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Protective effect of gelatin peptides from pacific cod skin against photoaging by inhibiting the expression of MMPs via MAPK signaling pathway
Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 34–41
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Protective effect of gelatin peptides from pacific cod skin against photoaging by inhibiting the expression of MMPs via MAPK signaling pathway Tiejun Chen, Hu Hou ⁎, Yan Fan, Shikai Wang, Qianru Chen, Leilei Si, Bafang Li College of Food Science and Engineering, Ocean University of China, No.5, Yu Shan Road, Qingdao, Shandong Province 266003, PR China
a r t i c l e
i n f o
Article history: Received 30 August 2016 Received in revised form 9 October 2016 Accepted 13 October 2016 Available online 14 October 2016 Keywords: Pacific cod Gelatin hydrolysate Matrix metalloproteinases Photoaging MAPK
a b s t r a c t Chronic exposure to ultraviolet (UV) irradiation causes skin photoaging. This study was undertaken to identify the anti-photoaging mechanisms of gelatin hydrolysate (CH) derived from pacific cod skin. Quantitative realtime reverse transcription-polymerase chain reaction (qRT-PCR) and ELISA assays were used to investigate the effects of CH on matrix metalloproteinases (MMPs) and the signaling pathways after UV irradiation by using a mice skin photoaging model. The average molecular weight of CH was 1200 Da, and 273/1000 residues were hydrophobic, Gly-Pro and Gly-Leu sequences and Arg at C-terminus appeared frequently in CH. CH improved pathological changes of collagen fibers and significantly inhibited collagen content reduction in photoaging skin. Moreover, CH blocked the up-regulated expression of interstitial collagenase (MMP-1), stromelysin 1 (MMP3), and gelatinase (MMP-9) in photoaging skin. Besides, CH suppressed the activities of MMPs by increasing the contents of tissue inhibitors of matrix metalloproteinases (TIMPs). CH significantly reduced the UV irradiation-dependent up-regulated phosphorylation of ERK and p38 in the mitogen-activated protein kinase (MAPK) signaling pathway. Furthermore, it inhibited the activation of activator protein 1 (AP-1) by down-regulating the mRNA level of c-Jun and c-Fos, which are the two transcription factors responsible for the regulation of MMPs expression. CH can effectively protect against UV irradiation-induced skin photoaging by inhibiting the expression and the activity of MMPs. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The skin aging process can be divided into intrinsic aging and photoaging. Intrinsic aging is an inevitable process highly correlated with genetic factors [1]. The chronic exposure of skin to ultraviolet (UV) irradiation causes sunburn, inflammation and immune suppression; it also disrupts the skin's normal architecture, which ultimately results in photoaging and even skin cancer [2,3]. Degradation of the skin's dermal collagen matrix by matrix metalloproteinases (MMPs) is the main reason for UV irradiation-induced photoaging [4]. MMPs are a family of structurally related zinc-dependent endopeptidases, collectively capable of degrading the extracellular matrix (ECM), and they can be divided into different subgroups, based on their structure and substrate specificity [5]. UV irradiation up-regulates the expression of interstitial collagenase (MMP-1) that initiates the degradation of type I and type III collagen, gelatinase (MMP-9) that further decomposes collagen fragments, and stromelysin 1 (MMP-3) that degrades type IV collagen and activates pro-MMP-1 [6]. In addition, the degraded collagen fragments produced by MMPs down-regulate new collagen synthesis in vitro and in vivo [7].
⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (H. Hou).
http://dx.doi.org/10.1016/j.jphotobiol.2016.10.015 1011-1344/© 2016 Elsevier B.V. All rights reserved.
The MMPs can be regulated at the transcription level. UV irradiationinduced reactive oxygen species (ROS) mediate the phosphorylation of protein kinases through mitogen-activated protein kinases (MAPK) signaling pathway [8]. The activation of the MAPK signaling pathway directly results in phosphorylation of proteins belonging to the activator protein-1 (AP-1) complex, which up-regulates the expression of MMPs [9]. MMPs activity can also be modulated by endogenous inhibitors. Tissue inhibitors of matrix metalloproteinases (TIMPs) are specific inhibitors of MMPs, and changes in TIMPs levels are considered to be important as they directly affect MMPs activities [10]. Pacific cod (Gadus macrocephalus) is one of the most important commercial food species used worldwide, and its skin is a good source of gelatin. It has been reported that an active peptide derived from pacific cod skin gelatin hydrolysate demonstrates antioxidant activity and angiotensin-I converting enzyme inhibitory ability [11]. Besides, polypeptides from pacific cod skin gelatin hydrolysate play a crucial role in the regulation of melanogenesis in B16 melanoma cells [12], and can alleviate UV irradiation-induced photodamages to the skin [13]. In our previous study, cod skin gelatin hydrolysate (CH) involved inhibiting inflammation and improving transforming growth factor-β/Smad signaling pathway in collagen metabolism of photoaging skin [14]. However, the direct target molecule(s) and protective mechanism(s) of cod skin gelatin hydrolysate in the photoaging process remain unclear.
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Therefore, in this study, we explored the mechanisms responsible for the anti-photoaging effects of CH in terms of matrix collagen degradation, using a mice skin photoaging model. A mixture of polypeptides with different molecular weights, rather than individual constituents, was used to investigate the efficacy of CH against photoaging skin, as some of the CH constituents possibly act synergistically and have better anti-photoaging effects than the individual constituents. 2. Materials and Methods 2.1. Materials Pacific cod skin was obtained from Qingdao Fusheng Food Co., Ltd. (Qingdao, China). Female ICR mice (4 to 6-week-old) were purchased from Lukang Pharmaceutical Group Co., Ltd. (Qingdao, China), permit number: scxk 20130001. All animal procedures involved in this study complied with international ethical principles and the Guide for the Care and Use of Laboratory Animals. Other chemicals used in this experiment were of analytical grade and commercially available. 2.2. Preparation of Pacific Cod Skin Gelatin Hydrolysate Pacific cod skins were pretreated with 0.05 mol/L NaOH at a ratio of 1:6 (w/v) to remove non-collagenous proteins, and were further soaked in 2 mol/L H2SO4 at a ratio of 1:6 (w/v) to obtain the swollen fish skin. Gelatin was then extracted with distilled water for 24 h at 75 °C under constant agitation. To prepare cod skin gelatin hydrolysate, enzymatic hydrolysis was performed using alkaline protease (Nanning Pangbo Biological Engineering Co., Ltd., Nanning, China) at an enzyme-to-substrate ratio of 2:100 (w/w) and trypsin (64008860 Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) at an enzyme-to-substrate ratio of 1:100 (w/w). The initial pH was adjusted to 7.5 and the enzymatic hydrolysis was conducted at 50 °C for 3 h. 2.3. Analysis of CH 2.3.1. Gel Permeation Chromatography of CH The molecular weight (MW) of CH was determined by gel permeation chromatography on a TSK GEL 2000 SWXL column (Tosoh, Tokyo, Japan) by using a high-performance liquid chromatography (HPLC) system (Agilent 1100, Palo Alto, USA). The mobile phase was acetonitrile-water (3:7, w/v) in the presence of 0.1% trifluoroacetic acid. The samples were eluted at a flow rate of 0.5 mL min−1 and monitored at 220 nm at room temperature. Bovine serum albumin (68,000 Da), cytochrome C (15,000 Da), insulin (5500 Da), bacillus (1422 Da), and L-glutathione (307 Da) (Sigma Chemical Co., Ltd. St. Louis, MO. USA) were used as standards. 2.3.2. Amino Acid Composition of CH CH was hydrolyzed with 6 M HCl at 110 °C for 24 h, and the hydrolysate was analyzed on a Hitachi 835-50 amino acid analyzer (Hitachi, Tokyo, Japan). 2.3.3. Mass Spectrometry Identification and Sequence Analysis of CH Fractions The fractions of CH were identified using a Waters Xevo G2 Q-TOF high-resolution mass spectrometer equipped with an electrospray ionization (ESI) source (Waters Corporation, Manchester, UK), operating in positive ionization modes from 50 to 2000 m/z. The mass spectrometer was operated under the following conditions: a capillary temperature of 200 °C, a capillary voltage of 30 V, a source temperature of 100 °C, and a desolvation gas temperature of 450 °C. Data acquisition and analysis were controlled using Waters Mass Lynx v 4.1 software.
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2.4. Animal Treatment and UV Irradiation All animals were allowed free access to food and water throughout the experimental period. Animals were maintained in a specific pathogen-free laboratory under standard conditions of temperature (23 ± 2 °C), humidity (55 ± 10%), and light (12 h light/12 h darkness). After acclimatization for a week, all animals were randomly divided into the following 5 groups (13 mice per group): normal group (NC); model group (MC); positive control group (PC), which was administered vitamin C at a dose of 100 mg kg−1 per day; CH-L group treated with CH at a dose of 100 mg kg−1 per day; and CH-H group treated with CH at a dose of 500 mg kg−1 per day. The NC and MC groups received distilled water at the same volume per day. All animals were administered chemicals or distilled water by oral gavage every morning, and half an hour later, with the exception of the NC group, all mice were irradiated with the same UV source for the same amount of time. UV irradiation-induced mice skin photoaging model was established according to previous study [14]. UVA and UVB tubes were used to irradiate the hairless dorsal regions without any filtering device. The distance from the lamps to the animals' backs was 30 cm. The irradiation intensity was measured using a UVA-radiometer and a UVB-radiometer (Photoelectric Instrument Factory of Beijing Normal University, China). The mice were sacrificed by cervical dislocation after the last UV irradiation, and dorsal skin samples were collected for further analyses. 2.5. Tissue Morphology Staining Dorsal skin samples were fixed in 10% buffered formalin for 24 h and embedded in paraffin. Serial sections (4 μm) were mounted onto glass slides and stained with Masson's trichrome and picrosirius red [15]. The images were recorded using an Olympus DP70 digital camera system (Olympus Optical Co., Ltd., Tokyo, Japan) at 200× magnification. 2.6. Determining Hydroxyproline Levels in the Skin The hydroxyproline (Hyp) level was determined after hydrolysis of the skin tissue in 6 M HCl for 4 h at 130 °C, using the colorimetric method recommended by ISO 3496(E). 2.7. Quantitative Real-Time PCR Analysis Total RNA was prepared from 0.1 g of skin tissue using Trizol reagent (Life Technologies Co., Ltd., Rockville, USA), according to the manufacturer's protocol. The integrity of isolated RNA samples was assessed by electrophoresis in 1% agarose gels, and the concentration and purity of RNA samples were checked with a Nanodrop ND-2000 (Thermo Science, USA). Total RNA (1 μg) was reverse-transcribed to cDNA using M-MLV and random primers (Promega Co., Ltd. Madison, USA). Quantitative real-time PCR was performed using Line-Gene 9600 plus Detection System (Bioer Technology, China) in 25 μL mixture containing SYBR Green Master Mix (Promega Co., Ltd. Madison, USA) and specific primer pairs (Sangon Biotech, China). Cycling conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The primer sequences used for amplification were as follows: MMP-1 forward 5′-TGCCTGATGTGGGTGAATAC-3′ and MMP-1 reverse 5′-GCCTTTGGAACTGCTTGTC-3′; MMP-3 forward 5′-GCCATCTCTTCC ATCCAACA-3′ and MMP-3 reverse 5′-GACAGCATCCACCCTTGAGT-3′; MMP-9 forward 5′-TGTCATCCAGTTTGGTGTCG-3′ and MMP-9 reverse 5′-TGCCGTCCT. TATCGTAGTCA-3′; c-Jun forward 5′-AGTCTCAGGAGCGGATCAAG-3′ and c-Jun reverse 5′-TGTCGCAACCAGTCAAGTTC-3′; c-Fos forward 5′CCGACTC CTTCTCCAGCAT-3′ and c-Fos reverse 5′-CCGTTTCTCTTCCTCTT CAGG-3′; β-actin forward 5′-GTCCCTCACCCTCCCAAAG-3′ and β-actin reverse 5′-GCTGCCTCAA CACCTCAACC-3′. Relative amounts of mRNA were calculated by the relative quantification (ΔΔCt) method. The β-
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actin served as housekeeping gene and the mRNA levels of specific genes were normalized to β-actin.
Table 1 Amino acid compositions of CH. Amino acid CH (residues/1000 residues) Amino acid CH (residues/1000 residues)
2.8. ELISA Analysis Dorsal skin samples were dissected after the last UV irradiation and stored at − 80 °C until needed. The quantitative analysis of MMP-1, MMP-3, MMP-9, TIMP-1, TIMP-2, AP-1, p-ERK, p-JNK, and p-p38 MAPK in 10% dorsal skin homogenate supernatant was conducted using ELISA kits (R&D, Minneapolis, USA). All kit regents were first removed from the refrigerator and allowed to reach room temperature (20–25 °C). The assays were performed according to manufacturer's instructions. The standard curve was constructed to determine the protein concentration in an unknown sample. The protein level in each sample was determined by comparing the absorbance of the sample to that of the standard curve, and then normalized against total protein concentrations.
2.9. Statistical Analysis Data were expressed as mean ± standard deviation. The results were analyzed by one-way analysis of variance (ANOVA) using the SPSS software (SPSS, Version 17.0, IBM Inc. USA). Differences were considered statistically significant when P b 0.05. 3. Results 3.1. Characteristics of CH 3.1.1. Molecular Weight Distribution of CH The molecular weight distribution of CH was determined using an HPLC system. There was a linear relationship between the retention time and the logarithm of the molecular weight in the range of 100,000–100 Da (R2 = 0.9994); the formula used for the calculation was as follows: lgMw = − 0.2088 t + 7.2579. As shown in Fig. 1, the main CH peak was at 1200 Da, which accounted for 97.06% of the total area.
3.1.2. Amino Acid Composition of CH The amino acid composition of CH is presented in Table 1. Glycine accounted for nearly one-third of the total amino acids (268/1000 residues), hydrophobic amino acids accounted for 273/1000 residues, and hydroxyproline accounted for 75 /1000 residues, cysteine and tryptophan were not detected.
Fig. 1. Molecular weight distribution profile of CH using a high-performance liquid chromatography system analysis (Column: TSK GEL 2000 SWXL; flow rate: 0.5 mL min−1; elution buffer: 30% acetonitrile in the presence of 0.1% trifluoroacetic acid).
Asp Thr Ser Glu Gly Ala Cys Val Met Ile Total
61 36 55 100 268 109 0 42 7 12 1000
Leu Tyr Phe Lys His Arg Pro Trp Hyp
40 20 33 43 6 63 30 0 75
3.1.3. Identification of Peptide Sequences from CH As shown in Table 2, Gly-Pro and Gly-Leu sequences and Arg at Cterminus appeared frequently in the isolated 23 polypeptides from CH. The number of amino acid residues ranged from 6 to 15, and the molecular weights ranged from 469.24 to 1387.69 Da. In addition, aromatic amino acid residues were found in some polypeptides of CH. 3.2. Masson's Trichrome Staining Masson's trichrome staining is a classic technique used for collagen fiber staining mainly used to distinguish collagen fibers from muscle fibers. Hematoxylin, ponceau, and aniline blue were used as anionic dyes. Hematoxylin stained the nuclear region dark blue and since the molecular weight of ponceau is lower than that of aniline blue, muscle fibers were stained red and collagen fibers were stained blue by Masson's trichrome staining. As shown in Fig. 2, compared with NC group, UV irradiation (MC group) resulted in the fracture and in the loose and uneven distribution of collagen fibers, as well as in the reduction of collagen levels. In the CH-treated groups, collagen levels increased in a dose-dependent manner and the distribution of collagen fibers was both dense and systematic. 3.3. Quantitative Analysis of Hydroxyproline in Photoaging Skin Hyp levels were measured to evaluate the effect of CH on UV irradiation-induced down-regulation of collagen synthesis and up-regulation Table 2 Main peptide sequences of CH. Peptide sequence
Molecular mass (Da)
Glu-Gly-Pro-Ala-Gly-Pro-Ser-Gly-Gln-Asp-Gly-Arg Gly-Pro-Ala-Gly-Pro-Ala Gly-Pro-Ala-Gly-Pro-Ser-Gly-Ile-Arg Gly-Pro-Ala-Gly-Ser-Pro-Gly-Leu-Arg Gly-Pro-Ala-Gly-Ala-Ser-Gly-Pro-Ala-Gly-Pro-Arg Gly-Ala-Pro-Gly-Pro-Gln Gly-Ser-Pro-Gly-Leu-Val-Gly-Pro-Lys Gly-Gln-Pro-Gly-Leu-Pro-Gly-Pro-Arg Gly-Pro-Ala-Gly-Thr-Pro-Gly-Pro-Lys-Gly-Asp-Arg Ala-Pro-Gly-Gly-Pro-Leu-Gly-Asp-Ala-Arg Gly-Leu-Pro-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Pro-Pro-Gly-Glu-Arg Gly-Glu-Thr-Gly-Asp-Ile-Gly-Pro-Met-Gly-Leu-Pro-Gly-Arg Gly-Pro-Ser-Gly-Pro-Ser-Gly-Pro-Ser-Gly-Pro-Ser-Gly-Pro-Lys Gly-Pro-Pro-Gly-Pro-Ala Gly-Phe-Ser-Gly-Leu-Asp-Gly-Ala-Lys Gly-Asp-Thr-Gly-Ala-Pro-Gly-Pro-Lys Gly-Glu-Ala-Gly-Ala-Lys Gly-Phe-Ser-Gly-Leu-Pro-Gly-Pro-Ala-Gly-Glu-Pro-Gly-Lys Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ser-Arg Ala-Asp-Gly-Leu-Ala-Pro-Ala-Arg Gly-Thr-Glu-Gly-Glu-Arg Gly-Pro-Glu-Gly-Gln-Arg Gly-Asp-Arg-Gly-Tyr-Glu-Pro-Arg
1117.53 469.24 811.44 811.44 994.51 526.26 811.47 878.49 1109.58 910.47 1387.69 1356.67 1265.62 495.26 851.43 799.4 532.28 1270.62 842.41 770.43 648.3 643.31 1006.47
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Fig. 2. Masson's trichrome staining (200×).
of collagen degradation in photoaging skin. In this study, vitamin C was used as a positive control because it is an essential component for the production of collagen in the body and a potent antioxidant that can help rejuvenate aged and photodamaged skin [16]. As shown in Fig. 3, UV irradiation caused a marked decrease of Hyp levels in the MC group (P b 0.01, vs. NC group). Moreover, CH clearly increased Hyp levels in a dose-dependent fashion (P b 0.01, vs. MC group), and there was no significant difference in Hyp levels between the CH-H and the NC groups (P N 0.05). Decreased Hyp levels reflect the loss of collagen in UV-irradiated skin. Hyp levels were consistent with the collagen deposition found by Masson's trichrome staining (Fig. 2). 3.4. Effect of CH on the mRNA and Protein Expression of MMPs in Photoaging Skin As shown in Fig. 4, chronic exposure to UV irradiation dramatically up-regulated both the mRNA and protein expressions of MMP-1, 3,
and 9 (P b 0.01, vs. NC group) in photoaging skin. The significant increase in mRNA expressions of MMPs was inhibited by CH in a dose-dependent manner, and there was a significant difference between the CH-H group and the MC group (P b 0.01, P b 0.05, vs. MC group). CH significantly suppressed the up-regulated protein expressions of MMPs in a dose-dependent manner (P b 0.01, vs. MC group).
3.5. Effect of CH on the MMPs Activities and TIMPs Levels in Photoaging Skin The activities of MMP-1, MMP-3, and MMP-9 were evidently increased in UV irradiation-induced photoaging skin in Fig. 5A, B, and C (P b 0.01, P b 0.05, vs. NC group). CH suppressed the increased activities of MMP-1, MMP-3, and MMP-9 in a dose-dependent manner. Besides, the CH-H group showed a significant inhibition of MMP-1 and MMP-3 activities in photoaging skin (P b 0.05, vs. MC group). TIMPs play an important role in inhibiting the activities of MMPs. As shown in Fig. 5D and E, UV irradiation significantly decreased TIMP-1 and TIMP-2 levels (P b 0.05, vs. NC group) and this decrease was inhibited by CH in a dose-dependent manner. Moreover, compared to the MC group, CH-H group could evidently control the reduction of TIMP-1 level (P b 0.05, vs. MC group).
3.6. Effect of CH on the mRNA Expressions of c-Jun and c-Fos and on the Protein Level of the Transcription Factor AP-1
Fig. 3. Effect of CH on hydroxyproline level of photoaging skin. Data are shown as mean ± standard deviation (n = 13). ##P b 0.01, #P b 0.05, compared with NC group, **P b 0.01, *P b 0.05, compared with MC group.
As shown in Fig. 6A and B, UV irradiation caused a significant increase in the mRNA expressions of c-Fos and c-Jun (P b 0.01, P b 0.05, vs. NC group), which resulted in a significant increase of the transcription factor AP-1 expression in photoaging skin (P b 0.01, vs. NC group, Fig. 6C). CH clearly suppressed the up-regulation of AP-1 in a dose-dependent manner (P b 0.01, vs. MC group), which was in line with its inhibitory effect on the mRNA expressions of c-Fos and c-Jun. Therefore, CH down-regulated the expressions of MMPs by decreasing AP-1 level, which has been implicated in the up-regulated expressions of MMPs [17].
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Fig. 4. Effect of CH on the mRNA expression and protein expression of MMPs in photoaging skin. A: Effect of CH on the mRNA expression of MMP-1; B: Effect of CH on the protein expression of MMP-1; C: Effect of CH on the mRNA expression of MMP-3; D: Effect of CH on the protein expression of MMP-3; E: Effect of CH on the mRNA expression of MMP-9; F: Effect of CH on the protein expression of MMP-9. Data are shown as mean ± standard deviation (n = 13). ##P b 0.01, #P b 0.05, compared with NC group; **P b 0.01, *P b 0.05, compared with MC group.
3.7. CH Suppresses AP-1 Expression by Blocking the Phosphorylation of the MAPK Signaling Pathway As shown in Fig. 7A and B, UV irradiation resulted in a significant increase in p-ERK and p-p38 levels in photoaging skin (P b 0.01, vs. NC group). CH evidently suppressed the phosphorylation of ERK and p38 in a dose-dependent manner (P b 0.01, vs. MC group). However, there was no significant difference in p-JNK level between CH treated groups and MC group (P N 0.05, Fig. 7C). 4. Discussion Chronic exposure to UV irradiation is thought to be the major cause of skin damage, which leads to photoaging. Photoaging skin is characterized by wrinkles, laxity and coarseness, which is closely related with reduction in collagen content and disorganization of collagen fibers [18,19]. CH had protective effect against UV irradiation-induced pathological changes of collagen fibers in photoaging skin, as observed by Masson's trichrome staining (Fig. 2). The results of Masson's trichrome staining indicated that CH was able to alleviate UV irradiation-induced photodamages to collagen fibers. UV irradiation-induced photoaging is the result of increased collagen
breakdown and decreased synthesis of new collagen, which results in the overall reduction of collagen levels in photoaged skin [20]. Masson's trichrome staining is generally used to determine the distribution of skin collagen and hydroxyproline content analysis of photoaging skin, reflected the changes of collagen content. As shown in Fig. 3, CH was able to inhibit the decrease in collagen levels in photoaging skin. UV irradiation disrupts the skin collagen matrix via two interdependent pathways: inhibiting procollagen production and stimulating collagen degradation [21]. Exposure to UV irradiation causes the upregulation of MMPs, which is associated with collagen degradation in photoaged skin [22,23]. It has been reported that marine collagen hydrolysate can inhibit collagen degradation by suppressing MMP-1 expression and increasing TIMP-1 expression [24]. In the present study, CH was not only involved in the down-regulated expression of MMPs, but also inhibited the activities of MMPs in photoaging skin (Fig. 4 and Fig. 5A, B, and C). TIMPs are specific inhibitors of MMPs activities and important regulators of ECM turnover, tissue remodeling, and cellular behavior [25]. The levels of TIMP-1 and TIMP-2 in photoaged skin were decreased, and this was inhibited by CH in a dose-dependent manner (Fig. 5D and E). Therefore, CH could down-regulate the activities of MMPs by elevating TIMPs levels in photoaged skin.
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Fig. 5. Effect of CH on the MMPs activities and TIMPs levels in photoaging skin. A: Effect of CH on the activity of MMP-1; B: Effect of CH on the activity of MMP-3; C: Effect of CH on the activity of MMP-9; D: Effect of CH on the level of TIMP-1; E: Effect of CH on the level of TIMP-2. Data are shown as mean ± standard deviation (n = 13). ##P b 0.01, #P b 0.05, compared with NC group; **P b 0.01, *P b 0.05, compared with MC group.
Furthermore, UV irradiation-induced ROS mediate the phosphorylation of protein kinases through the MAPK signaling pathway. The phosphorylation of MAPK signaling pathway directly activates the transcription factor AP-1, which up-regulates the expressions of MMPs [26]. MAPK signaling pathway includes ERK, JNK, and p38 MAPK. The phosphorylation of JNK activates phosphor-c-Jun, which is known to form AP-1 and further induce the expressions of MMPs thus leading to collagen deficiency [27]. In addition, phosphorylated ERK mediates the transcriptional activation of c-Fos and c-Jun, which are known to form AP-1 [28]. We investigated whether the up-regulated expressions of MMPs via the MAPK signaling pathway were influenced by CH. Results demonstrated that CH inhibited the phosphorylation of ERK and p38 in the MAPK signaling pathway (Fig. 7A and B), resulting in a down-regulation of the mRNA expressions of c-Jun and c-Fos in a dose-dependent manner, which triggered AP-1 level reduction in photoaging skin (Fig. 6). These results indicated that CH suppressed the up-regulated expression of MMPs caused by UV irradiation by blocking the MAPK signaling pathway. Bioactive peptides usually contain 3–20 amino acid residues, and their activities are closely related to their molecular weights, as well as their amino acid constituents and sequences [29]. Bioactive peptides with low molecular weight easily cross the intestinal barrier and exert biological effects [30]. The average molecular weight of CH was 1200 Da (Fig. 1), which was similar to that of the antioxidant peptide from hoki (Johnius belengerii) frame protein (1801 Da) [31] and tuna backbone protein (1519 Da) [32]. Besides, it has been demonstrated that gelatin hydrolysate with an abundance of hydrophobic amino
acids possesses antioxidant activity [33]. Furthermore, the amino acid sequences of bioactive peptides are closely related to their biological activities, and Gly-Leu and Gly-Pro sequences play important roles in the radical-scavenging potency of peptides [34]. In this study, we found that the hydrophobic amino acids in CH accounted for 27.3% of total amino acid residues, and Gly-Leu and Gly-Pro sequences were abundant (Table 2). Therefore, the protective effects of CH on photoaging skin may be closely related to its low molecular weight, abundance of hydrophobic amino acids, and specific amino acid sequences. 5. Conclusions CH had protective effects against UV irradiation-induced photodamages to collagen fibers and reduction of collagen content in skin. CH suppressed the activities and expressions of MMPs by elevating TIMPs levels and suppressing the activation of MAPK signaling pathway. These results indicated that CH is involved in the inhibition of collagen degradation in photoaging skin, which may be closely related to its low molecular weight, abundance of hydrophobic amino acids, and specific amino acid sequences. This study confirms that CH may be a potential active ingredient in anti-photoaging skin treatments. More extensive studies are needed for a thorough understanding of the protective effects of CH on skin. Conflict of Interest The authors state no conflict of interest.
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Fig. 6. Effect of CH on the mRNA expressions of c-Jun and c-Fos and on the protein level of transcription factor AP-1 in photoaging skin. A: Effect of CH on the mRNA expression of c-Jun; B: Effect of CH on the mRNA expression of c-Fos; C: Effect of CH on the protein level of AP-1. Data are shown as mean ± standard deviation (n = 13). ##P b 0.01, #P b 0.05, compared with NC group; **P b 0.01, *P b 0.05, compared with MC group.
Acknowledgements This work was supported by “National Natural Science Foundation of China (No. 31401476)”, “Specialized Research Fund for the Doctoral
Program of Higher Education (No. 20130132120024)”, “Regional Demonstration Project of Shandong Marine Economic Innovation and Development” and “the Fundamental Research Funds for the Central Universities (No. 201313002)”.
Fig. 7. Effect of CH on the phosphorylation of MAPK signaling pathway in photoaging skin. A: Effect of CH on the phosphorylation of ERK; B: Effect of CH on the phosphorylation of p38; C: Effect of CH on the phosphorylation of JNK. Data are shown as mean ± standard deviation (n = 13). ##P b 0.01, #P b 0.05, compared with NC group; **P b 0.01, *P b 0.05, compared with MC group.
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Protective effect of gelatin peptides from pacific cod skin against photoaging by inhibiting the expression of MMPs via MAPK signaling pathway Tiejun Chen, Hu Hou ⁎, Yan Fan, Shikai Wang, Qianru Chen, Leilei Si, Bafang Li College of Food Science and Engineering, Ocean University of China, No.5, Yu Shan Road, Qingdao, Shandong Province 266003, PR China
a r t i c l e
i n f o
Article history: Received 30 August 2016 Received in revised form 9 October 2016 Accepted 13 October 2016 Available online 14 October 2016 Keywords: Pacific cod Gelatin hydrolysate Matrix metalloproteinases Photoaging MAPK
a b s t r a c t Chronic exposure to ultraviolet (UV) irradiation causes skin photoaging. This study was undertaken to identify the anti-photoaging mechanisms of gelatin hydrolysate (CH) derived from pacific cod skin. Quantitative realtime reverse transcription-polymerase chain reaction (qRT-PCR) and ELISA assays were used to investigate the effects of CH on matrix metalloproteinases (MMPs) and the signaling pathways after UV irradiation by using a mice skin photoaging model. The average molecular weight of CH was 1200 Da, and 273/1000 residues were hydrophobic, Gly-Pro and Gly-Leu sequences and Arg at C-terminus appeared frequently in CH. CH improved pathological changes of collagen fibers and significantly inhibited collagen content reduction in photoaging skin. Moreover, CH blocked the up-regulated expression of interstitial collagenase (MMP-1), stromelysin 1 (MMP3), and gelatinase (MMP-9) in photoaging skin. Besides, CH suppressed the activities of MMPs by increasing the contents of tissue inhibitors of matrix metalloproteinases (TIMPs). CH significantly reduced the UV irradiation-dependent up-regulated phosphorylation of ERK and p38 in the mitogen-activated protein kinase (MAPK) signaling pathway. Furthermore, it inhibited the activation of activator protein 1 (AP-1) by down-regulating the mRNA level of c-Jun and c-Fos, which are the two transcription factors responsible for the regulation of MMPs expression. CH can effectively protect against UV irradiation-induced skin photoaging by inhibiting the expression and the activity of MMPs. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The skin aging process can be divided into intrinsic aging and photoaging. Intrinsic aging is an inevitable process highly correlated with genetic factors [1]. The chronic exposure of skin to ultraviolet (UV) irradiation causes sunburn, inflammation and immune suppression; it also disrupts the skin's normal architecture, which ultimately results in photoaging and even skin cancer [2,3]. Degradation of the skin's dermal collagen matrix by matrix metalloproteinases (MMPs) is the main reason for UV irradiation-induced photoaging [4]. MMPs are a family of structurally related zinc-dependent endopeptidases, collectively capable of degrading the extracellular matrix (ECM), and they can be divided into different subgroups, based on their structure and substrate specificity [5]. UV irradiation up-regulates the expression of interstitial collagenase (MMP-1) that initiates the degradation of type I and type III collagen, gelatinase (MMP-9) that further decomposes collagen fragments, and stromelysin 1 (MMP-3) that degrades type IV collagen and activates pro-MMP-1 [6]. In addition, the degraded collagen fragments produced by MMPs down-regulate new collagen synthesis in vitro and in vivo [7].
⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (H. Hou).
http://dx.doi.org/10.1016/j.jphotobiol.2016.10.015 1011-1344/© 2016 Elsevier B.V. All rights reserved.
The MMPs can be regulated at the transcription level. UV irradiationinduced reactive oxygen species (ROS) mediate the phosphorylation of protein kinases through mitogen-activated protein kinases (MAPK) signaling pathway [8]. The activation of the MAPK signaling pathway directly results in phosphorylation of proteins belonging to the activator protein-1 (AP-1) complex, which up-regulates the expression of MMPs [9]. MMPs activity can also be modulated by endogenous inhibitors. Tissue inhibitors of matrix metalloproteinases (TIMPs) are specific inhibitors of MMPs, and changes in TIMPs levels are considered to be important as they directly affect MMPs activities [10]. Pacific cod (Gadus macrocephalus) is one of the most important commercial food species used worldwide, and its skin is a good source of gelatin. It has been reported that an active peptide derived from pacific cod skin gelatin hydrolysate demonstrates antioxidant activity and angiotensin-I converting enzyme inhibitory ability [11]. Besides, polypeptides from pacific cod skin gelatin hydrolysate play a crucial role in the regulation of melanogenesis in B16 melanoma cells [12], and can alleviate UV irradiation-induced photodamages to the skin [13]. In our previous study, cod skin gelatin hydrolysate (CH) involved inhibiting inflammation and improving transforming growth factor-β/Smad signaling pathway in collagen metabolism of photoaging skin [14]. However, the direct target molecule(s) and protective mechanism(s) of cod skin gelatin hydrolysate in the photoaging process remain unclear.
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Therefore, in this study, we explored the mechanisms responsible for the anti-photoaging effects of CH in terms of matrix collagen degradation, using a mice skin photoaging model. A mixture of polypeptides with different molecular weights, rather than individual constituents, was used to investigate the efficacy of CH against photoaging skin, as some of the CH constituents possibly act synergistically and have better anti-photoaging effects than the individual constituents. 2. Materials and Methods 2.1. Materials Pacific cod skin was obtained from Qingdao Fusheng Food Co., Ltd. (Qingdao, China). Female ICR mice (4 to 6-week-old) were purchased from Lukang Pharmaceutical Group Co., Ltd. (Qingdao, China), permit number: scxk 20130001. All animal procedures involved in this study complied with international ethical principles and the Guide for the Care and Use of Laboratory Animals. Other chemicals used in this experiment were of analytical grade and commercially available. 2.2. Preparation of Pacific Cod Skin Gelatin Hydrolysate Pacific cod skins were pretreated with 0.05 mol/L NaOH at a ratio of 1:6 (w/v) to remove non-collagenous proteins, and were further soaked in 2 mol/L H2SO4 at a ratio of 1:6 (w/v) to obtain the swollen fish skin. Gelatin was then extracted with distilled water for 24 h at 75 °C under constant agitation. To prepare cod skin gelatin hydrolysate, enzymatic hydrolysis was performed using alkaline protease (Nanning Pangbo Biological Engineering Co., Ltd., Nanning, China) at an enzyme-to-substrate ratio of 2:100 (w/w) and trypsin (64008860 Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) at an enzyme-to-substrate ratio of 1:100 (w/w). The initial pH was adjusted to 7.5 and the enzymatic hydrolysis was conducted at 50 °C for 3 h. 2.3. Analysis of CH 2.3.1. Gel Permeation Chromatography of CH The molecular weight (MW) of CH was determined by gel permeation chromatography on a TSK GEL 2000 SWXL column (Tosoh, Tokyo, Japan) by using a high-performance liquid chromatography (HPLC) system (Agilent 1100, Palo Alto, USA). The mobile phase was acetonitrile-water (3:7, w/v) in the presence of 0.1% trifluoroacetic acid. The samples were eluted at a flow rate of 0.5 mL min−1 and monitored at 220 nm at room temperature. Bovine serum albumin (68,000 Da), cytochrome C (15,000 Da), insulin (5500 Da), bacillus (1422 Da), and L-glutathione (307 Da) (Sigma Chemical Co., Ltd. St. Louis, MO. USA) were used as standards. 2.3.2. Amino Acid Composition of CH CH was hydrolyzed with 6 M HCl at 110 °C for 24 h, and the hydrolysate was analyzed on a Hitachi 835-50 amino acid analyzer (Hitachi, Tokyo, Japan). 2.3.3. Mass Spectrometry Identification and Sequence Analysis of CH Fractions The fractions of CH were identified using a Waters Xevo G2 Q-TOF high-resolution mass spectrometer equipped with an electrospray ionization (ESI) source (Waters Corporation, Manchester, UK), operating in positive ionization modes from 50 to 2000 m/z. The mass spectrometer was operated under the following conditions: a capillary temperature of 200 °C, a capillary voltage of 30 V, a source temperature of 100 °C, and a desolvation gas temperature of 450 °C. Data acquisition and analysis were controlled using Waters Mass Lynx v 4.1 software.
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2.4. Animal Treatment and UV Irradiation All animals were allowed free access to food and water throughout the experimental period. Animals were maintained in a specific pathogen-free laboratory under standard conditions of temperature (23 ± 2 °C), humidity (55 ± 10%), and light (12 h light/12 h darkness). After acclimatization for a week, all animals were randomly divided into the following 5 groups (13 mice per group): normal group (NC); model group (MC); positive control group (PC), which was administered vitamin C at a dose of 100 mg kg−1 per day; CH-L group treated with CH at a dose of 100 mg kg−1 per day; and CH-H group treated with CH at a dose of 500 mg kg−1 per day. The NC and MC groups received distilled water at the same volume per day. All animals were administered chemicals or distilled water by oral gavage every morning, and half an hour later, with the exception of the NC group, all mice were irradiated with the same UV source for the same amount of time. UV irradiation-induced mice skin photoaging model was established according to previous study [14]. UVA and UVB tubes were used to irradiate the hairless dorsal regions without any filtering device. The distance from the lamps to the animals' backs was 30 cm. The irradiation intensity was measured using a UVA-radiometer and a UVB-radiometer (Photoelectric Instrument Factory of Beijing Normal University, China). The mice were sacrificed by cervical dislocation after the last UV irradiation, and dorsal skin samples were collected for further analyses. 2.5. Tissue Morphology Staining Dorsal skin samples were fixed in 10% buffered formalin for 24 h and embedded in paraffin. Serial sections (4 μm) were mounted onto glass slides and stained with Masson's trichrome and picrosirius red [15]. The images were recorded using an Olympus DP70 digital camera system (Olympus Optical Co., Ltd., Tokyo, Japan) at 200× magnification. 2.6. Determining Hydroxyproline Levels in the Skin The hydroxyproline (Hyp) level was determined after hydrolysis of the skin tissue in 6 M HCl for 4 h at 130 °C, using the colorimetric method recommended by ISO 3496(E). 2.7. Quantitative Real-Time PCR Analysis Total RNA was prepared from 0.1 g of skin tissue using Trizol reagent (Life Technologies Co., Ltd., Rockville, USA), according to the manufacturer's protocol. The integrity of isolated RNA samples was assessed by electrophoresis in 1% agarose gels, and the concentration and purity of RNA samples were checked with a Nanodrop ND-2000 (Thermo Science, USA). Total RNA (1 μg) was reverse-transcribed to cDNA using M-MLV and random primers (Promega Co., Ltd. Madison, USA). Quantitative real-time PCR was performed using Line-Gene 9600 plus Detection System (Bioer Technology, China) in 25 μL mixture containing SYBR Green Master Mix (Promega Co., Ltd. Madison, USA) and specific primer pairs (Sangon Biotech, China). Cycling conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The primer sequences used for amplification were as follows: MMP-1 forward 5′-TGCCTGATGTGGGTGAATAC-3′ and MMP-1 reverse 5′-GCCTTTGGAACTGCTTGTC-3′; MMP-3 forward 5′-GCCATCTCTTCC ATCCAACA-3′ and MMP-3 reverse 5′-GACAGCATCCACCCTTGAGT-3′; MMP-9 forward 5′-TGTCATCCAGTTTGGTGTCG-3′ and MMP-9 reverse 5′-TGCCGTCCT. TATCGTAGTCA-3′; c-Jun forward 5′-AGTCTCAGGAGCGGATCAAG-3′ and c-Jun reverse 5′-TGTCGCAACCAGTCAAGTTC-3′; c-Fos forward 5′CCGACTC CTTCTCCAGCAT-3′ and c-Fos reverse 5′-CCGTTTCTCTTCCTCTT CAGG-3′; β-actin forward 5′-GTCCCTCACCCTCCCAAAG-3′ and β-actin reverse 5′-GCTGCCTCAA CACCTCAACC-3′. Relative amounts of mRNA were calculated by the relative quantification (ΔΔCt) method. The β-
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actin served as housekeeping gene and the mRNA levels of specific genes were normalized to β-actin.
Table 1 Amino acid compositions of CH. Amino acid CH (residues/1000 residues) Amino acid CH (residues/1000 residues)
2.8. ELISA Analysis Dorsal skin samples were dissected after the last UV irradiation and stored at − 80 °C until needed. The quantitative analysis of MMP-1, MMP-3, MMP-9, TIMP-1, TIMP-2, AP-1, p-ERK, p-JNK, and p-p38 MAPK in 10% dorsal skin homogenate supernatant was conducted using ELISA kits (R&D, Minneapolis, USA). All kit regents were first removed from the refrigerator and allowed to reach room temperature (20–25 °C). The assays were performed according to manufacturer's instructions. The standard curve was constructed to determine the protein concentration in an unknown sample. The protein level in each sample was determined by comparing the absorbance of the sample to that of the standard curve, and then normalized against total protein concentrations.
2.9. Statistical Analysis Data were expressed as mean ± standard deviation. The results were analyzed by one-way analysis of variance (ANOVA) using the SPSS software (SPSS, Version 17.0, IBM Inc. USA). Differences were considered statistically significant when P b 0.05. 3. Results 3.1. Characteristics of CH 3.1.1. Molecular Weight Distribution of CH The molecular weight distribution of CH was determined using an HPLC system. There was a linear relationship between the retention time and the logarithm of the molecular weight in the range of 100,000–100 Da (R2 = 0.9994); the formula used for the calculation was as follows: lgMw = − 0.2088 t + 7.2579. As shown in Fig. 1, the main CH peak was at 1200 Da, which accounted for 97.06% of the total area.
3.1.2. Amino Acid Composition of CH The amino acid composition of CH is presented in Table 1. Glycine accounted for nearly one-third of the total amino acids (268/1000 residues), hydrophobic amino acids accounted for 273/1000 residues, and hydroxyproline accounted for 75 /1000 residues, cysteine and tryptophan were not detected.
Fig. 1. Molecular weight distribution profile of CH using a high-performance liquid chromatography system analysis (Column: TSK GEL 2000 SWXL; flow rate: 0.5 mL min−1; elution buffer: 30% acetonitrile in the presence of 0.1% trifluoroacetic acid).
Asp Thr Ser Glu Gly Ala Cys Val Met Ile Total
61 36 55 100 268 109 0 42 7 12 1000
Leu Tyr Phe Lys His Arg Pro Trp Hyp
40 20 33 43 6 63 30 0 75
3.1.3. Identification of Peptide Sequences from CH As shown in Table 2, Gly-Pro and Gly-Leu sequences and Arg at Cterminus appeared frequently in the isolated 23 polypeptides from CH. The number of amino acid residues ranged from 6 to 15, and the molecular weights ranged from 469.24 to 1387.69 Da. In addition, aromatic amino acid residues were found in some polypeptides of CH. 3.2. Masson's Trichrome Staining Masson's trichrome staining is a classic technique used for collagen fiber staining mainly used to distinguish collagen fibers from muscle fibers. Hematoxylin, ponceau, and aniline blue were used as anionic dyes. Hematoxylin stained the nuclear region dark blue and since the molecular weight of ponceau is lower than that of aniline blue, muscle fibers were stained red and collagen fibers were stained blue by Masson's trichrome staining. As shown in Fig. 2, compared with NC group, UV irradiation (MC group) resulted in the fracture and in the loose and uneven distribution of collagen fibers, as well as in the reduction of collagen levels. In the CH-treated groups, collagen levels increased in a dose-dependent manner and the distribution of collagen fibers was both dense and systematic. 3.3. Quantitative Analysis of Hydroxyproline in Photoaging Skin Hyp levels were measured to evaluate the effect of CH on UV irradiation-induced down-regulation of collagen synthesis and up-regulation Table 2 Main peptide sequences of CH. Peptide sequence
Molecular mass (Da)
Glu-Gly-Pro-Ala-Gly-Pro-Ser-Gly-Gln-Asp-Gly-Arg Gly-Pro-Ala-Gly-Pro-Ala Gly-Pro-Ala-Gly-Pro-Ser-Gly-Ile-Arg Gly-Pro-Ala-Gly-Ser-Pro-Gly-Leu-Arg Gly-Pro-Ala-Gly-Ala-Ser-Gly-Pro-Ala-Gly-Pro-Arg Gly-Ala-Pro-Gly-Pro-Gln Gly-Ser-Pro-Gly-Leu-Val-Gly-Pro-Lys Gly-Gln-Pro-Gly-Leu-Pro-Gly-Pro-Arg Gly-Pro-Ala-Gly-Thr-Pro-Gly-Pro-Lys-Gly-Asp-Arg Ala-Pro-Gly-Gly-Pro-Leu-Gly-Asp-Ala-Arg Gly-Leu-Pro-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Pro-Pro-Gly-Glu-Arg Gly-Glu-Thr-Gly-Asp-Ile-Gly-Pro-Met-Gly-Leu-Pro-Gly-Arg Gly-Pro-Ser-Gly-Pro-Ser-Gly-Pro-Ser-Gly-Pro-Ser-Gly-Pro-Lys Gly-Pro-Pro-Gly-Pro-Ala Gly-Phe-Ser-Gly-Leu-Asp-Gly-Ala-Lys Gly-Asp-Thr-Gly-Ala-Pro-Gly-Pro-Lys Gly-Glu-Ala-Gly-Ala-Lys Gly-Phe-Ser-Gly-Leu-Pro-Gly-Pro-Ala-Gly-Glu-Pro-Gly-Lys Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ser-Arg Ala-Asp-Gly-Leu-Ala-Pro-Ala-Arg Gly-Thr-Glu-Gly-Glu-Arg Gly-Pro-Glu-Gly-Gln-Arg Gly-Asp-Arg-Gly-Tyr-Glu-Pro-Arg
1117.53 469.24 811.44 811.44 994.51 526.26 811.47 878.49 1109.58 910.47 1387.69 1356.67 1265.62 495.26 851.43 799.4 532.28 1270.62 842.41 770.43 648.3 643.31 1006.47
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Fig. 2. Masson's trichrome staining (200×).
of collagen degradation in photoaging skin. In this study, vitamin C was used as a positive control because it is an essential component for the production of collagen in the body and a potent antioxidant that can help rejuvenate aged and photodamaged skin [16]. As shown in Fig. 3, UV irradiation caused a marked decrease of Hyp levels in the MC group (P b 0.01, vs. NC group). Moreover, CH clearly increased Hyp levels in a dose-dependent fashion (P b 0.01, vs. MC group), and there was no significant difference in Hyp levels between the CH-H and the NC groups (P N 0.05). Decreased Hyp levels reflect the loss of collagen in UV-irradiated skin. Hyp levels were consistent with the collagen deposition found by Masson's trichrome staining (Fig. 2). 3.4. Effect of CH on the mRNA and Protein Expression of MMPs in Photoaging Skin As shown in Fig. 4, chronic exposure to UV irradiation dramatically up-regulated both the mRNA and protein expressions of MMP-1, 3,
and 9 (P b 0.01, vs. NC group) in photoaging skin. The significant increase in mRNA expressions of MMPs was inhibited by CH in a dose-dependent manner, and there was a significant difference between the CH-H group and the MC group (P b 0.01, P b 0.05, vs. MC group). CH significantly suppressed the up-regulated protein expressions of MMPs in a dose-dependent manner (P b 0.01, vs. MC group).
3.5. Effect of CH on the MMPs Activities and TIMPs Levels in Photoaging Skin The activities of MMP-1, MMP-3, and MMP-9 were evidently increased in UV irradiation-induced photoaging skin in Fig. 5A, B, and C (P b 0.01, P b 0.05, vs. NC group). CH suppressed the increased activities of MMP-1, MMP-3, and MMP-9 in a dose-dependent manner. Besides, the CH-H group showed a significant inhibition of MMP-1 and MMP-3 activities in photoaging skin (P b 0.05, vs. MC group). TIMPs play an important role in inhibiting the activities of MMPs. As shown in Fig. 5D and E, UV irradiation significantly decreased TIMP-1 and TIMP-2 levels (P b 0.05, vs. NC group) and this decrease was inhibited by CH in a dose-dependent manner. Moreover, compared to the MC group, CH-H group could evidently control the reduction of TIMP-1 level (P b 0.05, vs. MC group).
3.6. Effect of CH on the mRNA Expressions of c-Jun and c-Fos and on the Protein Level of the Transcription Factor AP-1
Fig. 3. Effect of CH on hydroxyproline level of photoaging skin. Data are shown as mean ± standard deviation (n = 13). ##P b 0.01, #P b 0.05, compared with NC group, **P b 0.01, *P b 0.05, compared with MC group.
As shown in Fig. 6A and B, UV irradiation caused a significant increase in the mRNA expressions of c-Fos and c-Jun (P b 0.01, P b 0.05, vs. NC group), which resulted in a significant increase of the transcription factor AP-1 expression in photoaging skin (P b 0.01, vs. NC group, Fig. 6C). CH clearly suppressed the up-regulation of AP-1 in a dose-dependent manner (P b 0.01, vs. MC group), which was in line with its inhibitory effect on the mRNA expressions of c-Fos and c-Jun. Therefore, CH down-regulated the expressions of MMPs by decreasing AP-1 level, which has been implicated in the up-regulated expressions of MMPs [17].
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Fig. 4. Effect of CH on the mRNA expression and protein expression of MMPs in photoaging skin. A: Effect of CH on the mRNA expression of MMP-1; B: Effect of CH on the protein expression of MMP-1; C: Effect of CH on the mRNA expression of MMP-3; D: Effect of CH on the protein expression of MMP-3; E: Effect of CH on the mRNA expression of MMP-9; F: Effect of CH on the protein expression of MMP-9. Data are shown as mean ± standard deviation (n = 13). ##P b 0.01, #P b 0.05, compared with NC group; **P b 0.01, *P b 0.05, compared with MC group.
3.7. CH Suppresses AP-1 Expression by Blocking the Phosphorylation of the MAPK Signaling Pathway As shown in Fig. 7A and B, UV irradiation resulted in a significant increase in p-ERK and p-p38 levels in photoaging skin (P b 0.01, vs. NC group). CH evidently suppressed the phosphorylation of ERK and p38 in a dose-dependent manner (P b 0.01, vs. MC group). However, there was no significant difference in p-JNK level between CH treated groups and MC group (P N 0.05, Fig. 7C). 4. Discussion Chronic exposure to UV irradiation is thought to be the major cause of skin damage, which leads to photoaging. Photoaging skin is characterized by wrinkles, laxity and coarseness, which is closely related with reduction in collagen content and disorganization of collagen fibers [18,19]. CH had protective effect against UV irradiation-induced pathological changes of collagen fibers in photoaging skin, as observed by Masson's trichrome staining (Fig. 2). The results of Masson's trichrome staining indicated that CH was able to alleviate UV irradiation-induced photodamages to collagen fibers. UV irradiation-induced photoaging is the result of increased collagen
breakdown and decreased synthesis of new collagen, which results in the overall reduction of collagen levels in photoaged skin [20]. Masson's trichrome staining is generally used to determine the distribution of skin collagen and hydroxyproline content analysis of photoaging skin, reflected the changes of collagen content. As shown in Fig. 3, CH was able to inhibit the decrease in collagen levels in photoaging skin. UV irradiation disrupts the skin collagen matrix via two interdependent pathways: inhibiting procollagen production and stimulating collagen degradation [21]. Exposure to UV irradiation causes the upregulation of MMPs, which is associated with collagen degradation in photoaged skin [22,23]. It has been reported that marine collagen hydrolysate can inhibit collagen degradation by suppressing MMP-1 expression and increasing TIMP-1 expression [24]. In the present study, CH was not only involved in the down-regulated expression of MMPs, but also inhibited the activities of MMPs in photoaging skin (Fig. 4 and Fig. 5A, B, and C). TIMPs are specific inhibitors of MMPs activities and important regulators of ECM turnover, tissue remodeling, and cellular behavior [25]. The levels of TIMP-1 and TIMP-2 in photoaged skin were decreased, and this was inhibited by CH in a dose-dependent manner (Fig. 5D and E). Therefore, CH could down-regulate the activities of MMPs by elevating TIMPs levels in photoaged skin.
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Fig. 5. Effect of CH on the MMPs activities and TIMPs levels in photoaging skin. A: Effect of CH on the activity of MMP-1; B: Effect of CH on the activity of MMP-3; C: Effect of CH on the activity of MMP-9; D: Effect of CH on the level of TIMP-1; E: Effect of CH on the level of TIMP-2. Data are shown as mean ± standard deviation (n = 13). ##P b 0.01, #P b 0.05, compared with NC group; **P b 0.01, *P b 0.05, compared with MC group.
Furthermore, UV irradiation-induced ROS mediate the phosphorylation of protein kinases through the MAPK signaling pathway. The phosphorylation of MAPK signaling pathway directly activates the transcription factor AP-1, which up-regulates the expressions of MMPs [26]. MAPK signaling pathway includes ERK, JNK, and p38 MAPK. The phosphorylation of JNK activates phosphor-c-Jun, which is known to form AP-1 and further induce the expressions of MMPs thus leading to collagen deficiency [27]. In addition, phosphorylated ERK mediates the transcriptional activation of c-Fos and c-Jun, which are known to form AP-1 [28]. We investigated whether the up-regulated expressions of MMPs via the MAPK signaling pathway were influenced by CH. Results demonstrated that CH inhibited the phosphorylation of ERK and p38 in the MAPK signaling pathway (Fig. 7A and B), resulting in a down-regulation of the mRNA expressions of c-Jun and c-Fos in a dose-dependent manner, which triggered AP-1 level reduction in photoaging skin (Fig. 6). These results indicated that CH suppressed the up-regulated expression of MMPs caused by UV irradiation by blocking the MAPK signaling pathway. Bioactive peptides usually contain 3–20 amino acid residues, and their activities are closely related to their molecular weights, as well as their amino acid constituents and sequences [29]. Bioactive peptides with low molecular weight easily cross the intestinal barrier and exert biological effects [30]. The average molecular weight of CH was 1200 Da (Fig. 1), which was similar to that of the antioxidant peptide from hoki (Johnius belengerii) frame protein (1801 Da) [31] and tuna backbone protein (1519 Da) [32]. Besides, it has been demonstrated that gelatin hydrolysate with an abundance of hydrophobic amino
acids possesses antioxidant activity [33]. Furthermore, the amino acid sequences of bioactive peptides are closely related to their biological activities, and Gly-Leu and Gly-Pro sequences play important roles in the radical-scavenging potency of peptides [34]. In this study, we found that the hydrophobic amino acids in CH accounted for 27.3% of total amino acid residues, and Gly-Leu and Gly-Pro sequences were abundant (Table 2). Therefore, the protective effects of CH on photoaging skin may be closely related to its low molecular weight, abundance of hydrophobic amino acids, and specific amino acid sequences. 5. Conclusions CH had protective effects against UV irradiation-induced photodamages to collagen fibers and reduction of collagen content in skin. CH suppressed the activities and expressions of MMPs by elevating TIMPs levels and suppressing the activation of MAPK signaling pathway. These results indicated that CH is involved in the inhibition of collagen degradation in photoaging skin, which may be closely related to its low molecular weight, abundance of hydrophobic amino acids, and specific amino acid sequences. This study confirms that CH may be a potential active ingredient in anti-photoaging skin treatments. More extensive studies are needed for a thorough understanding of the protective effects of CH on skin. Conflict of Interest The authors state no conflict of interest.
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Fig. 6. Effect of CH on the mRNA expressions of c-Jun and c-Fos and on the protein level of transcription factor AP-1 in photoaging skin. A: Effect of CH on the mRNA expression of c-Jun; B: Effect of CH on the mRNA expression of c-Fos; C: Effect of CH on the protein level of AP-1. Data are shown as mean ± standard deviation (n = 13). ##P b 0.01, #P b 0.05, compared with NC group; **P b 0.01, *P b 0.05, compared with MC group.
Acknowledgements This work was supported by “National Natural Science Foundation of China (No. 31401476)”, “Specialized Research Fund for the Doctoral
Program of Higher Education (No. 20130132120024)”, “Regional Demonstration Project of Shandong Marine Economic Innovation and Development” and “the Fundamental Research Funds for the Central Universities (No. 201313002)”.
Fig. 7. Effect of CH on the phosphorylation of MAPK signaling pathway in photoaging skin. A: Effect of CH on the phosphorylation of ERK; B: Effect of CH on the phosphorylation of p38; C: Effect of CH on the phosphorylation of JNK. Data are shown as mean ± standard deviation (n = 13). ##P b 0.01, #P b 0.05, compared with NC group; **P b 0.01, *P b 0.05, compared with MC group.
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