The protective effects of long-term oral administration of marine collagen hydrolysate from chum salmon on collagen matrix homeostasis in the chronological aged skin of Sprague-Dawley male rats

To investigate the long-term effects of marine collagen hydrolysate (MCH) from Chum Salmon skin on the aberrant collagen matrix homeostasis in chronological aged skin, Sprague-Dawley male rats of 4-wk-old were orally administrated with MCH at the diet concentrations of 2.25% and 4.5% for 24 mo. Histological and biochemical analysis revealed that MCH had the potential to inhibit the collagen loss and collagen fragmentation in chronological aged skin. Based on immunohistochemistry and western blot analysis, collagen type I and III protein expression levels in MCH-treated groups significantly increased as compared with the aged control group. Furthermore, quantitative real-time polymerase chain reaction and western blot analysis showed MCH was able to increase the expressions of procollagen type I and III mRNA (COL1A2 and COL3A1) through activating Smad signaling pathway with up-regulated TGF-βRII (TβRII) expression level. Meanwhile, MCH was shown to inhibit the age-related increased collagen degradation through attenuating MMP-1 expression and increasing tissue inhibitor of metalloproteinases-1 expression in a dose-dependent manner. Moreover, MCH could alleviate the oxidative stress in chronological aged skin, which was revealed from the data of superoxide dismutase activity and the thiobarbituric acid reactive substances level in skin homogenates. Therefore, MCH was demonstrated to have the protective effects on chronological skin aging due to the influence on collagen matrix homeostasis. And the antioxidative property of MCH might play an important role in the process.     

Identification and Characterization of Molecular Species of Collagen in Fish Skin

ABSTRACT: Pepsin-solubilized collagen (PSC) prepared from the skin of 3 fish species—common horse mackerel, yellow sea bream, and tiger puffer—were separated into 2 fractions, major and minor, by ammonium sulfate precipitation. These collagen fractions were further purified by phosphocellulose column chromatography. From the results of SDS-PAGE, peptide mapping, and amino-acid analysis, the purified major and minor collagens were identified to be type I and V collagens, respectively. These results suggest that type V collagen might be widely present in fish skin as a minor collagen.     

一种胶原蛋白寡肽促进皮肤胶原蛋白与透明质酸合成的研究

研究罗非鱼鱼鳞胶原蛋白寡肽对人皮肤成纤维细胞透明质酸分泌以及对小鼠真皮中胶原蛋白合成的影响。采用WST-8法测定了该胶原蛋白寡肽对人皮肤成纤维细胞增殖的影响,ELISA法检测了胶原蛋白寡肽对人皮肤成纤维细胞透明质酸分泌的影响;以D-半乳糖衰老模型小鼠为实验对象,考察胶原蛋白寡肽对小鼠真皮中胶原蛋白合成的影响。结果表明,胶原蛋白寡肽能促进人皮肤成纤维细胞增殖（p<0.01）,能促进人皮肤成纤维细胞透明质酸和小鼠真皮中胶原蛋白的合成（p<0.05）。说明该胶原蛋白寡肽能有效促进透明质酸和胶原蛋白的合成。      

Cosmetic efficacy claims in vitro using a three-dimensional human skin model

A tissue engineered human skin equivalent is successfully used for the testing of raw materials and cosmetic formulations. This reconstructed skin is supported by a collagen-glycosaminoglycan-chitosan biopolymer in which human keratinocytes and dermal fibroblasts were co-cultured to form a tissue that closely reproduces the in vivo architecture of normal human skin and takes into account the complex interactions between epidermis and dermis. On the other hand, dermal and epidermal responses can be assessed separately in the dermal or skin equivalent. The three-dimensional model has important advantages compared to monolayer cell cultures and epidermis models in efficacy testing: (i) the possibility of long-term cultivation with repeated application of cream formulations containing bioactives and (ii) the similarity to human skin concerning the interaction between dermis and epidermis. These similarities include the expression of keratinocyte differentiation markers such as cytokeratin 10, filaggrin and transglutaminase, as well as proteins of the basal lamina (laminin, collagen type IV) and extracellular matrix proteins such as elastin. The efficacy of selected bioactives was determined using different endpoints, for example, stimulation of collagen synthesis in the dermal and skin equivalents was shown in comparison to vitamin C as a positive control. On skin equivalents using immunofluorescence techniques we also demonstrated stimulation of the differentiation marker filaggrin, which is important for skin moisturization. The results could be used for claim substantiation, e.g. for the treatment of dry and aged skin.     

黑咖啡提取物对人皮肤成纤维细胞胶原蛋白及衰老相关基因的影响

目的研究黑咖啡提取物对人皮肤成纤维细胞(human skin fibroblast,HSF)胶原蛋白合成及衰老相关基因的影响。方法体外培养HSF细胞,通过CCK-8(cell counting kit-8)法筛选黑咖啡提取物安全剂量;实验分为样品组和空白对照组,用ELISA技术检测黑咖啡提取物对细胞中Ⅰ型胶原蛋白(collagenⅠ,ColⅠ)和基质金属蛋白酶-1(matrix metalloproteinase-1, MMP-1)含量的影响,用实时荧光定量聚合酶链式反应(quantitative real-time PCR, qRT-PCR)技术检测黑咖啡提取物作用后细胞中ColⅠ和MMP-1 mRNA的表达水平。结果与空白对照组比较,黑咖啡提取物可抑制HSF细胞中MMP-1合成(P0.05),促进ColⅠ合成(P0.05);使ColⅠmRNA表达水平升高(P0.01),而MMP-1 mRNA表达水平降低(P0.01)。结论黑咖啡提取物通过促进ColⅠ和抑制MMP-1表达,对胶原蛋白代谢具有改善和调节作用,可延缓皮肤衰老。     

超高压及酶解对虹鳟鱼I型胶原蛋白抗原性的影响

采用超高压及酶解两种方法处理虹鳟鱼过敏原I型胶原蛋白,并研究对其抗原性的影响。结果表明：经超 高压处理后,I型胶原蛋白抗原性随着压力的升高呈现先降低后上升的趋势,200 MPa、25 ℃超高压处理10 min时, I型胶原蛋白抗原性最高降低42.66%。比较木瓜蛋白酶、α-糜蛋白酶以及胰蛋白酶酶解对I型胶原蛋白抗原性的影 响,得到最佳酶解条件为胰蛋白酶在45 ℃、pH 8.0,酶与底物质量比为1∶10时,I型胶原蛋白抗原性消减97.69%。 上述结果表明超高压及酶解均能有效降低虹鳟鱼I型胶原蛋白抗原性,且酶解更为有效。本研究结果为消减过敏原 提供借鉴。     

Propriétés antiélastasiques des propionylaminoacides

Antielastase activity of derivatives like 'propionylaminoacid'(C₃ prolin, C₃ hydroxyprolin, C₃ collagen) was examined for pancreatic elastase, and fibroblastic elastase production. Essential metabolic variations of normal dermal fibroblasts were evaluated: adhesion, proliferation capacity, total protein biosynthesis and collagen type I and type III production. Possible other factors such as cellular nutrients were examined by oxygen consumption evaluation.
Propionylaminoacid derivatives have antielastase activities. Pancreatic elastase showed dose related inhibition (20% to 50% inhibition for concentration from 5 to 80 mg ml⁻¹.
Moreover, fibroblastic elastase production was inhibited, cellular respiration was enhanced. A very good tolerance in vitro was observed for concentration 0–1 mg ml⁻¹ range: adhesion, proliferation capacity and collagen (type I and type III) production were not altered, and oxygen consumption was enhanced.     

Comparison of the biomechanical and biosynthetic behavior of normal human fibroblasts and fibroblasts from a forehead wrinkle

Wrinkles are the most obvious expression of skin aging and are manifested by numerous changes in the organization and structure of the dermis. To better understand if this tissue modification could be linked to a modification of cell function, contractile and synthesis capacities of normal aged human fibroblasts and those obtained from a biopsy of a forehead wrinkle were studied and compared. The capacity of fibroblasts to adhere to the collagen network and to maintain a three-dimensional structure of the dermis was studied using a three-dimensional model of a collagen gel. The metabolic activity of both cell types was determined immunochemically by quantifying collagen I synthesis. Human fibroblasts from the wrinkle contracted the collagen gel less than normal aged human fibroblasts and synthesized less collagen I. The results show that the metabolic activity of aging fibroblasts decelerates and that aging fibroblasts lose their capacity to adhere to collagen fibers, thus limiting the possibility of organizing dermal tissue.
The potential of an active ingredient to compensate for the reduction of metabolic activity and to restore the contractile capacity of fibroblasts from the wrinkle was investigated. This effect was compared with a reference molecule, vitamin C.     

Temperature effects on type I pepsin‐solubilised collagen extraction from silver‐line grunt skin and its in vitro fibril self‐assembly

BACKGROUND: Fish skin is a potential source of collagen. Increasing the extraction temperature increases the yield of collagen. However, it may also result in degradation of the peptide chains, thus damaging the 3D structure of collagen that is vital for its application as a biomaterial. This work investigated the effects of extraction temperature on the yield and characteristics, including fibril self‐assembly, of type I pepsin‐solubilised fish skin collagen. RESULTS: Pepsin‐solubilised collagens were extracted from fresh skin of silver‐line grunt at 4, 10, 20 and 28 °C for 6 h. Extraction at 10 °C gave the highest yield of collagens (439.32 ± 96.43 mg g^?1 fresh skin, dry basis), which were identified as type I and comprised β, α1 and α2 subunits. Extraction at higher temperatures (20 and 28 °C) resulted in the formation of low‐molecular‐weight peptide fragments, thus reducing the yield of the resultant type I collagen. The denaturation temperatures of collagens extracted at 4 and 10 °C, as determined by thermal analysis using differential scanning calorimetry, were 39.5 and 37.5 °C respectively. In vitro fibril self‐assembly of 1 mg mL^?1 collagen solution (pH 6) incubated at 25 °C was only observed with collagens extracted at 4 and 10 °C. The 10 °C collagen not only showed a higher rate of self‐assembly, but its matrix also had a larger fibril diameter of 0.50 ± 0.07 µm (compared with 0.41 ± 0.07 µm for the 4 °C collagen) after 4 h of incubation. CONCLUSION: The results indicated strong effects of extraction temperature on the yield and characteristics of the collagen obtained. Extraction of pepsin‐solubilised collagen from silver‐line grunt skin at 4–10 °C gave a high yield of type I collagen with molecular integrity suitable for tissue‐engineering applications. Copyright © 2010 Society of Chemical Industry     

Zinc l-pyrrolidone carboxylate inhibits the UVA-induced production of matrix metalloproteinase-1 by in vitro cultured skin fibroblasts, whereas it enhances their collagen synthesis

Reduced collagen matrix in the dermis constitutes one of the characteristic features of chronologically aged skin, which is further enhanced on the sun-exposed portions of the body by chronic ultraviolet light (UV) irradiation, inducing the unique changes associated with skin photoageing. The zinc salt of l-pyrrolidone carboxylate (Zinc PCA) has long been used as a cosmetic ingredient, because of its astringent and anti-microbial properties. In the present study, by employing cultured normal human dermal fibroblasts, we found that Zinc PCA suppressed UVA-induced activation of activator protein-1 (AP-1) and reduced matrix metalloproteinase-1 production in these cells, which is thought to be involved in collagen degradation in photoaged skin. Moreover, Zinc PCA treatment of the cells increased the expression of an ascorbic acid transporter mRNA, SVCT2, but not SVCT1, resulting in the enhanced production of type I collagen. Based on these in vitro findings, we consider Zinc PCA to be a promising candidate for an anti-skin ageing agent.     

胃蛋白酶提取马面鱼皮胶原蛋白及结构分析

以马面鱼皮为原料,优化胃蛋白酶提取马面鱼皮胶原蛋白工艺,并对所提胶原蛋白进行结构分析,为马面鱼皮胶原蛋白的制备提供实验基础。得到提取最优条件为胃蛋白酶加酶量16?800?U/g、缓冲液pH?3.0、料液比1∶30（g/mL）、提取时间21.5?h,马面鱼皮胶原蛋白提取率为27.6%。氨基酸分析、紫外光谱分析、傅里叶变换红外光谱分析、垂直电泳分析表明,所提胶原蛋白符合I型胶原蛋白特征,保持胶原蛋白的三螺旋结构。     

皮胶原的结构、性质与提取方法

简要介绍了动物皮胶原的结构组成和物化性质,着重综述了最近几年国内外从猪皮、牛皮和鱼皮中提取胶原的工艺方法。     

狮子鱼皮胶原蛋白的提取及理化性质研究

利用酸提法从狮子鱼(Pterois anennata)的鱼皮中提取了酸溶性胶原蛋白(ASC),并对其理化性质作了研究。其氨基酸组成和聚丙烯酰胺凝胶电泳(SDS-PAGE)均符合Ⅰ型胶原蛋白的特征。傅氏红外线光谱(FTIR)表明其中存在三螺旋的结构。由乌氏黏度法测定它的变性温度(Td)是34.1℃,差示量热扫描法(DSC)测定它的热稳定性温度(Ts)为66.7℃,它的Td明显高于阿拉斯加狭鳕(Td,24.6℃)、马哈鱼(Td,19.4℃)、刀鱼(Td,23.0℃)等一些常见的鱼皮胶原蛋白的变性温度。     

膳食补充n-3多不饱和脂肪酸保护大鼠衰老皮肤胶原的作用及机制研究

目的:探索膳食补充n-3多不饱和脂肪酸(n-3 PUFAs)对大鼠自然老化皮肤真皮胶原的保护作用及机制。方法:选取9月龄大鼠为年轻组,22月龄大鼠作为老化n-3 PUFAs组(膳食补充n-3 PUFAs)及老化对照组(膳食中脂质成分模拟日常饮食中成分作为对照)。通过HE染色、Masson染色及Weigert染色观察年轻组大鼠、老化n-3 PUFAs组和老化对照组大鼠真皮结构变化,ELISA检测Ⅰ、Ⅲ型胶原表达量,RT-qPCR检测Ⅰ、Ⅲ、Ⅳ、Ⅴ型胶原和MMP1、MMP3、MMP9、MMP10、MMP13的mRNA表达量。结果:老化大鼠表皮较年轻大鼠变平变薄,真皮胶原排列紊乱,Ⅰ、Ⅲ型胶原含量显著减少(P<0.05),Ⅰ、Ⅲ、Ⅳ、Ⅴ型胶原mRNA表达量显著下调(P<0.05)。老化n-3 PUFAs组中Ⅰ、Ⅲ型胶原含量及mRNA相对表达量均较老化对照组显著提高(P<0.05)。老化大鼠皮肤中MMP1、MMP9、MMP10、MMP13 mRNA相对表达量均较年轻大鼠显著增多(P<0.05),老化n-3 PUFAs组中MMP10、MMP13 mRNA相对表达量较老化对照组显著降低(P<0.05)。结论:膳食补充n-3 PUFAs可促进老化大鼠皮肤Ⅰ、Ⅲ型胶原合成,减少MMP10、MMP13的表达,增加皮肤中Ⅰ、Ⅲ型胶原含量,延缓皮肤自然老化进程。     

罗非鱼皮Ⅰ型胶原蛋白的抗原反应特性分析

目的：确定罗非鱼皮Ⅰ型胶原蛋白的抗原性,为进一步研究罗非鱼皮Ⅰ型胶原蛋白生物相容性提供依据。方法：采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE）对分离提取的罗非鱼皮胶原蛋白进行鉴定,共免疫ICR小鼠3 次。采用酶联免疫吸附反应（enzymelinked immune sorbent assay,ELISA）分别于每次免疫后第7天测定小鼠产生的总胶原蛋白抗体,并在第21天测定小鼠血清中内免疫球蛋白G（immunoglobulin G,IgG）、免疫球蛋白A（IgA）和免疫球蛋白M（IgM）的含量。结果：罗非鱼皮胶原蛋白样品是Ⅰ型胶原蛋白。所有经罗非鱼皮Ⅰ型胶原蛋白免疫的小鼠体内均产生抗体,3 次免疫后罗非鱼皮Ⅰ型胶原蛋白抗体质量浓度范围为160.50～164.25 μg/L,小鼠IgG、IgA和IgM的质量浓度范围分别为424.81～437.59 ng/mL、46.86～49.53 μg/mL、1.81～1.89 ng/mL。结论：罗非鱼皮Ⅰ型胶原蛋白对ICR小鼠呈现较弱的抗原性。     

Characteristics of collagen from the skin of clown featherback (Chitala ornata)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of clown featherback (Chitala ornata) were isolated and characterised. Yields of ASC and PSC were 27.64 and 44.63% (dry weight basis) with total collagen recovery of 82.08%. Both collagens contained glycine as the major amino acid with relatively high content of proline, hydroxyproline and glutamic acid/glutamine. Nevertheless, they had the low content of cysteine, histidine and tryrosine. The collagen was characterised as type I, comprising (α1)₂α2‐heterotrimer. Pepsin‐aided process did not affect triple‐helical structure of PSC as determined by FTIR spectra. Thermal transition temperature of ASC (36.28 °C) was slightly higher than that of PSC (35.23 °C). However, no differences in isoelectric point (5.54–5.68) between ASC and PSC were observed. Therefore, collagen from the skin of clown featherback could be successfully extracted for further applications.     

Use of pepsin for collagen extraction from the skin of bigeye snapper (Priacanthus tayenus)

Extraction and some properties of pepsin-solubilised collagens from the skin of bigeye snapper (Priacanthus tayenus) were investigated. Addition of bigeye snapper pepsin (BSP) at a level of 20 kUnits/g of defatted skin resulted in an increased content of collagen extracted from bigeye snapper skin. The yields of collagen from bigeye snapper skin extracted for 48 h with acid and with BSP were 5.31% and 18.74% (dry basis), respectively. With pre-swelling in acid for 24 h, collagen extracted with BSP at a level of 20 kUnits/g of defatted skin for 48 h had a yield of 19.79%, which was greater than that of collagen extracted using porcine pepsin at the same level (13.03%). The skin collagen was characterised to be type I with no disulfide bond. Electrophoretic study revealed slight differences in molecular weight between acid-solubilised collagen and all pepsin-solubilised collagens. The molecular weights of α1 and α2 chains in acid-solubilised collagen were estimated to be 120 and 112 kDa, respectively, whereas α1 and α2 chains of pepsin-solubilised collagens had molecular weights of 118 and 111 kDa, respectively. The result suggested that these pepsin-solubilised collagens might undergo partial cleavage in the telopeptide region by pepsin treatment. The maximum transition temperature (T_max) of acid-solubilised collagen was observed at 32.5 °C, which was slightly higher than that of pepsin-solubilised collagens (by about 1 °C). Generally, all collagens were highly solubilised in the pH range of 2–5 and sharply decreased at the neutral pH. No changes in solubility were observed in the presence of NaCl up to 3% (w/v) and the decrease was more pronounced with increasing NaCl concentration.     

黑线鳕鱼皮胶原蛋白胰蛋白酶酶解多肽对UVB诱导HaCaT光损伤的抑制作用

目的：研究黑线鳕鱼（Melanogrammus aeglefinus）皮胶原蛋白胰蛋白酶酶解多肽的抗氧化、抑制光损伤作用,提高鱼皮的再利用价值。方法：利用单因素试验和响应面试验设计,探讨黑线鳕鱼皮胶原蛋白的最佳酶解工艺。将制得的酶解多肽通过基质辅助激光解离飞行时间串联质谱仪（matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry,MALDI-TOF MS/MS）测定多肽序列。利用Discovery Studio中CDOCKER模块将所得的优势肽与抗氧化相关的Kelch样环氧氯丙烷相关蛋白-1（Keap1）进行分子对接。用不同质量浓度的多肽预处理人表皮角质形成细胞（HaCaT）后,使用中波紫外线诱导HaCaT细胞构建光损伤模型,通过超氧化歧化酶（superoxide dismutase,SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）活性、丙二醛（malondialdehyde,MDA）含量及细胞凋亡与周期来研究酶解多肽抑制光损伤的能力。结果：黑线鳕鱼皮胶原蛋白在胰蛋白酶质量分数1.23%、酶解温度52.95?℃、酶解时间1.75?h条件下,水解度为78.33%。MALDI-TOF MS/MS测定出酶解液中的优势肽序列为V-IFFVTMGTP-R、L-SIVPV-R和L-MHT-T。通过分子对接发现V-IFFVTMGTP-R与Keap1结合最稳定,从而预测其具有抗氧化的作用。用250?mg/mL酶解液处理光损伤的HaCaT细胞,其SOD、GSH-Px活性较模型组极显著提高（P＜0.01）,MDA含量极显著降低（P＜0.01）。结论：黑线鳕鱼皮胶原蛋白胰蛋白酶酶解多肽具有抗氧化活性,对光损伤具有一定的抑制作用。     

Connective tissue and aging

Human skin is composed of epidermal and dermal layers, each of which has its own functional importance. Dermis consist of a fine network of collagen fibers, elastic fibers, and other components of the extracellular matrix (ECM). ECM consist primarily of proteins and complex sugars, which form fibrillar networks and a ground substance. Collagen is an important structural component of skin connective tissue and provides the tensile strength of skin. Approximately 70–80% of the dry weight of skin consists of collagen. The most abundant collagen types in skin are types I and III; the former accounts for 80% of the total collagen content of skin and the latter for approximately 15%. The other collagen types present in skin include type IV collagen, which is abundant in the basement membrane (BM); type V collagen, which is located pericellularly; type VI collagen, which plays a role in matrix assembly and is present as microfibrils between collagen fibers; and type VII collagen, which is a structural component of anchoring fibrils. Elastin accounts for only about 1–2% of the dry weight of skin but is important for the maintenance of skin elasticity and resilience. Glycosaminoglycans are of central importance for the maintenance of a water balance in skin, even though the quantities in ECM are small (0.1–0.3% of the dry weight of skin). In the dermis fibroblasts are responsible for the synthesis of ECM proteins. The fibroblasts in the dermis spend majority of time in quiescent state. However in response to activation, the fibroblasts can be reactivated, and certain pool of cell is able to differentiate into myofibroblasts which have important role in repairing skin defects such as during wound healing. During aging the number of fibroblasts is markedly reduced. Also the response of fibroblasts to various growth factors and mechanical or pathological stimulates (wound healing) is diminished. Skin collagen synthesis declines with aging and as the result of such external factors as long‐term sun exposure and medications, for example, topical corticosteroids. In aging skin, collagen fibers become thicker and less soluble and the synthesis of collagen declines. Skin thickness remains quite constant between 20 and 70 years of age, after which a marked decrease in skin thickness occurs. During aging the expression of collagenases are increased and inhibitors of collagenases are reduced leading to increased proteolysis of connective tissue. Recent studies have shown that collagen synthesis is declined in the skin of heavy smokers, while collagenases are increased inducing premature skin aging. The elastic properties of skin are also affected by aging. Along with increasing age, dermal elastic fibers become thicker and fragmented and oxytalan fibers appear fragmented and shortened. Disintegration of elastic fibers is already seen in a minority of fibers between ages 30 and 70, but the changes become more profound after the age of 70 years, affecting a majority of the fibers. As a result of the decreased number of elastic fibers in aged skin, the elastic recovery of skin decreases in elderly people. Even though the content of GAGs and proteoglycans is relatively small, they have significant role in collagen fibril formation, water content of dermis and in mechanical properties. During aging there are marked alterations in different proteoglycans. The amount and synthesis of versican (high molecular size) is decreased and small molecular size decorin is increased. In photoaged skin versican is increased and is closely associated to elastin while decorin is decreased.