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Identification of MMP-1 inhibitory peptides from cod skin gelatin hydrolysates and the inhibition mechanism by MAPK signaling pathway
Journal of Functional Foods 33 (2017) 251–260
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Identification of MMP-1 inhibitory peptides from cod skin gelatin hydrolysates and the inhibition mechanism by MAPK signaling pathway Jiaohan Lu, Hu Hou ⇑, Yan Fan, Tingting Yang, 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 17 November 2016 Received in revised form 23 January 2017 Accepted 25 March 2017 Available online 3 April 2017 Keywords: Cod skin gelatin hydrolysates Matrix metalloproteinases Photoaging Fibroblasts Ultraviolet radiation
a b s t r a c t Cod skin gelatin hydrolysates (CGH) could serve as a source of peptides with anti-photoaging activity. To prepare CGH, alkaline protease and trypsin were used for enzymatic hydrolysis. The anti-photoaging effects of CGH were evaluated using a UVB-induced mouse skin fibroblasts photoaging model in vitro. CGH could efficiently inhibit the expression of MMP-1, especially at the concentration of 0.1 mg/mL, in the fibroblasts irradiated with UVB of 20 mJ/cm2. In addition, the MMP-1 inhibitory peptides were purified using ion-exchange chromatography and RP-HPLC. The fractions which exhibited the highest activity, were identified using LC-ESI-MS/MS. Finally, two peptides GEIGPSGGRGKPGKDGDAGPK and GFSGLDGAKGD were found to exhibit a significant inhibition of MMP-1, p-ERK and p-p38 and the first purified peptide significantly inhibited p-JNK in MAPK signaling pathways. Therefore, this study provided a scientific basis for the preparation of MMP-1 inhibitory peptides and bioactive peptides can be used as functional supplements for marine skin protection products. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Photoaging, an extrinsic aging of the skin, occurs as a consequence of exposure to plenty of environmental stresses, such as ultraviolet (UV) radiation (Chung et al., 2002; Gilchrest, 1996). Increase in the UV radiation in earth’s atmosphere because of the breaches in the stratospheric ozone layer has increased the risk of photoaging to skin, which is the feature of wrinkling, irregular pigmentation and poor elasticity (Ichihashi et al., 2003). Ultraviolet radiation B (UVB, 280–320 nm), the main cause of skin photodamage, can penetrate the epidermis and reach the upper dermis, which consist mostly of fibroblasts and extracellular matrix (ECM), and leads to the generation of reactive oxygen species (ROS) and activation of signaling pathways, which further induce the up-regulation of matrix metalloproteinases (MMPs) in human skin (Helenius, Mäkeläinen, & Salminen, 1999; Keyse, 1993; Rittié & Fisher, 2002). The dermal fibroblasts, suffering accumulated damage during UV irradiation, play a key role in photoaging due to their effect on maintaining the metabolic balance of dermal collagenous ECM (Fisher, Varani, & Voorhees, 2008). Reports suggest that collagen degradation by MMPs secreted by various cells (e.g., fibroblasts) largely accounts for UV-induced photoaging
⇑ Corresponding author. E-mail addresses: [email protected], [email protected] (H. Hou). http://dx.doi.org/10.1016/j.jff.2017.03.049 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.
(Chung et al., 2002; Rabe, Mamelak, McElgunn, Morison, & Sauder, 2006). MMPs, a family of zinc-dependent endopeptidases, are related with the degradation of ECM in connective tissues. MMPs can be divided into different subgroups, according to their structure and substrate specificity (Egeblad & Werb, 2002). MMP-1 is known to induce dermal collagen degradation and preferentially degrades type I collagen, the most abundant structural protein in the skin, which is synthesized primarily by dermal fibroblasts and related with resiliency and strength of the skin (Gelse, Pöschl, & Aigner, 2003). Hence, it is not surprising that UVB-induced photodamage is an emerging area of research, with special focus on ways of arresting collagen degradation and collagenase activation (Bae et al., 2008; Katiyar, 2003). The accumulation of ROS and the up-regulation of MMPs expression in fibroblasts caused by UV irradiation reflect the central influences of photoaging process (Kohl, Steinbauer, Landthaler, & Szeimies, 2011). ROS causes oxidative damage to major cellular components, and mediates the phosphorylation of protein kinases through mitogen-activated protein kinases (MAPK) pathway (Bickers & Athar, 2006; Sharma, Meeran, & Katiyar, 2007; Yang, Sharrocks, & Whitmarsh, 2003), which includes ERK, p38 MAPK and JNK. The phosphorylation of ERK mediates the activation of c-Fos and c-Jun, which are known to form AP-1 and further induce the expressions of MMPs thus leading to collagen degradation (Mehta & Griendling, 2007).
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Moreover, phosphorylated JNK activates phosphor-c-Jun (Kang et al., 2003). In recent years, most researches payed attention to peptides derived from plant sources and marine animal sources, which have been found to possess good antioxidant activity, such as sea urchin gonad (Qin et al., 2011), sea cucumber (Zhou, Wang, & Jiang, 2012), and pacific hake (Cheung, Cheung, Tan, & Li-Chan, 2012). Collagen polypeptides from Apostichopus japonicus (Wang et al., 2008) and collagen peptides from Chlamys farreri (Yu, Li, Liu, & Wang, 2004) showed protective effects on UV-induced skin photoaging. Hou et al. (2009) proposed that gelatin polypeptides extracted from Pacific cod (Gadus macrocephalus) skin and hydrolyzed with alkaline protease and pepsin could protect collagen fibers in skin, which indicated that Pacific cod skin hydrolysates had the peptides of anti-photoaging activity. Chen, Hou, Lu, Zhang, and Li (2016) pointed out that Pacific cod skin hydrolysates blocked the UV-induced up-regulation of MMPs expression in photoaging skin. These researches show that cod skin hydrolysates could protect against UV-induced damage in skin. But, the mechanisms of the anti-photoaging activities of peptides are not fully understood. Several researches have indicated biological activities of peptides are related to their composition, structure and hydrophobicity (Chen, Muramoto, Yamauchi, Fujimoto, & Nokihara, 1998). It was reported that peptides containing Glu, Lys, Thr, Asp, Tyr, Leu, Ala and Pro exhibited a strong antioxidant capacity (Liu et al., 2013). The MMP-1 inhibitory activity of cod skin gelatin hydrolysates prepared with alkaline protease and trypsin has not been investigated systematically. There have been no reports on the purification and identification of MMP-1 inhibitory peptides from CGH. Therefore, in this study, CGH was chosen as a potential source of MMP-1 inhibitory peptides, and peptides with high MMP-1 inhibiting activity were isolated using ion-exchange chromatography and reversed-phase HPLC (RP-HPLC). The amino acid sequence of the MMP-1 inhibitory peptides was determined using Q-TOF ESI mass spectroscopy. Besides, we explored the MAPK signaling pathway responsible for the expressions of MMP-1 in terms of matrix collagen degradation, using a UVB-induced mouse skin fibroblasts photoaging model in vitro. The results of this study could be important in further research into the mechanism of matrix collagen degradation and for the development of functional foods and marine skin protection products. 2. Materials and methods 2.1. Materials Fresh Pacific cod (Gadus Macrocephalus) skins were obtained from Qingdao Fusheng Foods Co. Ltd. (Qingdao, China) and stored at 20 °C until used. Alkaline protease (CID: 455168) and trypsin (CID: 16130295) were bought from Pangbo Biological Engineering Co., Ltd. (Nanning, China). Dulbecco’s modified Eagle’s medium (DMEM), Fetal bovine serum (FBS), Dulbecco’s Phosphate Buffered Saline (DPBS), and 0.25% Trypsin-EDTA were purchased from Gibcob (New York, USA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) were bought from Sigma Chemical Co. (USA). ICR fetal mice (1–3 g) were obtained from Lukang Pharmaceutical Group Co., Ltd. (Qingdao, China). SP Sephadex C-25 was provided by GE Healthcare (Uppsala, Sweden). 2.2. Preparation of CGH Frozen cod skins were thawed under running tap water and the residual adherent tissues and scales were removed manually. The cleaned fish skins were cut into pieces (8 cm 3 cm), and
soaked in 0.01 M H2SO4 for 30 min at a solid to acid ratio of 1:6 (w/v). The skins were then washed with distilled water until pH 5.0 was reached. Next, the swollen skins were extracted with distilled water at a solid to solution ratio of 1:2 (w/v) for 2 h at 75 °C with stirring. The gelatin solution was filtered through 0.45 lm filter membrane and lyophilized in a vacuum freeze dryer. Alkaline protease and trypsin were used to prepare CGH. The pH of the gelatin solution was adjusted to 7.5 using 1 M NaOH and the gelatin was hydrolyzed by alkaline protease and trypsin at 50 °C with stirring. The reaction was stopped by heating the mixtures at 100 °C for 10 min. The hydrolysates were filtered through 0.45 lm filter membrane and desalted by dialysis. The freezedried CGH was stored at 20 °C until further used. 2.3. Fibroblasts preparation Fibroblasts were isolated from dermis of ICR fetal mice. Briefly, mouse was killed by cervical dislocation and soaked in 75% ethyl alcohol for 1 min. The back skin was removed under aseptic conditions and treated with 0.25% trypsin-EDTA for 12–14 h. DMEM containing FBS (10%, v/v), streptomycin (100 g/L) and penicillin (100 kU/L) was used to stop the reaction. Dermal skin was separated from the epidermis and further trypsinized for 60 min with stirring. After terminating the reaction, dermis was washed twice with DMEM containing FBS (10%, v/v), and centrifuged at 342g for 5 min to remove the suspension. The cells were subsequently dissociated through a 200 mesh sterile metal sieve, and then seeded into 25-cm2 cell culture flasks in DMEM supplemented with FBS (10%, v/ v), streptomycin (100 g/L) and penicillin (100 kU/L) and incubated at 37 °C in a humidified, 5% CO2 atmosphere. 2.4. UVB irradiation Fibroblasts (passages 6–8) were seeded at 2 104 cells/well in 24-well plates containing DMEM with 10% (v/v) FBS and incubated at 37 °C in a humidified, 5% CO2 atmosphere for 24 h. Subsequently, the cells were incubated in culture medium containing samples for an additional 24 h. The cells were then placed in a thin layer of DPBS and exposed to UVB, using two lamps (Beijing Zhongyiboteng-tech Co., Ltd.) that emitted UVB peaking at 313 nm and delivered uniform irradiation at a distance of 30 cm. The energy output was measured by a UVB radiometer with a UVB sensor (Photoeletric Instrument Factory of Beijing Normal University, China). The output of the lamps was 33.5 ± 0.9 lw/cm2. After exposure to UVB, DPBS was replaced by the same culture medium containing samples and incubated at 37 °C for 12 h. Then, cells and culture supernatants were collected and used for ELISA analysis. The normal control (NC) group was incubated in culture medium at the same conditions without UVB and samples prior to analysis and the model control (MC) group treated without samples was exposed to UVB at the same dose.
2.5. Cell viability assay Cell viability was measured by the MTT assay. Fibroblasts (passages 6–8) cultured on 96-well plates and treated with CGH, were exposed to the UVB source. After incubating in culture medium with CGH at 37 °C in a humidified, 5% CO2 atmosphere for 24 h, cells were washed with DPBS and kept in DMEM with MTT (0.5 mg/mL) for 4 h. The MTT solution was then replaced with dimethyl sulfoxide and the absorbance at 490 nm was determined by a microplate reader.
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2.6. Purification of MMP-1 inhibitory peptides 2.6.1. SP Sephadex C25 cation exchange column chromatography The CGH solution was applied to a SP Sephadex C-25 (2.6 cm 50 cm) cation exchange column pre-equilibrated with 0.02 M acetic acid buffer solution (pH 4.0) and eluted with a linear gradient of NaCl (0–1.0 M). Every 8.5 mL of the separated fractions was collected using a flow rate of 1.7 mL/min and absorption at 220 nm was monitored to determine the peptide elution profile. The collected samples were desalted by dialysis and their MMP-1 inhibitory activity was determined. 2.6.2. RP-HPLC using SB-C18 Fractions with high MMP-1 inhibitory activity were further purified using a Zorbax SB-C18 semi-preparative column (9.4 mm 250 mm) and eluted using a linear acetonitrile gradient
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(5–30%, v/v) and a flow rate of 2.0 mL/min. The absorbance of the eluate was measured at 220 nm. The fraction with the highest MMP-1 inhibitory activity was collected and further purified under different linear gradient conditions. 2.7. Analysis of amino acid sequence of purified MMP-1 inhibiting peptide The amino acid sequence and accurate molecular mass of the purified peptides were determined by a Waters Xevo G2 Q-TOF high-resolution mass spectrometer coupled with an electrospray ionization (ESI) source (Waters Corporation, Manchester, UK). MaxEnt3 and Peptide Sequencing software were applied to data acquisition and analysis. The purified peptides (95% purity) were synthesized by ChinaPeptides Co. Ltd. (Suzhou, China), and their MMP-1 inhibitory activity was tested.
Fig. 1. Effects on morphology of mouse skin fibroblasts (photographed under 100 magnifications) (A) and the concentration and activity of MMP-1 (B) and viability of mouse skin fibroblasts irradiated by UVB (C). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
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2.8. ELISA analysis
3. Results and discussion
MMP-1 inhibitory activity was evaluated by measuring its effect on MMP-1 concentration and activity in vitro. And the quantitative analysis of MMP-1, p-ERK, p-JNK, and p-p38 MAPK was conducted using ELISA kits (R&D, USA). The assays were operated on the basis of manufacturer’s instructions. Cells were lysed by rapid freezing and thawing, and then used for the test of p-ERK, p-JNK, and pp38. Cell culture supernatants were used for the test of MMP-1.
3.1. Determination of UVB dose that induces photoaging and CGH concentration that inhibits MMP-1 expression
2.9. Statistical analysis All results were expressed as means ± standard deviation and analyzed by SPSS (version 17.0, IBM Inc., USA). All data were submitted to one-way analysis of variance and statistical differences were considered significantly at P < 0.05.
To determine the appropriate dose of UVB and concentration of CGH for the subsequent experiments, the biological effects of UVB and CGH on the expression of MMP-1 and cell viability effect were first examined. Fibroblasts were irradiated with increasing doses of UVB (0–120 mJ/cm2) and treated with increasing concentrations of CGH (0–10 mg/mL), respectively. As shown in Fig. 1b, UVB exposure increased the concentration and activity of MMP-1, which reached the highest level at 20 mJ/cm2 of UVB. A progressive decrease in viability of mouse skin fibroblasts was illustrated in Fig. 1c and the viability of fibroblasts was expressed as a percentage of live cells compared with the 0 mJ/cm2 group, which were considered to be 100% viable. The UVB dose of 20 mJ/cm2 group
Fig. 2. Effects on morphology of mouse skin fibroblasts (photographed under x100 magnifications) (A) and the concentration and activity of MMP-1 (B) and viability of mouse skin fibroblasts treated with CGH (C). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
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was approximately 80% viable and a remarkable decrease in cell viability was at UVB dose of 60 mJ/cm2 (74%). This dose is nearly equivalent to that received by human skin exposed to a clear midday, midsummer sun, for a short period of time (Besaratinia, Kim, & Pfeifer, 2008). Similar results were shown in Fig. 1a that an obvious increase in cell death was observed starting from 60 mJ/cm2 UVB. Compared with normal cells (0 mJ/cm2), some fibroblasts became round and floated in the supernatants under UVB irradiation. The number of floating (dead) cells (Froissard, Monrocq, & Duval, 1997) in the culture supernatant increased starting from UVB dose of 60 mJ/cm2 by microscopic observation (magnification 100), especially at UVB dose of 90 mJ/cm2 and 120 mJ/cm2. Based on these results, a UVB dose of 20 mJ/cm2 was chosen for subsequent experiments. As shown in the 0 mg/mL group of Fig. 2a, a single exposure to a 20 mJ/cm2 dose of UVB resulted in premature senescence of cells. Treatment with 0.1 mg/mL CGH strongly inhibit the expression of
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MMP-1 (Fig. 2b). Additionally, the cell viability was expressed as a percentage of live cells compared with the 0 mg/mL group, which were considered to be 100% viable and a remarkable increase in cell viability was indicated starting from 0.1 mg/mL CGH; at this concentration, cell viability was approximately 151% (Fig. 2c). Similar results were obtained from morphology that an obvious decrease in cell death was observed at 0.1 mg/mL CGH. (Fig. 2a). Floating cells decreased in the supernatants after treating with CGH by microscopic observation (magnification 100), especially at 0.001 mg/ mL, 0.01 mg/mL, and 0.1 mg/mL CGH. Therefore, a CGH concentration of 0.1 mg/mL was chosen for subsequent experiments. 3.2. Purification of MMP-1 inhibitory peptides using ion-exchange chromatography Ion-exchange chromatography separates proteins or peptides based on their differences of charge (Bouhallab, Henry, &
Fig. 3. The elution profile of CGH by the SP Sephadex C-25 (A) and the concentration and activity of MMP-1 in each of the separated fractions (B). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
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Fig. 4. The RP-HPLC profile of C5 by the Zorbax SB-C18 semi-prep column (A) and the concentration and activity of MMP-1 in each of the separated fractions (B) and the RPHPLC profile of P8 and P10 by the Zorbax SB-C18 semi-prep column (C). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
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Boschetti, 1996). As one of the strongest cation exchangers, SP Sephadex C-25 (main functional group: sulfopropyl) is used widely for separating bioactive peptides. In order to isolate the MMP-1 inhibitory peptides, CGH was dissolved in 0.02 M acetic acid buffer solution (pH 4.0) and separated by SP Sephadex C-25 cation exchange column chromatography (Fig. 3). Six components were obtained, named C1, C2, C3, C4, C5 and C6, respectively (Fig. 3a). The C2, C4, C5, and C6 were the main components of CGH. It was reported that the activity of peptide was associated with the content of positive charge (Kong, Guo, Hua, Cao, & Zhang, 2008). The MMP-1 inhibiting activity of each component (10 lg/mL, 50 lg/ mL and 100 lg/mL) was assayed. At the concentration of 50 lg/ mL, the concentration of MMP-1 decreased by 26% in the cells treated with component C5 (38.7 ng/mL), compared with MC (52.3 ng/ mL). MMP-1 activity values were 4.7 U/L, 5.7 U/L, and 3.5 U/L at C5 concentration of 10 lg/mL, 50 lg/mL, and 100 lg/mL, respectively. These values approximately indicated a 0.61-fold down-regulation
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in activity, compared with MC (8.9 U/L) (Fig. 3b). Since C5 showed remarkably stronger MMP-1 inhibitory activity than any of the other components, it was further purified by RP-HPLC. 3.3. Purification of MMP-1 inhibitory peptides using RP-HPLC RP-HPLC can be utilized for separating peptides based on their hydrophobicity (Pownall, Udenigwe, & Aluko, 2010). The RPHPLC profile showed a large number of peaks, indicating the abundance of the peptides generated. Component C5 was separated into 10 fractions (P1-P10) by the Zorbax SB-C18 semi-prep column (Fig. 4a). The eigth (P8) and tenth (P10) peaks showed the highest MMP-1 inhibitory activity at concentrations of 10 lg/mL and 50 lg/mL. MMP-1 concentration values were 60.6 ng/mL and 60.5 ng/mL at P8 and P10 concentration of 10 lg/mL, which showed nearly a 0.22-fold decrease, compared with MC (77.3 ng/ mL). The MMP-1 activity of cells treated with P8 and P10 (10 lg/
Fig. 5. The mass spectrum of M1 (A) and M2 (B).
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mL) was 31.6 U/L and 28.8 U/L. Compared with MC (60.2 U/L), the MMP-1 activity decreased by 47% and 52%, respectively (Fig. 4b). Fraction P8 was further purified on the Zorbax SB-C18 column with the following increasing acetonitrile treatment: 0–5 min, 5–45% acetonitrile; 5–25 min, 45–60% acetonitrile; 25–30 min, 60–80% acetonitrile. P10 purification was performed with 0–5 min, 5–40% acetonitrile; 5–25 min, 40–55% acetonitrile; 25–30 min, 55–80% acetonitrile gradient. Fig. 4c showed a single peak at 9 min (M1) and 10 min (M2) derived from P8 and P10, respectively. Moreover, these fractions were identified by Q-TOF mass spectrometer.
3.4. Determination of the amino acid sequence of MMP-1 inhibitory peptides
(Qian, Jung, Byun, & Kim, 2008). Rajapakse, Mendis, Jung, Je, and Kim (2005) pointed out that presence of Asp seemed to play an important role in antioxidant peptide sequences irrespective of its position. In addition, aliphatic amino acids such as Asn and Glu had great effect on antioxidant activity (Park, Jung, Nam, Shahidi, & Kim, 2001; Zhang et al., 2010). The MMP-1 inhibitory peptide M1 and M2 obtained from CGH contained amino acids, such as Asp, Lys and Gly, and the presence of Glu in M1 and Leu in M2 also showed similar results with previous studies. Jang, Liceaga, and Yoon (2016) reported that Ala-Thr-Ser-His-His (551.25 Da) derived from sandfish protein hydrolysates showed a significant antioxidant activity. Peptides ACGT and ATAGT from Tibpia frame protein (Fan, He, Zhuang, & Sun, 2012), LEELEEELEGCE
Liquid chromatography-mass spectrometry is commonly used to determine peptide sequences. As shown in Fig. 5, the molecular weights of M1 and M2 obtained from CGH were determined to be 1936 Da and 1022 Da, respectively. The amino acid sequences of M1 and M2 were found to be GEIGPSGGRGKPGKDGDAGPK and GFSGLDGAKGD, respectively. Both the presence of amino acids and the sequence of the peptide chain have a great effect on antioxidant power (Rajapakse, Mendis, Byun, & Kim, 2005). The presence of the amino acids Tyr, Trp, Phe, Met, Lys and Cys was reported to be an important contributor to the antioxidant activities of the peptides (Carrasco-Castilla et al., 2012; Huang et al., 2010; Sarmadi & Ismail, 2010). It was reported that peptides with Gly, Glu and Asp exhibited strong antioxidant activity due to they are very important to the radical-scavenging activity of peptides
Fig. 6. Effects of M1 and M2 on the concentration (A) and activity (B) of MMP-1. NC, normal group; MC, model group; PC, positive control group; M1-L, synthesized peptide M1 treatment (12.5 lg/mL); M1-M, synthesized peptide M1 treatment (50 lg/mL); M1-H, synthesized peptide M1 treatment (200 lg/mL); M2-L, synthesized peptide M2 treatment (12.5 lg/mL); M2-M, synthesized peptide M2 treatment (50 lg/mL); M2-H, synthesized peptide M2 treatment (200 lg/mL). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
Fig. 7. Effects of M1 and M2 on the phosphorylation of ERK (A), p38 (B) and JNK (C) of MAPK pathway. NC, normal group; MC, model group; PC, positive control group; M1-L, synthesized peptide M1 treatment (12.5 lg/mL); M1-M, synthesized peptide M1 treatment (50 lg/mL); M1-H, synthesized peptide M1 treatment (200 lg/mL); M2-L, synthesized peptide M2 treatment (12.5 lg/mL); M2-M, synthesized peptide M2 treatment (50 lg/mL); M2-H, synthesized peptide M2 treatment (200 lg/mL). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
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from Bullfrog skin (Qian, Jung, & Kim, 2008), LPHSGY from Alaska Pollock protein hydrolysate (Je, Park, & Kim, 2005), have been reported to possess antioxidant activity. Twenty-three polypeptides containing Ala-Gly-Pro, Gly-Pro, and Gly-Leu repeated sequences, which also presented in M1 and M2, were identified in the hydrolysates of Pacific cod skin gelatin, which was found to improve the morphology of collagen fibers and significantly mitigate the reduction of collagen content in photoaging skin. Although the complete mechanism of MMP-1 inhibition by bioactive peptides remains unclear, it has been confirmed that MMP-1 inhibitory activity is related to the mitogen-activated protein kinase pathway (Chen et al., 2016). Moreover, Liang et al. (2010) revealed the effects of long-term oral administration of collagen hydrolysate on the chronological skin aging in vivo by inhibiting the collagen loss and collagen fragmentation. Koyama (2016) pointed out that di-peptide Pro-Hyp and Hyp-Gly normally were found in the blood after collagen hydrolysate intake. And Pro-Hyp stimulated the growth of fibroblasts (Shigemura et al., 2009). Pro-Hyp presented in vivo after the intake of the cod skin gelatin hydrolysate, which was also reported by Chen et al. (2016). 3.5. Effect of synthesized peptides on the expression of MMP-1 Fibroblasts were divided into the following 9 groups: normal group (NC), model group (MC), positive control group (PC) treated with vitamin C at a dose of 12.5 lg/mL, M1-L group treated with M1 at a dose of 12.5 lg/mL, M1-M group treated with M1 at a dose of 50 lg/mL, M1-H group treated with M1 at a dose of 200 lg/mL, M2-L group treated with M2 at a dose of 12.5 lg/mL, M2-M group treated with M2 at a dose of 50 lg/mL, and M2-H group treated with M2 at a dose of 200 lg/mL. UV irradiation could cause the up-regulation of MMPs, which is related with collagen degradation in skin photodamage (Jung et al., 2014). As shown in Fig. 6, chronic exposure to UVB dramatically up-regulated both the concentration and activity of MMP-1. It has been reported that marine collagen hydrolysate can inhibit collagen degradation by suppressing MMP-1 expression (Liang et al., 2010). The significant increase in protein expression of MMP-1 was inhibited by M1 and M2 in a dose-dependent manner, and there was a significant difference between the group treated with M1 and MC (p < 0.05). The M2-H group significantly suppressed the up-regulated protein level of MMP-1, which decreased by 16% compared with MC (p < 0.05). The activity of MMP-1 was observably suppressed by the M1-H and M2-H group compared with MC in Fig. 6b, which decreased by 16% and 15%. However, there was no significant difference of inhibitory effects on MMP-1 concentration and activity between M1 and M2 (p > 0.05). 3.6. Effects of synthesized peptides on the MAPK signaling pathway As shown in Fig. 7, UVB irradiation resulted in a significant increase in p-ERK, p-p38 and p-JNK levels in photoaging fibroblasts. M1 and M2 evidently suppressed the phosphorylation of ERK (p < 0.05), and M2 showed a dose-dependent manner in Fig. 7a. It was clear that 50 lg/mL M1 dramatically attenuated pERK expression, which decreased by 23% compared with MC. M1 significantly showed better inhibitory effect on p-ERK expression than M2 (p < 0.05). Both the M1-H group and the M2-H group showed a significant inhibition of phosphorylated p38 (Fig. 7b). However, there was no significant difference in p-JNK level in photoaging fibroblasts treated with M2 in Fig. 7c (p > 0.05). The M1-M and M1-H group significantly suppressed the up-regulated expression of p-JNK (p < 0.05). But the difference of inhibitory effects on p-p38 and p-JNK expression between M1 and M2 was not significant.
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4. Conclusions The expression of MMP-1 in the fibroblasts was high when they were irradiated with UVB doses of 20 mJ/cm2, and CGH exhibited high MMP-1 inhibitory activity when its concentration was 0.1 mg/mL. Two peptides GEIGPSGGRGKPGKDGDAGPK (1935 Da) and GFSGLDGAKGD (1022 Da), isolated from CGH by ionexchange chromatography and RP-HPLC and identified using QTOF mass spectrometer, were found to exhibit high MMP-1 inhibitory activity with the MMP-1 activity decreased by approximately 16% and 15%, respectively. The results of this study showed that MMP-1 inhibitory activity of the peptides is greatly influenced by their physicochemical characteristics, such as positive charge and hydrophobicity. The purified peptides showed a significant inhibition of p-ERK and p-p38 in the MAPK pathway, which further decreased the expressions of MMP-1. These results could be used to further investigate the inhibiting mechanism and the binding sites of the MMP-1 inhibitory peptides. Acknowledgements This work was supported by National Natural Science Foundation of China (Nos. 31401476 and 31471606), Specialized Research Fund for the Doctoral Program of Higher Education (No. 20130132120024), and Key Research and Development Program of Shandong Province (No. 2016YYSP005). References Bae, J.-Y., Choi, J.-S., Choi, Y.-J., Shin, S.-Y., Kang, S.-W., Han, S. J., & Kang, Y.-H. (2008). ( ) Epigallocatechin gallate hampers collagen destruction and collagenase activation in ultraviolet-B-irradiated human dermal fibroblasts: Involvement of mitogen-activated protein kinase. Food and Chemical Toxicology, 46, 1298–1307. Besaratinia, A., Kim, S.-I., & Pfeifer, G. P. (2008). Rapid repair of UVA-induced oxidized purines and persistence of UVB-induced dipyrimidine lesions determine the mutagenicity of sunlight in mouse cells. The FASEB Journal, 22, 2379–2392. Bickers, D. R., & Athar, M. (2006). Oxidative stress in the pathogenesis of skin disease. Journal of Investigative Dermatology, 126, 2565–2575. Bouhallab, S., Henry, G., & Boschetti, E. (1996). Separation of small cationic bioactive peptides by strong ion-exchange chromatography. Journal of Chromatography A, 724, 137–145. Carrasco-Castilla, J., Hernández-Álvarez, A. J., Jiménez-Martínez, C., JacintoHernández, C., Alaiz, M., Girón-Calle, J., & Dávila-Ortiz, G. (2012). Antioxidant and metal chelating activities of peptide fractions from phaseolin and bean protein hydrolysates. Food Chemistry, 135, 1789–1795. Chen, T., Hou, H., Lu, J., Zhang, K., & Li, B. (2016). Protective effect of gelatin and gelatin hydrolysate from salmon skin on UV irradiation-induced photoaging of mice skin. Journal of Ocean University of China, 15, 711–718. Chen, H.-M., Muramoto, K., Yamauchi, F., Fujimoto, K., & Nokihara, K. (1998). Antioxidative properties of histidine-containing peptides designed from peptide fragments found in the digests of a soybean protein. Journal of Agricultural and Food Chemistry, 46, 49–53. Cheung, I. W., Cheung, L. K., Tan, N. Y., & Li-Chan, E. C. (2012). The role of molecular size in antioxidant activity of peptide fractions from Pacific hake (Merluccius productus) hydrolysates. Food Chemistry, 134, 1297–1306. Chung, J. H., Seo, J. Y., Lee, M. K., Eun, H. C., Lee, J. H., Kang, S., ... Voorhees, J. J. (2002). Ultraviolet modulation of human macrophage metalloelastase in human skin in vivo. Journal of Investigative Dermatology, 119, 507–512. Egeblad, M., & Werb, Z. (2002). New functions for the matrix metalloproteinases in cancer progression. Nature Reviews Cancer, 2, 161–174. Fan, J., He, J., Zhuang, Y., & Sun, L. (2012). Purification and identification of antioxidant peptides from enzymatic hydrolysates of tilapia (Oreochromis niloticus) frame protein. Molecules, 17, 12836–12850. Fisher, G. J., Varani, J., & Voorhees, J. J. (2008). Looking older: Fibroblast collapse and therapeutic implications. Archives of Dermatology, 144, 666–672. Froissard, P., Monrocq, H., & Duval, D. (1997). Role of glutathione metabolism in the glutamate-induced programmed cell death of neuronal-like PC12 cells. European Journal of Pharmacology, 326, 93–99. Gelse, K., Pöschl, E., & Aigner, T. (2003). Collagens—structure, function, and biosynthesis. Advanced Drug Delivery Reviews, 55, 1531–1546. Gilchrest, B. A. (1996). A review of skin ageing and its medical therapy. British Journal of Dermatology, 135, 867–875. Helenius, M., Mäkeläinen, L., & Salminen, A. (1999). Attenuation of NF-jB signaling response to UVB light during cellular senescence. Experimental Cell Research, 248, 194–202.
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Identification of MMP-1 inhibitory peptides from cod skin gelatin hydrolysates and the inhibition mechanism by MAPK signaling pathway Jiaohan Lu, Hu Hou ⇑, Yan Fan, Tingting Yang, 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 17 November 2016 Received in revised form 23 January 2017 Accepted 25 March 2017 Available online 3 April 2017 Keywords: Cod skin gelatin hydrolysates Matrix metalloproteinases Photoaging Fibroblasts Ultraviolet radiation
a b s t r a c t Cod skin gelatin hydrolysates (CGH) could serve as a source of peptides with anti-photoaging activity. To prepare CGH, alkaline protease and trypsin were used for enzymatic hydrolysis. The anti-photoaging effects of CGH were evaluated using a UVB-induced mouse skin fibroblasts photoaging model in vitro. CGH could efficiently inhibit the expression of MMP-1, especially at the concentration of 0.1 mg/mL, in the fibroblasts irradiated with UVB of 20 mJ/cm2. In addition, the MMP-1 inhibitory peptides were purified using ion-exchange chromatography and RP-HPLC. The fractions which exhibited the highest activity, were identified using LC-ESI-MS/MS. Finally, two peptides GEIGPSGGRGKPGKDGDAGPK and GFSGLDGAKGD were found to exhibit a significant inhibition of MMP-1, p-ERK and p-p38 and the first purified peptide significantly inhibited p-JNK in MAPK signaling pathways. Therefore, this study provided a scientific basis for the preparation of MMP-1 inhibitory peptides and bioactive peptides can be used as functional supplements for marine skin protection products. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Photoaging, an extrinsic aging of the skin, occurs as a consequence of exposure to plenty of environmental stresses, such as ultraviolet (UV) radiation (Chung et al., 2002; Gilchrest, 1996). Increase in the UV radiation in earth’s atmosphere because of the breaches in the stratospheric ozone layer has increased the risk of photoaging to skin, which is the feature of wrinkling, irregular pigmentation and poor elasticity (Ichihashi et al., 2003). Ultraviolet radiation B (UVB, 280–320 nm), the main cause of skin photodamage, can penetrate the epidermis and reach the upper dermis, which consist mostly of fibroblasts and extracellular matrix (ECM), and leads to the generation of reactive oxygen species (ROS) and activation of signaling pathways, which further induce the up-regulation of matrix metalloproteinases (MMPs) in human skin (Helenius, Mäkeläinen, & Salminen, 1999; Keyse, 1993; Rittié & Fisher, 2002). The dermal fibroblasts, suffering accumulated damage during UV irradiation, play a key role in photoaging due to their effect on maintaining the metabolic balance of dermal collagenous ECM (Fisher, Varani, & Voorhees, 2008). Reports suggest that collagen degradation by MMPs secreted by various cells (e.g., fibroblasts) largely accounts for UV-induced photoaging
⇑ Corresponding author. E-mail addresses: [email protected], [email protected] (H. Hou). http://dx.doi.org/10.1016/j.jff.2017.03.049 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.
(Chung et al., 2002; Rabe, Mamelak, McElgunn, Morison, & Sauder, 2006). MMPs, a family of zinc-dependent endopeptidases, are related with the degradation of ECM in connective tissues. MMPs can be divided into different subgroups, according to their structure and substrate specificity (Egeblad & Werb, 2002). MMP-1 is known to induce dermal collagen degradation and preferentially degrades type I collagen, the most abundant structural protein in the skin, which is synthesized primarily by dermal fibroblasts and related with resiliency and strength of the skin (Gelse, Pöschl, & Aigner, 2003). Hence, it is not surprising that UVB-induced photodamage is an emerging area of research, with special focus on ways of arresting collagen degradation and collagenase activation (Bae et al., 2008; Katiyar, 2003). The accumulation of ROS and the up-regulation of MMPs expression in fibroblasts caused by UV irradiation reflect the central influences of photoaging process (Kohl, Steinbauer, Landthaler, & Szeimies, 2011). ROS causes oxidative damage to major cellular components, and mediates the phosphorylation of protein kinases through mitogen-activated protein kinases (MAPK) pathway (Bickers & Athar, 2006; Sharma, Meeran, & Katiyar, 2007; Yang, Sharrocks, & Whitmarsh, 2003), which includes ERK, p38 MAPK and JNK. The phosphorylation of ERK mediates the activation of c-Fos and c-Jun, which are known to form AP-1 and further induce the expressions of MMPs thus leading to collagen degradation (Mehta & Griendling, 2007).
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Moreover, phosphorylated JNK activates phosphor-c-Jun (Kang et al., 2003). In recent years, most researches payed attention to peptides derived from plant sources and marine animal sources, which have been found to possess good antioxidant activity, such as sea urchin gonad (Qin et al., 2011), sea cucumber (Zhou, Wang, & Jiang, 2012), and pacific hake (Cheung, Cheung, Tan, & Li-Chan, 2012). Collagen polypeptides from Apostichopus japonicus (Wang et al., 2008) and collagen peptides from Chlamys farreri (Yu, Li, Liu, & Wang, 2004) showed protective effects on UV-induced skin photoaging. Hou et al. (2009) proposed that gelatin polypeptides extracted from Pacific cod (Gadus macrocephalus) skin and hydrolyzed with alkaline protease and pepsin could protect collagen fibers in skin, which indicated that Pacific cod skin hydrolysates had the peptides of anti-photoaging activity. Chen, Hou, Lu, Zhang, and Li (2016) pointed out that Pacific cod skin hydrolysates blocked the UV-induced up-regulation of MMPs expression in photoaging skin. These researches show that cod skin hydrolysates could protect against UV-induced damage in skin. But, the mechanisms of the anti-photoaging activities of peptides are not fully understood. Several researches have indicated biological activities of peptides are related to their composition, structure and hydrophobicity (Chen, Muramoto, Yamauchi, Fujimoto, & Nokihara, 1998). It was reported that peptides containing Glu, Lys, Thr, Asp, Tyr, Leu, Ala and Pro exhibited a strong antioxidant capacity (Liu et al., 2013). The MMP-1 inhibitory activity of cod skin gelatin hydrolysates prepared with alkaline protease and trypsin has not been investigated systematically. There have been no reports on the purification and identification of MMP-1 inhibitory peptides from CGH. Therefore, in this study, CGH was chosen as a potential source of MMP-1 inhibitory peptides, and peptides with high MMP-1 inhibiting activity were isolated using ion-exchange chromatography and reversed-phase HPLC (RP-HPLC). The amino acid sequence of the MMP-1 inhibitory peptides was determined using Q-TOF ESI mass spectroscopy. Besides, we explored the MAPK signaling pathway responsible for the expressions of MMP-1 in terms of matrix collagen degradation, using a UVB-induced mouse skin fibroblasts photoaging model in vitro. The results of this study could be important in further research into the mechanism of matrix collagen degradation and for the development of functional foods and marine skin protection products. 2. Materials and methods 2.1. Materials Fresh Pacific cod (Gadus Macrocephalus) skins were obtained from Qingdao Fusheng Foods Co. Ltd. (Qingdao, China) and stored at 20 °C until used. Alkaline protease (CID: 455168) and trypsin (CID: 16130295) were bought from Pangbo Biological Engineering Co., Ltd. (Nanning, China). Dulbecco’s modified Eagle’s medium (DMEM), Fetal bovine serum (FBS), Dulbecco’s Phosphate Buffered Saline (DPBS), and 0.25% Trypsin-EDTA were purchased from Gibcob (New York, USA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) were bought from Sigma Chemical Co. (USA). ICR fetal mice (1–3 g) were obtained from Lukang Pharmaceutical Group Co., Ltd. (Qingdao, China). SP Sephadex C-25 was provided by GE Healthcare (Uppsala, Sweden). 2.2. Preparation of CGH Frozen cod skins were thawed under running tap water and the residual adherent tissues and scales were removed manually. The cleaned fish skins were cut into pieces (8 cm 3 cm), and
soaked in 0.01 M H2SO4 for 30 min at a solid to acid ratio of 1:6 (w/v). The skins were then washed with distilled water until pH 5.0 was reached. Next, the swollen skins were extracted with distilled water at a solid to solution ratio of 1:2 (w/v) for 2 h at 75 °C with stirring. The gelatin solution was filtered through 0.45 lm filter membrane and lyophilized in a vacuum freeze dryer. Alkaline protease and trypsin were used to prepare CGH. The pH of the gelatin solution was adjusted to 7.5 using 1 M NaOH and the gelatin was hydrolyzed by alkaline protease and trypsin at 50 °C with stirring. The reaction was stopped by heating the mixtures at 100 °C for 10 min. The hydrolysates were filtered through 0.45 lm filter membrane and desalted by dialysis. The freezedried CGH was stored at 20 °C until further used. 2.3. Fibroblasts preparation Fibroblasts were isolated from dermis of ICR fetal mice. Briefly, mouse was killed by cervical dislocation and soaked in 75% ethyl alcohol for 1 min. The back skin was removed under aseptic conditions and treated with 0.25% trypsin-EDTA for 12–14 h. DMEM containing FBS (10%, v/v), streptomycin (100 g/L) and penicillin (100 kU/L) was used to stop the reaction. Dermal skin was separated from the epidermis and further trypsinized for 60 min with stirring. After terminating the reaction, dermis was washed twice with DMEM containing FBS (10%, v/v), and centrifuged at 342g for 5 min to remove the suspension. The cells were subsequently dissociated through a 200 mesh sterile metal sieve, and then seeded into 25-cm2 cell culture flasks in DMEM supplemented with FBS (10%, v/ v), streptomycin (100 g/L) and penicillin (100 kU/L) and incubated at 37 °C in a humidified, 5% CO2 atmosphere. 2.4. UVB irradiation Fibroblasts (passages 6–8) were seeded at 2 104 cells/well in 24-well plates containing DMEM with 10% (v/v) FBS and incubated at 37 °C in a humidified, 5% CO2 atmosphere for 24 h. Subsequently, the cells were incubated in culture medium containing samples for an additional 24 h. The cells were then placed in a thin layer of DPBS and exposed to UVB, using two lamps (Beijing Zhongyiboteng-tech Co., Ltd.) that emitted UVB peaking at 313 nm and delivered uniform irradiation at a distance of 30 cm. The energy output was measured by a UVB radiometer with a UVB sensor (Photoeletric Instrument Factory of Beijing Normal University, China). The output of the lamps was 33.5 ± 0.9 lw/cm2. After exposure to UVB, DPBS was replaced by the same culture medium containing samples and incubated at 37 °C for 12 h. Then, cells and culture supernatants were collected and used for ELISA analysis. The normal control (NC) group was incubated in culture medium at the same conditions without UVB and samples prior to analysis and the model control (MC) group treated without samples was exposed to UVB at the same dose.
2.5. Cell viability assay Cell viability was measured by the MTT assay. Fibroblasts (passages 6–8) cultured on 96-well plates and treated with CGH, were exposed to the UVB source. After incubating in culture medium with CGH at 37 °C in a humidified, 5% CO2 atmosphere for 24 h, cells were washed with DPBS and kept in DMEM with MTT (0.5 mg/mL) for 4 h. The MTT solution was then replaced with dimethyl sulfoxide and the absorbance at 490 nm was determined by a microplate reader.
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2.6. Purification of MMP-1 inhibitory peptides 2.6.1. SP Sephadex C25 cation exchange column chromatography The CGH solution was applied to a SP Sephadex C-25 (2.6 cm 50 cm) cation exchange column pre-equilibrated with 0.02 M acetic acid buffer solution (pH 4.0) and eluted with a linear gradient of NaCl (0–1.0 M). Every 8.5 mL of the separated fractions was collected using a flow rate of 1.7 mL/min and absorption at 220 nm was monitored to determine the peptide elution profile. The collected samples were desalted by dialysis and their MMP-1 inhibitory activity was determined. 2.6.2. RP-HPLC using SB-C18 Fractions with high MMP-1 inhibitory activity were further purified using a Zorbax SB-C18 semi-preparative column (9.4 mm 250 mm) and eluted using a linear acetonitrile gradient
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(5–30%, v/v) and a flow rate of 2.0 mL/min. The absorbance of the eluate was measured at 220 nm. The fraction with the highest MMP-1 inhibitory activity was collected and further purified under different linear gradient conditions. 2.7. Analysis of amino acid sequence of purified MMP-1 inhibiting peptide The amino acid sequence and accurate molecular mass of the purified peptides were determined by a Waters Xevo G2 Q-TOF high-resolution mass spectrometer coupled with an electrospray ionization (ESI) source (Waters Corporation, Manchester, UK). MaxEnt3 and Peptide Sequencing software were applied to data acquisition and analysis. The purified peptides (95% purity) were synthesized by ChinaPeptides Co. Ltd. (Suzhou, China), and their MMP-1 inhibitory activity was tested.
Fig. 1. Effects on morphology of mouse skin fibroblasts (photographed under 100 magnifications) (A) and the concentration and activity of MMP-1 (B) and viability of mouse skin fibroblasts irradiated by UVB (C). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
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2.8. ELISA analysis
3. Results and discussion
MMP-1 inhibitory activity was evaluated by measuring its effect on MMP-1 concentration and activity in vitro. And the quantitative analysis of MMP-1, p-ERK, p-JNK, and p-p38 MAPK was conducted using ELISA kits (R&D, USA). The assays were operated on the basis of manufacturer’s instructions. Cells were lysed by rapid freezing and thawing, and then used for the test of p-ERK, p-JNK, and pp38. Cell culture supernatants were used for the test of MMP-1.
3.1. Determination of UVB dose that induces photoaging and CGH concentration that inhibits MMP-1 expression
2.9. Statistical analysis All results were expressed as means ± standard deviation and analyzed by SPSS (version 17.0, IBM Inc., USA). All data were submitted to one-way analysis of variance and statistical differences were considered significantly at P < 0.05.
To determine the appropriate dose of UVB and concentration of CGH for the subsequent experiments, the biological effects of UVB and CGH on the expression of MMP-1 and cell viability effect were first examined. Fibroblasts were irradiated with increasing doses of UVB (0–120 mJ/cm2) and treated with increasing concentrations of CGH (0–10 mg/mL), respectively. As shown in Fig. 1b, UVB exposure increased the concentration and activity of MMP-1, which reached the highest level at 20 mJ/cm2 of UVB. A progressive decrease in viability of mouse skin fibroblasts was illustrated in Fig. 1c and the viability of fibroblasts was expressed as a percentage of live cells compared with the 0 mJ/cm2 group, which were considered to be 100% viable. The UVB dose of 20 mJ/cm2 group
Fig. 2. Effects on morphology of mouse skin fibroblasts (photographed under x100 magnifications) (A) and the concentration and activity of MMP-1 (B) and viability of mouse skin fibroblasts treated with CGH (C). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
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was approximately 80% viable and a remarkable decrease in cell viability was at UVB dose of 60 mJ/cm2 (74%). This dose is nearly equivalent to that received by human skin exposed to a clear midday, midsummer sun, for a short period of time (Besaratinia, Kim, & Pfeifer, 2008). Similar results were shown in Fig. 1a that an obvious increase in cell death was observed starting from 60 mJ/cm2 UVB. Compared with normal cells (0 mJ/cm2), some fibroblasts became round and floated in the supernatants under UVB irradiation. The number of floating (dead) cells (Froissard, Monrocq, & Duval, 1997) in the culture supernatant increased starting from UVB dose of 60 mJ/cm2 by microscopic observation (magnification 100), especially at UVB dose of 90 mJ/cm2 and 120 mJ/cm2. Based on these results, a UVB dose of 20 mJ/cm2 was chosen for subsequent experiments. As shown in the 0 mg/mL group of Fig. 2a, a single exposure to a 20 mJ/cm2 dose of UVB resulted in premature senescence of cells. Treatment with 0.1 mg/mL CGH strongly inhibit the expression of
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MMP-1 (Fig. 2b). Additionally, the cell viability was expressed as a percentage of live cells compared with the 0 mg/mL group, which were considered to be 100% viable and a remarkable increase in cell viability was indicated starting from 0.1 mg/mL CGH; at this concentration, cell viability was approximately 151% (Fig. 2c). Similar results were obtained from morphology that an obvious decrease in cell death was observed at 0.1 mg/mL CGH. (Fig. 2a). Floating cells decreased in the supernatants after treating with CGH by microscopic observation (magnification 100), especially at 0.001 mg/ mL, 0.01 mg/mL, and 0.1 mg/mL CGH. Therefore, a CGH concentration of 0.1 mg/mL was chosen for subsequent experiments. 3.2. Purification of MMP-1 inhibitory peptides using ion-exchange chromatography Ion-exchange chromatography separates proteins or peptides based on their differences of charge (Bouhallab, Henry, &
Fig. 3. The elution profile of CGH by the SP Sephadex C-25 (A) and the concentration and activity of MMP-1 in each of the separated fractions (B). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
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Fig. 4. The RP-HPLC profile of C5 by the Zorbax SB-C18 semi-prep column (A) and the concentration and activity of MMP-1 in each of the separated fractions (B) and the RPHPLC profile of P8 and P10 by the Zorbax SB-C18 semi-prep column (C). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
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Boschetti, 1996). As one of the strongest cation exchangers, SP Sephadex C-25 (main functional group: sulfopropyl) is used widely for separating bioactive peptides. In order to isolate the MMP-1 inhibitory peptides, CGH was dissolved in 0.02 M acetic acid buffer solution (pH 4.0) and separated by SP Sephadex C-25 cation exchange column chromatography (Fig. 3). Six components were obtained, named C1, C2, C3, C4, C5 and C6, respectively (Fig. 3a). The C2, C4, C5, and C6 were the main components of CGH. It was reported that the activity of peptide was associated with the content of positive charge (Kong, Guo, Hua, Cao, & Zhang, 2008). The MMP-1 inhibiting activity of each component (10 lg/mL, 50 lg/ mL and 100 lg/mL) was assayed. At the concentration of 50 lg/ mL, the concentration of MMP-1 decreased by 26% in the cells treated with component C5 (38.7 ng/mL), compared with MC (52.3 ng/ mL). MMP-1 activity values were 4.7 U/L, 5.7 U/L, and 3.5 U/L at C5 concentration of 10 lg/mL, 50 lg/mL, and 100 lg/mL, respectively. These values approximately indicated a 0.61-fold down-regulation
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in activity, compared with MC (8.9 U/L) (Fig. 3b). Since C5 showed remarkably stronger MMP-1 inhibitory activity than any of the other components, it was further purified by RP-HPLC. 3.3. Purification of MMP-1 inhibitory peptides using RP-HPLC RP-HPLC can be utilized for separating peptides based on their hydrophobicity (Pownall, Udenigwe, & Aluko, 2010). The RPHPLC profile showed a large number of peaks, indicating the abundance of the peptides generated. Component C5 was separated into 10 fractions (P1-P10) by the Zorbax SB-C18 semi-prep column (Fig. 4a). The eigth (P8) and tenth (P10) peaks showed the highest MMP-1 inhibitory activity at concentrations of 10 lg/mL and 50 lg/mL. MMP-1 concentration values were 60.6 ng/mL and 60.5 ng/mL at P8 and P10 concentration of 10 lg/mL, which showed nearly a 0.22-fold decrease, compared with MC (77.3 ng/ mL). The MMP-1 activity of cells treated with P8 and P10 (10 lg/
Fig. 5. The mass spectrum of M1 (A) and M2 (B).
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mL) was 31.6 U/L and 28.8 U/L. Compared with MC (60.2 U/L), the MMP-1 activity decreased by 47% and 52%, respectively (Fig. 4b). Fraction P8 was further purified on the Zorbax SB-C18 column with the following increasing acetonitrile treatment: 0–5 min, 5–45% acetonitrile; 5–25 min, 45–60% acetonitrile; 25–30 min, 60–80% acetonitrile. P10 purification was performed with 0–5 min, 5–40% acetonitrile; 5–25 min, 40–55% acetonitrile; 25–30 min, 55–80% acetonitrile gradient. Fig. 4c showed a single peak at 9 min (M1) and 10 min (M2) derived from P8 and P10, respectively. Moreover, these fractions were identified by Q-TOF mass spectrometer.
3.4. Determination of the amino acid sequence of MMP-1 inhibitory peptides
(Qian, Jung, Byun, & Kim, 2008). Rajapakse, Mendis, Jung, Je, and Kim (2005) pointed out that presence of Asp seemed to play an important role in antioxidant peptide sequences irrespective of its position. In addition, aliphatic amino acids such as Asn and Glu had great effect on antioxidant activity (Park, Jung, Nam, Shahidi, & Kim, 2001; Zhang et al., 2010). The MMP-1 inhibitory peptide M1 and M2 obtained from CGH contained amino acids, such as Asp, Lys and Gly, and the presence of Glu in M1 and Leu in M2 also showed similar results with previous studies. Jang, Liceaga, and Yoon (2016) reported that Ala-Thr-Ser-His-His (551.25 Da) derived from sandfish protein hydrolysates showed a significant antioxidant activity. Peptides ACGT and ATAGT from Tibpia frame protein (Fan, He, Zhuang, & Sun, 2012), LEELEEELEGCE
Liquid chromatography-mass spectrometry is commonly used to determine peptide sequences. As shown in Fig. 5, the molecular weights of M1 and M2 obtained from CGH were determined to be 1936 Da and 1022 Da, respectively. The amino acid sequences of M1 and M2 were found to be GEIGPSGGRGKPGKDGDAGPK and GFSGLDGAKGD, respectively. Both the presence of amino acids and the sequence of the peptide chain have a great effect on antioxidant power (Rajapakse, Mendis, Byun, & Kim, 2005). The presence of the amino acids Tyr, Trp, Phe, Met, Lys and Cys was reported to be an important contributor to the antioxidant activities of the peptides (Carrasco-Castilla et al., 2012; Huang et al., 2010; Sarmadi & Ismail, 2010). It was reported that peptides with Gly, Glu and Asp exhibited strong antioxidant activity due to they are very important to the radical-scavenging activity of peptides
Fig. 6. Effects of M1 and M2 on the concentration (A) and activity (B) of MMP-1. NC, normal group; MC, model group; PC, positive control group; M1-L, synthesized peptide M1 treatment (12.5 lg/mL); M1-M, synthesized peptide M1 treatment (50 lg/mL); M1-H, synthesized peptide M1 treatment (200 lg/mL); M2-L, synthesized peptide M2 treatment (12.5 lg/mL); M2-M, synthesized peptide M2 treatment (50 lg/mL); M2-H, synthesized peptide M2 treatment (200 lg/mL). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
Fig. 7. Effects of M1 and M2 on the phosphorylation of ERK (A), p38 (B) and JNK (C) of MAPK pathway. NC, normal group; MC, model group; PC, positive control group; M1-L, synthesized peptide M1 treatment (12.5 lg/mL); M1-M, synthesized peptide M1 treatment (50 lg/mL); M1-H, synthesized peptide M1 treatment (200 lg/mL); M2-L, synthesized peptide M2 treatment (12.5 lg/mL); M2-M, synthesized peptide M2 treatment (50 lg/mL); M2-H, synthesized peptide M2 treatment (200 lg/mL). Data are expressed as means ± standard deviation (n = 4) and the different letters indicate significant differences (p < 0.05).
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from Bullfrog skin (Qian, Jung, & Kim, 2008), LPHSGY from Alaska Pollock protein hydrolysate (Je, Park, & Kim, 2005), have been reported to possess antioxidant activity. Twenty-three polypeptides containing Ala-Gly-Pro, Gly-Pro, and Gly-Leu repeated sequences, which also presented in M1 and M2, were identified in the hydrolysates of Pacific cod skin gelatin, which was found to improve the morphology of collagen fibers and significantly mitigate the reduction of collagen content in photoaging skin. Although the complete mechanism of MMP-1 inhibition by bioactive peptides remains unclear, it has been confirmed that MMP-1 inhibitory activity is related to the mitogen-activated protein kinase pathway (Chen et al., 2016). Moreover, Liang et al. (2010) revealed the effects of long-term oral administration of collagen hydrolysate on the chronological skin aging in vivo by inhibiting the collagen loss and collagen fragmentation. Koyama (2016) pointed out that di-peptide Pro-Hyp and Hyp-Gly normally were found in the blood after collagen hydrolysate intake. And Pro-Hyp stimulated the growth of fibroblasts (Shigemura et al., 2009). Pro-Hyp presented in vivo after the intake of the cod skin gelatin hydrolysate, which was also reported by Chen et al. (2016). 3.5. Effect of synthesized peptides on the expression of MMP-1 Fibroblasts were divided into the following 9 groups: normal group (NC), model group (MC), positive control group (PC) treated with vitamin C at a dose of 12.5 lg/mL, M1-L group treated with M1 at a dose of 12.5 lg/mL, M1-M group treated with M1 at a dose of 50 lg/mL, M1-H group treated with M1 at a dose of 200 lg/mL, M2-L group treated with M2 at a dose of 12.5 lg/mL, M2-M group treated with M2 at a dose of 50 lg/mL, and M2-H group treated with M2 at a dose of 200 lg/mL. UV irradiation could cause the up-regulation of MMPs, which is related with collagen degradation in skin photodamage (Jung et al., 2014). As shown in Fig. 6, chronic exposure to UVB dramatically up-regulated both the concentration and activity of MMP-1. It has been reported that marine collagen hydrolysate can inhibit collagen degradation by suppressing MMP-1 expression (Liang et al., 2010). The significant increase in protein expression of MMP-1 was inhibited by M1 and M2 in a dose-dependent manner, and there was a significant difference between the group treated with M1 and MC (p < 0.05). The M2-H group significantly suppressed the up-regulated protein level of MMP-1, which decreased by 16% compared with MC (p < 0.05). The activity of MMP-1 was observably suppressed by the M1-H and M2-H group compared with MC in Fig. 6b, which decreased by 16% and 15%. However, there was no significant difference of inhibitory effects on MMP-1 concentration and activity between M1 and M2 (p > 0.05). 3.6. Effects of synthesized peptides on the MAPK signaling pathway As shown in Fig. 7, UVB irradiation resulted in a significant increase in p-ERK, p-p38 and p-JNK levels in photoaging fibroblasts. M1 and M2 evidently suppressed the phosphorylation of ERK (p < 0.05), and M2 showed a dose-dependent manner in Fig. 7a. It was clear that 50 lg/mL M1 dramatically attenuated pERK expression, which decreased by 23% compared with MC. M1 significantly showed better inhibitory effect on p-ERK expression than M2 (p < 0.05). Both the M1-H group and the M2-H group showed a significant inhibition of phosphorylated p38 (Fig. 7b). However, there was no significant difference in p-JNK level in photoaging fibroblasts treated with M2 in Fig. 7c (p > 0.05). The M1-M and M1-H group significantly suppressed the up-regulated expression of p-JNK (p < 0.05). But the difference of inhibitory effects on p-p38 and p-JNK expression between M1 and M2 was not significant.
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4. Conclusions The expression of MMP-1 in the fibroblasts was high when they were irradiated with UVB doses of 20 mJ/cm2, and CGH exhibited high MMP-1 inhibitory activity when its concentration was 0.1 mg/mL. Two peptides GEIGPSGGRGKPGKDGDAGPK (1935 Da) and GFSGLDGAKGD (1022 Da), isolated from CGH by ionexchange chromatography and RP-HPLC and identified using QTOF mass spectrometer, were found to exhibit high MMP-1 inhibitory activity with the MMP-1 activity decreased by approximately 16% and 15%, respectively. The results of this study showed that MMP-1 inhibitory activity of the peptides is greatly influenced by their physicochemical characteristics, such as positive charge and hydrophobicity. The purified peptides showed a significant inhibition of p-ERK and p-p38 in the MAPK pathway, which further decreased the expressions of MMP-1. These results could be used to further investigate the inhibiting mechanism and the binding sites of the MMP-1 inhibitory peptides. Acknowledgements This work was supported by National Natural Science Foundation of China (Nos. 31401476 and 31471606), Specialized Research Fund for the Doctoral Program of Higher Education (No. 20130132120024), and Key Research and Development Program of Shandong Province (No. 2016YYSP005). References Bae, J.-Y., Choi, J.-S., Choi, Y.-J., Shin, S.-Y., Kang, S.-W., Han, S. J., & Kang, Y.-H. (2008). ( ) Epigallocatechin gallate hampers collagen destruction and collagenase activation in ultraviolet-B-irradiated human dermal fibroblasts: Involvement of mitogen-activated protein kinase. Food and Chemical Toxicology, 46, 1298–1307. Besaratinia, A., Kim, S.-I., & Pfeifer, G. P. (2008). Rapid repair of UVA-induced oxidized purines and persistence of UVB-induced dipyrimidine lesions determine the mutagenicity of sunlight in mouse cells. The FASEB Journal, 22, 2379–2392. Bickers, D. R., & Athar, M. (2006). Oxidative stress in the pathogenesis of skin disease. Journal of Investigative Dermatology, 126, 2565–2575. Bouhallab, S., Henry, G., & Boschetti, E. (1996). Separation of small cationic bioactive peptides by strong ion-exchange chromatography. Journal of Chromatography A, 724, 137–145. Carrasco-Castilla, J., Hernández-Álvarez, A. J., Jiménez-Martínez, C., JacintoHernández, C., Alaiz, M., Girón-Calle, J., & Dávila-Ortiz, G. (2012). Antioxidant and metal chelating activities of peptide fractions from phaseolin and bean protein hydrolysates. Food Chemistry, 135, 1789–1795. Chen, T., Hou, H., Lu, J., Zhang, K., & Li, B. (2016). Protective effect of gelatin and gelatin hydrolysate from salmon skin on UV irradiation-induced photoaging of mice skin. Journal of Ocean University of China, 15, 711–718. Chen, H.-M., Muramoto, K., Yamauchi, F., Fujimoto, K., & Nokihara, K. (1998). Antioxidative properties of histidine-containing peptides designed from peptide fragments found in the digests of a soybean protein. Journal of Agricultural and Food Chemistry, 46, 49–53. Cheung, I. W., Cheung, L. K., Tan, N. Y., & Li-Chan, E. C. (2012). The role of molecular size in antioxidant activity of peptide fractions from Pacific hake (Merluccius productus) hydrolysates. Food Chemistry, 134, 1297–1306. Chung, J. H., Seo, J. Y., Lee, M. K., Eun, H. C., Lee, J. H., Kang, S., ... Voorhees, J. J. (2002). Ultraviolet modulation of human macrophage metalloelastase in human skin in vivo. Journal of Investigative Dermatology, 119, 507–512. Egeblad, M., & Werb, Z. (2002). New functions for the matrix metalloproteinases in cancer progression. Nature Reviews Cancer, 2, 161–174. Fan, J., He, J., Zhuang, Y., & Sun, L. (2012). Purification and identification of antioxidant peptides from enzymatic hydrolysates of tilapia (Oreochromis niloticus) frame protein. Molecules, 17, 12836–12850. Fisher, G. J., Varani, J., & Voorhees, J. J. (2008). Looking older: Fibroblast collapse and therapeutic implications. Archives of Dermatology, 144, 666–672. Froissard, P., Monrocq, H., & Duval, D. (1997). Role of glutathione metabolism in the glutamate-induced programmed cell death of neuronal-like PC12 cells. European Journal of Pharmacology, 326, 93–99. Gelse, K., Pöschl, E., & Aigner, T. (2003). Collagens—structure, function, and biosynthesis. Advanced Drug Delivery Reviews, 55, 1531–1546. Gilchrest, B. A. (1996). A review of skin ageing and its medical therapy. British Journal of Dermatology, 135, 867–875. Helenius, M., Mäkeläinen, L., & Salminen, A. (1999). Attenuation of NF-jB signaling response to UVB light during cellular senescence. Experimental Cell Research, 248, 194–202.
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