Hawthorn polyphenol extract inhibits UVB-induced skin photoaging by

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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

4 5

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

6 7

†

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Technology, Qinhuangdao, Hebei, 066004, China

9

‡

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

11

§

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Hebei, 067000, China

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

13 14 15 16

*Corresponding author:

17

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

18

Science

19

[email protected]

and

Technology,

Qinhuangdao,

Hebei,

1 ACS Paragon Plus Environment

066004,

China.

E-mail:


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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,

51

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

57

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.

99

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

107

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


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

133 134

Cell viability

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

136

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

140

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

141

VT, USA).

bromide

tetrazolium

(MTT;

142 143

Animal experiments

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

145

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.

182 183

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.

192 193

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

203

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

228

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%

236

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

238

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;



240

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

241

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

242 243

Statistical analysis

244

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

245

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

247

< 0.05 was considered statistically significant.

248 249

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.

255 256

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

258

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

259

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

261

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

264

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

265

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

266 267

HPE decreases ROS production after UVB irradiation

268

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

269

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

270

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

271

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

272

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

273

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

274

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

275 276

HPE decreases MMP production in vitro and in vivo

277

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

279

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

280

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

281

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

282

or 0.01; Fig. 3b).

283

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



284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

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

300

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

301

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

302

control groups (P > 0.05).

303 304 305

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

307

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

308

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

309

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

310

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

311 312

HPE inhibits UVB-induced histological changes

313

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

314

exposed to UVB radiation. Weigert staining revealed uniform distribution and

315

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

316

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

317

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

318

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

319

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

320

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

321

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

322

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

323

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

324

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

325

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

326

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

327

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



328

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

329

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

330

These results demonstrate that the mouse model of photoaging was successfully

331

established. HPE treatment significantly alleviated cuticle thickening compared with

332

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

333

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

334

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

335

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

Hawthorn polyphenol extract inhibits UVB-induced skin photoaging by

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