Purification and Characterization of Peptides Inhibiting MMP-1 Activity

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Purification and characterization of peptides inhibiting MMP-1 activity with C-terminate of Gly-Leu from simulated gastrointestinal digestion hydrolysates of tilapia (Oreochromis niloticus) skin gelatin Sun Liping, Liu Qiuming, Fan Jian, Li Xiao, and Zhuang Yongliang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04196 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 28, 2017

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Journal of Agricultural and Food Chemistry

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Purification and characterization of peptides inhibiting MMP-1 activity with

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C-terminate of Gly-Leu from simulated gastrointestinal digestion hydrolysates of

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tilapia (Oreochromis niloticus) skin gelatin

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Sun Liping, Liu Qiuming, Fan Jian, Li Xiao, Zhuang Yongliang *

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Yunnan Institute of Food Safety, Kunming University of Science and Technology, No. 727 South

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Jingming Road, Kunming, Yunnan 650500, China

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*To whom correspondence should be addressed. Tel/Fax: +86 871 65920216

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E-mail address: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Tilapia skin gelatin hydrolysates (TSGH) were prepared by simulated

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gastrointestinal digestion and separated by gel filtration and semipreparative reverse

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phase-high performance liquid chromatography. The antiphotoaging effects were

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evaluated using an ultraviolet radiation B (UVB)-induced mouse embryonic

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fibroblasts (MEFs) photoaging model in vitro. Three fractions from TSGH with high

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inhibitory intercellular matrix metalloproteinases-1 (MMP-1) activities and reactive

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oxygen species (ROS) production were obtained. Three key peptides, GYTGL,

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LGATGL, and VLGL, were identified, and their C-terminate was Gly-Leu. Three

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peptides were synthesized, and they exhibited a significant inhibition of intercellular

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MMP-1 activity and ROS production. Furthermore, three peptides inhibiting MMP-1

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activities were evaluated through their docking of S1’ and S3’ active pockets of

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MMP-1. Hydrogen bonds and C-terminate Gly-Leu played important roles. Finally,

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the protective effects of three peptides on intercellular collagen in UVB-induced

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MEFs were compared. Our results indicated that tilapia gelatin peptides exhibited

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potential activities to prevent and regulate photoaging.

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Keywords: UV radiation B; oxidant stress; matrix metalloproteinases; molecular

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docking; collagen type I

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Introduction Photoaging occurs as a consequence of exposure to large amount of UV radiation.

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radiation can penetrate the epidermis and reach the upper dermis. UVB-exposed

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epidermal keratinocytes should be the initiator of a molecular crosstalk with dermal

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fibroblasts through diffusible signaling molecules.2 Reactive oxygen species (ROS)

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are normal products of metabolism, but they are significantly triggered with UVB

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irradiation. Excessive ROS causes oxidative damage, which is a major factor of

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photoaging.3 ROS can increase the matrix metalloproteinases-1 (MMP-1) expression

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in cells.4 MMP-1, an interstitial collagenase, degrades the collagen fibers. The

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increased MMP-1 expression can damage the skin’s connective tissue.5 The balance

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of ROS production and MMP-1 levels plays a key role in photoaging process. ROS

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scavengers can inhibit ROS production and UV-induced MMP-1 expression, thereby

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regulating the photoaging damages.

UV radiation B (UVB, 280–320 nm) is the main cause of photodamage; this

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Bioactive peptides commonly contain 2–20 residues. Marine-derived bioactive

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peptides are recently emerging in the nutraceutical field as supplements in health

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functional food.6 Marine-derived peptides exhibit high bioactivities, including

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antioxidant, inhibitory angiotensin I-converting enzyme effect, free radical

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scavenging activity, anti-inflammation, and antimicrobial activity.7 Peptide bioactivity

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of peptides depends on their amino acid composition, sequence, length, and polarity.8

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Some ROS scavenging peptides are prepared and obtained from marine animals, such

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as sea cucumber,9 jumbo squid10 and pacific hake.11 Marine-derived peptides display

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good inhibitory photoaging activities. Zhuang et al.12 proposed that the collagen

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polypeptides extracted from jellyfish and hydrolyzed with alkaline protease and

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pepsin could protect collagen fibers from photoaging. Chen et al.13 pointed out that 3



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Pacific cod skin hydrolysates block the UV-induced up-regulation of MMP

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expression in photoaging skin. Lu et al.14 reported that cod skin gelatin peptide exerts

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MMP-1 inhibitory activity in UVB-induced production of mouse skin fibroblasts.

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Many natural compounds are considered potential sources of MMPs inhibitors. 15

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The free radical scavenging activities are one part of inhibitory mechanisms; the other

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part is docking the active site of MMPs. A structure–activity relationship study

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requires molecular docking simulation to investigate the interaction between the

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MMP-1 and active peptides and subsequently evaluate the effect of peptides on

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MMP-1 activities and photoaging process. Therefore, bioactivity peptides

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identification and the study of their structure–activity relationship would be necessary.

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Tilapia (Oreochromis niloticus) is an important species in freshwater aquaculture.

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Deep-water aquaculture of tilapia is the leading industry of plateau-characteristic

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agricultural aquaculture in China. Most of tilapia is being processed into fillets and

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dices, thereby discharging a large amount of byproducts, such as heads, bones, and

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skins.16 In our previous studies, we determined that tilapia skin gelatin hydrolysates

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(TSGH) exhibit good free radical scavenging activities.17 The antiphotoaging

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activities in vivo of TSGH were also evaluated.16 However, no reports are available

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regarding the purification and identification of photoaging inhibitory peptides from

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TSGH. Considering the antiphotoaging activity in vivo, TSGH was selected as a

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potential source of ROS production and MMP-1 activity inhibitory peptides. Key

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peptides with high MMP-1 inhibiting activity were isolated using gel filtration

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chromatography and reversed-phase HPLC. The amino acid sequence of the MMP-1

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inhibitory peptides was also determined. The structure–activity relationship of key

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peptides was assessed by molecular docking simulation. Furthermore, the key

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peptides were synthesized, and the antiphotoaging activities were assessed. The 4


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identified novel peptides from TSGH could be considered as potential nutraceuticals

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fro development of functional foods.

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Materials and methods

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Materials and regents

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Tilapia skin was obtained from Ocean King Co. (Kunming, Yunnan, China).

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Tilapia skin gelatin (TSG) was prepared according to our previous study.17 Sephadex

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G-25 was purchased from GE Healthcare (Fairfield, CT, USA). Dulbecco’s modified

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eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS),

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and 0.25% trypsin were obtained from Gibco (New York, USA). Peptides identified

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were synthesized by Shanghai Synpeptide Co.; Ltd (Shanghai, China). 3-(4,5

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Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) was bought from

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Sigma-Aldrich (St. Louis, MO, USA). 2′,7′-Dichlorodihydrofluorescein diacetate

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(DCFH-DA) ROS assay kits were purchased from Beyotime Biotechnology Co. Ltd.

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(Shanghai, China). The ELISA kits of Collagen I and MMP-1 were bought from R&D

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(USA). Trifluoroacetic acid (TFA) and acetonitrile (HPLC grade) were obtained from

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Merck (Darmstadt, Germany).

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Preparation of TSG hydrolysates (TSGH)

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Approximately 2 g of TSG was dissolved in 150 mL of distilled water, and the

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pH was adjusted to 2.5 with HCl (6 M). The solution was hydrolyzed with pepsin

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with an enzyme-to-substrate ratio of 1:35 (w/w) and incubated at 37 °C for 1 h with

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shaking. Afterward, the mixture was adjusted to pH 7.5 using NaOH (2 M) and

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subsequently hydrolyzed with pancreatin with an enzyme-to-substrate ratio of 1:25

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(w/w) at 37 °C for 2 h with shaking. The reaction was stopped by heating the mixtures

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at 100 °C for 10 min. The hydrolysates (TSGH) were centrifuged at 5000 rpm for 20

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min and desalted by dialysis. The lyophilized TSGH was stored at −20 °C until 5



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further use.

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Purification of bioactive peptides in TSGH

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The TSGH was dissolved in distilled water. The peptides in TSGH were

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separated by a Sephadex G-25 gel filtration column (Ф 1.6 cm × 80 cm). The column

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was first equilibrated with distilled water, subsequently eluted with distilled water at a

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flow rate of 0.5 mL/min, and monitored at 220 nm. The fraction showing high

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inhibitory MMP-1 activity and ROS production was collected and concentrated. The

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RP-HPLC analysis was used for further separation of the fraction collected on a

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Zorbax semipreparative C18 (Ф 9.4 mm ×250 mm) column (Agilent Technologies,

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USA) with a gradient elution of solvent A (0.1% TFA in distilled water) and solvent

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B (0.1% TFA in acetonitrile). A linear gradient of solvent B (5%–30%, in 30 min)

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was used at a flow rate of 2.0 mL/min. Finally, the fractions showing high inhibitory

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MMP-1 activity and ROS production were collected. The purification procedures

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were repeated until sufficient amount of samples was collected for sequence

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identification and bioactivity assay.

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Identification of key peptides

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The amino acid sequences and molecular weights of purified fractions were

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identified by a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometry. The

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molecular weight and amino acid sequence of the mass date were processed using de

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novo software (Peak studio 7.5, Bioinformatics Solutions Inc., Waterloo, Canada).

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The peptides were automatically selected for fragmentation. Peptide identifications

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were accepted when they could be established at higher than 80% probability.

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Cell culture

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Mouse embryonic fibroblasts (MEFs) were isolated from the dermis of ICR fetal

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mice according to the method of Lu et al.14 and Ren et al.18 These MEFs were cultured 6


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in 5% CO2 at 37 °C in DMEM supplemented with 10% (v/v) FBS, 100 U/mL

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penicillin, and 100 mg/L streptomycin. Generations 5–7 of MEFs were selected for

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further study.

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UVB irradiation

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MEFs were seeded at 2×104 cells/well in a plate that contained DMEM with 10%

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(v/v) FBS and incubated in 5% CO2 at 37 °C for 24 h. Subsequently, the cells were

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incubated in a culture medium that contained the samples for an additional 24 h. The

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cells were then placed in a thin layer of PBS and exposed to UVB using two lamps

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(Beijing Zhongyi Boteng Technology Co., Ltd.). These lamps emitted UVB peak at

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313 nm and delivered uniform irradiation at a distance of 30 cm. The cells were

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irradiated with a dose of 30 mJ/cm2. After exposure to UVB radiation, PBS was

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replaced with the same culture medium containing samples and incubated at 37 °C for

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12 h. Afterward, cells and culture supernatants were collected for further study. The

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normal control (NC) group was incubated in culture medium at the same conditions

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without UVB radiation and samples. The model control (MC) group was treated with

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UVB radiation without samples.

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Cell viability assay

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Cell viability was evaluated with the MTT assay.19 The MEFs were cultured on

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96-well plates and incubated with TGSH. This process was treated with or without

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UVB radiation. After incubation in culture medium with TGSH at 37 °C in a

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humidified, 5% CO2 atmosphere for 24 h, the collected cells were treated with 150 µL

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of MTT reagent (0.5 mg/mL) for 4 h at 37 °C. Afterward, the MTT reagent was

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removed. The amount of MTT formazan dissolved in 150 µL of DMSO was measured

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by a microplate reader at 570 nm. The cell viability percentage was determined on the

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basis of the comparison with the formazan level of the NC group. 7



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Intracellular ROS production assay

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Intracellular ROS production was determined by a fluorescence assay using a

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DCFH-DA ROS assay kit.19 MEFs were seeded at 2×104 cells/well in 12-well plates.

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The UVB radiation was same as that in Section “UVB irradiation”. The collected cells

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were resuspended in freshly prepared, serum-free medium that contained DCFH-DA

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(10 µM) at 37 °C for 20 min in the dark. Cells were harvested and washed with PBS.

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Intracellular ROS was immediately examined at λex of 485 nm and λem of 535 nm

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using a flow cytometer (Guava easyCyte 6-2L; EMD Millipore, Hayward, CA, USA).

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The result is expressed by intercellular ROS relative level compared with the NC

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group.

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Collagen type I and matrix metalloproteinase (MMP-1) assay

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MEFs were seeded at 2×104 cells/well in six-well plates, and the UVB radiation

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was same as that in Section “UVB irradiation”. The supernatants were collected and

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subjected to ELISA for collagen type I content and MMP-1 activities. ELISA assays

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were performed according to the manufacturer’s protocol for collagen type I and

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MMP-1 assay kits.

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Molecular docking analysis

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The 3D structure of MMP-1 (966c.pdb) was obtained from the Protein Data

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Bank. The ligand structure was constructed by using SYBYL 2.1.1 software (Tripos

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Associates, St. Louis, MO). Hydrogen atoms were added to crystal structures 966c.

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Sample ligands were docked into the active site of MMP-1 by molecular visualization.

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T-score, C-score, hydrogen bond, and distance were calculated by SYBYL software.

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T-scores and C-scores were accepted when their numerical value were higher than 6.0

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and 4, respectively.

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Statistical analysis 8


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Data were expressed as mean ± standard deviation. Data were statistically

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analyzed using SPSS (version 17.0, IBM Inc., USA). Statistical differences were

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considered significant at p

Purification and Characterization of Peptides Inhibiting MMP-1 Activity

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