability

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Cells were cultured at 37°C with 5% CO2 for 48 h in 100 µl medium (HaCaT

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cells with DMEM, HDFs with DMEM/F12). A 10-µl volume of 0.5 mg/ml

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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Sigma-Aldrich, M-2128) was added to each well followed by incubation at 37°C, with

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150 µl dimethyl sulfoxide added after 4 h. Absorbance at 570 nm was measured by

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spectrophotometry on a microplate reader (ELX-800; Bio-Tek Instruments, Winooski,

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VT, USA).

bromide

tetrazolium

(MTT;

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Animal experiments

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Animal experiments were carried out as previously reported,15,25 with some

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modification. Female Balb/c mice (Wanleibio, Shenyang, China) aged 5–6 weeks

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were housed in a temperature- and humidity-controlled room (22°C ± 1°C, 45%–55%

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humidity) on a 12:12-h light/dark cycle with free access to food and water. All

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experimental procedures, animal care, and handling were carried out in accordance

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with the guidelines of the national standards outlined in “Laboratory Animal

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Requirements of Environment and Housing Facilities” (GB 14925-2010). The

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experiments were approved by the “Management and use of laboratory animals”

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Committee of Hebei Normal University of Science and Technology and provided by

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Beijing HFK biotechnology co., LTD. (License No. SCXK (Jing) 2014-0004). The mice

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were divided into five groups of eight mice each: normal and hairless control groups

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(without exposure to UVB radiation), UVB model, and HPE treatment groups. The

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dorsal skin of each mouse was removed with an electric shaver. The UVB source was

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a UVB lamp (JY-T58; Zhongshan OURI Optoelectronic Technology, Zhongshan,

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China) with an irradiance peak of 312 nm. The lamp was positioned 15 cm above the

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mice. UVB radiation intensity was 0.75 mW/cm2. Irradiance was measured by

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ultraviolet illumination (UV-340A; Luchang Electronic Enterprise). Before irradiation,

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mice in the UVB radiation, HPE-L, and HPE-H groups were treated with distilled

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saline or 100 or 300 mg/kg body weight/day HPE by oral administration, respectively.

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After 1 h, the dorsal skin of all hairless but not control mice was irradiated daily for the

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first week at 100 mJ/cm2 UVB. Starting from week 2, the mice were irradiated three

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times a week with 200 mJ/cm2 UVB up to 12 weeks. Mice in each group were housed

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separately. The animals were euthanized after the final UVB exposure, and biopsies

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were obtained from the dorsal skin for histological analysis. The remaining skin

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specimens were stored at −80°C. The wet skin was weighed, dried in an oven

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(QH01-9030A; Jinghong Experimental Equipment, Shanghai, China), and weighed a

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second time. The two values were used to calculate skin moisture content.

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Histological skin analysis

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Hematoxylin and eosin (H&E) staining

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Fixed skin tissue was dehydrated in a graded series of alcohol from low to high

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concentration and then incubated in xylene for 30 min. After embedding in paraffin,

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the tissue block was cut into 5-µm sections using a microtome (RM2235; Leica,

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Wetzlar, Germany). The sections were collected in a warm dish and then dried at

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60°C for 2 h. After deparaffinization in ethanol, the sections were stained with H&E,

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dehydrated, clarified, mounted, and photographed under a light microscope (DP73;

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Olympus, Tokyo, Japan) at 200× magnification. Photoshop v.16.1.0 software was

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used to evaluate and quantify epidermal thickness.

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Weigert staining

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Skin tissue sections (5 µm thick) were deparaffinized in ethanol and oxidized

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with potassium permanganate for 3 min, bleached with oxalic acid solution for 3 min,

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and rinsed with water for 5–10 min. The sections were stained with Weigert resorcinol

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magenta for 1–3 h, followed by incubation in acid differentiation liquid. After washing

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with water for 5–10 min, sections were stained with Verhoeff–Van Gieson for 30 s,

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quickly washed, and then rapidly differentiated in 95% ethanol. The sections were

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dehydrated, clarified, and mounted, and three sections from each group were

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photographed under the light microscope at 200× magnification.

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Enzyme-linked immunosorbent assay (ELISA)

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After UVB irradiation, HaCaT cells and HDFs were cultured for 24 h at 37°C

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with 5% CO2. Total protein was extracted and quantified with a bicinchoninic acid

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(BCA) assay kit (WLA004a; Wanleibio). MMP-1 and type I procollagen levels in the

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culture supernatant were determined by ELISA using commercial kits (SEA097Hu and

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SEA955Hu, respectively; USCN Life Science, Wuhan, China) according to the

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manufacturer’s instructions.

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Determination of ROS levels

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ROS levels were measured as previously described.26 Briefly, 10 µmol/l

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dihydrofluorescein diacetate was added to the culture medium at 37°C followed by

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incubation for 30 min. ROS production was measured on a microplate reader

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(M200PRO; TECAN, Mannedorf, Switzerland) at excitation and emission wavelengths

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of 485 and 530 nm. The results are expressed as fluorescence intensity. Skin tissue

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specimens were weighed and combined with normal saline at a 1:9 (w/v) ratio,

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homogenized on ice, and then centrifuged at 421 × g for 10 min. ROS levels in 10%

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homogenate supernatant were measured with an ROS assay kit (WLA070;

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Wanleibio).

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Determination of antioxidant enzyme levels and malondialdehyde (MDA)

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Antioxidant enzyme levels and MDA activity in the skin were detected with

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commercial kits, including the MDA (WLA048b) and superoxide dismutase (SOD)

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(WLA110) assay kits (both from Wanleibio), glutathione peroxidase (GSH-Px) (A005)

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and catalase (CAT) (A007-1) assay kits (both from Nanjing Jiancheng Bioengineering

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Institute, Nanjing, China), according to the manufacturers’ instructions. Skin tissue

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was weighed and combined with normal saline at a 1:9 (w/v) ratio, homogenized on

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ice, and centrifuged at 421 × g for 10 min. Antioxidant enzyme levels and MDA activity

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in 10% homogenate supernatant were measured.

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Western blot analysis

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Western blotting was performed using HaCaT cells, HDFs, and skin tissue

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lysates. Following treatment, cells and tissues were harvested and washed in

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phosphate-buffered saline (PBS). Total and nuclear proteins were extracted using

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commercial kits (WLA019 and WLA020, respectively; Wanleibio) according to the

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manufacturer’s instructions. The protein concentration was determined with a BCA

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assay kit, and 40 µg/sample was separated by sodium dodecyl sulfate polyacrylamide

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gel electrophoresis (DYY-7C; Beijing Liuyi Biotechnology Co., Beijing, China). The

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concentration of the spacer gel was 5%, and the concentration of the separation gel

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was 8% or 10%. Samples diluted in 5× loading buffer and PBS were boiled for 5 min.

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A 20-µl volume of sample was transferred to a polyvinylidene difluoride membrane

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(IPVH00010; Millipore, Billerica, MA, USA) that was blocked in skim milk powder

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solution for 1 h followed by overnight incubation at 4°C with primary antibody (5% w/v)

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in the same solution. The membrane was washed in Tris-buffered saline with 0.1%

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Tween-20 and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG

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(1:5000) at 37°C for 45 min. Protein bands were detected by applying enhanced

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chemiluminescence reagent in the dark. The film was scanned, and optical density

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values of target bands were analyzed with Gel-pro Analyzer software (wd-9413b;

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Beijing Liuyi Biotechnology Co.). The results are expressed as the ratio of average

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relative intensity of each band to that of β-actin or histone H3.27

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Statistical analysis

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All experiments were performed at least three times. Results are presented as

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the mean ± standard deviation (SD). Means of different treatment groups were

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compared by one-way analysis of variance with Tukey’s multiple comparisons test. P

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< 0.05 was considered statistically significant.

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RESULTS

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Polyphenol component analysis

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The polyphenol content of hawthorn fruits is shown in Table 1. A total of 11

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compounds were identified by HPLC-ESI-MS/MS. The total polyphenol content of

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HPE was 67.5%; the main components were CA, PRB, and EP, representing 13.5%,

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19.2%, and 18.8% of the total, respectively.

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HPE enhances the viability of UVB-irradiated cells

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The effects of HPE on the viability of HaCaT cells and HDFs exposed to UVB

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radiation (30 mJ/cm2) were evaluated with the MTT assay. The experimental

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components had no significant effect on cell viability, as shown in Figure 1a, c. Cell

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survival was significantly reduced by irradiation (P < 0.01), but this effect was

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abrogated by treatment with 5 or 10 µg/ml HPE (P < 0.05). The viability of

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UVB-irradiated cells was similar to that of non-irradiated control cells upon

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co-treatment with HPE and 20 µmol/l PRB, PR, or CA (P < 0.01; Fig. 1d). On the other

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hand, the viability of the EP group was higher than that of the UVB group (P < 0.05)

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but lower than that of the normal control group of HaCaT cells (Fig. 1b; P < 0.05).

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HPE decreases ROS production after UVB irradiation

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After irradiation at 30 mJ/cm2, ROS production increased; however, HPE (5 or

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10 µg/ml) abrogated this effect in HaCaT cells and HDFs (P < 0.05; Fig. 2). A similar

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effect was observed in HaCaT cells (Fig. 2a) and HDFs (P < 0.01; Fig. 2b) co-treated

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with HPE (5 or 10 µg/ml) and PRB, EP, or CA (20 µmol) after UVB exposure. In animal

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experiments, 100 or 300 mg/kg body weight HPE reduced ROS production by 16.1%

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and 31.4%, respectively, compared with that in the UVB group (P < 0.01; Fig. 2c).

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Therefore, HPE can suppress ROS production induced by UVB radiation.

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HPE decreases MMP production in vitro and in vivo

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HPE (5 or 10 µg/ml) suppressed MMP-1 expression in HaCaT cells and HDFs

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compared with that in the UVB group (P < 0.05 or 0.01). Treatment with 10 µg/ml HPE

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and PRB or EP (20 µmol/l) inhibited MMP-1 levels in UVB-irradiated HaCaT cells (P >

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0.05); MMP-1 was downregulated in the CA group as compared with the PRB and EP

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groups by 15.6% (P < 0.05; Fig. 3a). A similar effect was observed in HDFs (P < 0.05

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or 0.01; Fig. 3b).

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HPE (10 µg/ml) increased the level of type I procollagen by 80.5% as

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compared with that in the UVB group (P < 0.01). Type I procollagen level was 34.8%

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higher in the CA than in the EP group (P < 0.05). Similar trends were observed for the

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protein expression. Transforming growth factor (TGF)-β1 is the main regulator of type

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I collagen synthesis in human skin and is highly expressed in somatic cells.28 In this

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study, UVB irradiation for 24 h resulted in the downregulation of TGF-β1 relative to

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that in non-irradiated cells (P < 0.01; Fig. 3C). HPE treatment (5 or 10 µg/ml)

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upregulated TGF-β1 protein expression (80.9% and 181.4%, respectively) as

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compared with that in the UVB group. The PRB group (20 µmol/l) showed the highest

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level of TGF-β1, followed by the PR and EP groups (20 µmol/l) in that order (P < 0.05).

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In HDFs, the trend of TGF-β1 expression was mirrored by that of type I procollagen.

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UVB irradiation increased MMP expression (Fig. 4). However, HPE-L (100

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mg/kg body weight) and especially HPE-H (300 mg/kg body weight) treatment

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reduced MMP-1, -3, and -9 protein levels (P < 0.01) relative to those in the UVB group.

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TGF-β1 expression was downregulated in mice following UVB exposure, but this was

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reversed by treatment with 100 or 300 mg/kg body weight HPE, resulting in protein

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levels that were 139.6% and 194.0% of those in the control group, respectively. Thus,

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HPE stimulates type I procollagen production following UVB irradiation. There were

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no differences in MMP and TGF-β1 levels between the negative control and hairless

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control groups (P > 0.05).

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HPE increases antioxidant capacity in UVB-irradiated mice MDA production was increased, whereas the levels of antioxidant enzymes

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(SOD, CAT, and GSH-Px) were decreased in the dorsal skin following UVB irradiation

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(P < 0.01). However, HPE improved UVB-induced oxidative stress (P < 0.01; Table 2),

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an effect that was greater in the HPE-H group (300 mg/kg body weight) than in the

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HPE-L group (100 mg/kg body weight), with MDA production reduced by 23.0% and

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SOD and CAT activities increased by 16.1% and 23.4%, respectively.

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HPE inhibits UVB-induced histological changes

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To investigate the effects of HPE in vivo, hairless mouse dorsal skin was

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exposed to UVB radiation. Weigert staining revealed uniform distribution and

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thickness of skin elastic fibers in the negative and hairless control groups (Fig. 5a).

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UVB radiation increased elastic fiber thickening, disorganization, and damage, with

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hyperplasia observed in some areas. Treatment with low or high doses of HPE

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reversed these histopathological changes. H&E staining showed that the skin

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structure was intact in mice in the negative and hairless control groups; the epidermis

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was covered by thin cuticle with a uniform thickness, and the thickness of the corium

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layer was normal and enriched in sediment (i.e., collagen fibers) (Fig. 5b).

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In normal skin, collagen fiber bundles are wavy, with an orderly arrangement

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and uniform size. We did not observe any infiltration of inflammatory cells in the

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dermis, suggesting that the absence of hair did not alter skin tissue structure. The skin

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of mice in the UVB group showed epidermal thickening (P < 0.01; Fig. 5c). In addition,

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the thickness was non-uniform, and there was evidence of acanthocyte hypertrophy,

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growth of epidermal papillae, and a reduction in dermal papillae. The dermal layer

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was disorganized, elastic fibers showed basophilic degeneration, and the reticule was

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loosely distributed, with abnormal proliferation of cells in the sebaceous glands.

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These results demonstrate that the mouse model of photoaging was successfully

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established. HPE treatment significantly alleviated cuticle thickening compared with

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that in the UVB model group (P < 0.01): epidermal thickness was decreased by 24.0%

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and 46.7% in the HPE-L and -H groups, respectively. Collagen fiber damage was also

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markedly improved, especially in the high-dose group. Epidermal thickness reflects

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UV-induced epidermal hyperplasia, which is considered the cause of skin wrinkles.

336

After UVB irradiation, dorsal skin moisture in the mice decreased significantly (P