几种海洋特征寡糖—胶原蛋白复配物对紫外辐射损伤的保护作用及机制研究
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摘要
21世纪是海洋的世纪,海洋中富含的寡糖及生物活性肽,因其良好的抗氧化、抗紫外、抑癌、促生长等生理活性和药用价值已引起人们的广泛关注。海洋寡糖及活性肽生物活性的研究与开发,将成为海水养殖产业高值化利用的突破口。近年来,随着配伍理论的引入,人们逐渐将目光聚焦于海洋四大特征多糖褐藻胶、卡拉胶、琼胶、壳聚糖与鱼皮胶原蛋白的复配,并已开发出性能良好的改性生物材料,成功地应用于组织工程学领域。但目前,关于低分子量海洋四大特征寡糖与鱼皮胶原蛋白活性肽间的复配却鲜有报道,特别是针对其抗紫外作用的研究。紫外线广泛存在于自然界中,其引起的过量氧自由基会诱导光老化、癌症及神经退行性疾病的发生和发展,在病理学过程中扮演了重要角色。因此,无论从经济角度还是人类健康角度出发,对海洋四大特征寡糖与胶原蛋白活性肽的复配及其改性复配物在抗紫外辐照作用方面的研究,均具有深远的科学意义。本论文旨在将不同电荷性质、不同聚合度的海洋四大特征寡糖与低分子量鱼皮胶原蛋白肽进行复配,结合化学分析,药理研究对二者间的配伍规律、抗紫外作用及机制进行研究。主要研究内容如下：
1.复配物的制备与鉴定。低聚甘露糖酸、k-卡拉胶、琼脂糖,壳聚糖经降解制得不同聚合度的寡糖片段,与低分子量罗非鱼鱼皮胶原蛋白活性肽复配,通过HPLC和红外光谱分析二者间的相互作用,结果表明非共价键作用是复配物形成的内在驱动力,离子间缔合、分子间氢键以及疏水基间的相互作用均对二者的结合起到促进作用。
2.复配物抗氧化、抗紫外作用评价。于分子水平和细胞水平分别测定复配物对氧自由基的清除能力、细胞活力以及胞内抗氧化酶活力和脂质过氧化水平的影响.并将复配物与相应单一组分相比较,确定复配对其单一组分活性的提高作用。经筛选含羧酸根、具阴离子电荷性质、分子量1000左右的甘露糖醛酸与胶原蛋白活性肽复配产物的抗氧化、抗紫外作用最强。
3.复配物抗紫外辐照机理研究。同时建立低剂量和高剂量的紫外辐照细胞损伤模型。通过检测筛选所得复配物对胞内活性氧水平,胶原酶、Ⅰ型胶原蛋白含量,细胞凋亡以及MAPKs信号转导通路的影响,并结合FITC标记复配物的胞内定位,对复配物抗辐射机制进行研究。结果表明复配物能够定位于胞内线粒体,清除胞内氧自由基,提高抗氧化酶活性；通过抑制MAPKs信号转导途径激活,抑制辐照后的胶原降解、线粒体上游凋亡分子JNK,p38以及线粒体下游凋亡酶Caspase-3,9的表达,从而抑制光老化和凋亡的发生,降低光损伤程度,达到对皮肤细胞的保护作用。
4.复配物间作用方式及其微观结构分析。结合筛选得复配物及相应单一组分的Zeta-电势分析以及AFM对三者微观结构及状态变化的观察,为寡糖与肽间的离子间作用提供有力的数据支持及更为直观的证据。
以上研究提示,寡糖的电荷性质、聚合度以及连接与其上的极性基团,均会对其与鱼皮胶原蛋白活性肽间的作用方式产生不同程度的影响,引起复配物抗紫外活性的不同,其能够定位于线粒体,抑制皮肤细胞的光老化和凋亡,起到抗紫外辐照作用。本论文首次将不同电荷性质,不同聚合度的寡糖与胶原蛋白活性肽复配后的抗紫外活性进行比较,为海洋四大多糖以及海水养殖产业的高值化利用提供了有力的技术支撑,并为具有类似结构寡糖-肽复配物药物的设计与开发提供了新的线索和科学思路。
The 21st century is the age of ocean. Oceans are important natural resources for several peptides and oligosaccharides with potential antioxidant properties. Recently, there is a growing demand from the food, pharmaceutical and the cosmetics industry for the development of efficient antioxidants, especially from marine active extracts. The specific properties of each collagen peptide and marine oligosaccahride offer possibilities to produce complexes that can confer unique structural properties. The protein-polysaccharide complexes have been extensively studied and applied in tissue engineering. However, only few studies are available that relate to the improvement of protection agasint UV radiation of such complexes, in terms of their ROS scavenging capability. ROS are generated by UV radiation, resulting in photoaging, cancer and neurological degeneration. Thus, to satisfied with the possible economic and the treatment of disease, study on peptide-oligosaccharide complexes has both scientific significance and application values. In this study, we combined the different (isolated and purified) marine oligosaccharides with collagen peptides of the deep-sea fish skin, and evaluated their antioxidant activities and photoprotective effects, with the investigation of their protective mechanisms against UV-irradiated skin damage and the regular pattern of their cross-linking. The main ideas of this paper are listed below:
1. The peptide-oligosaccharide complexes were prepared by combining the different (isolated and purified) marine oligosaccharides with bioactive tilapia skin collagen peptides. The intermolecular interactions between the peptide and oligosaccharide have been investigated by Fourier transform infrared spectroscopy (FI-IR) and size exclusion chromatography (SE-HPLC). The results demonstrate that the intermolecular chain associations were formed between oligosaccharide chains and peptide molecules driven by the electrostatic, intermolecule hydrogen bond and hydrophobic interactions.
2. Chemical and cellular level assays were performed to measure the antioxidant activity and protection against UV radiation of some peptide-oligosaccharide complexes vis-a-vis the activity of its original or native molecules. Results indicated that the antioxidant activities and protective effects against UV radiation of all the complexes were stronger than that of their individual native peptides, especially for MA1000+CP and k-ca3000+CP.
3. Analysis of the protective mechanisms of peptide-oligosaccharide complexes was performed by FCM, ELISA, western blot and confocal. Results indicated that the complex targeted mitochondria after penetration of cell membrane, and suppressed intracellular ROS production as well as increased the antioxidant enzymes activities. The antioxidant properties of the complex can attenuate UV-induced skinphoto-damage by modulating MMP-1 and collagen I expression, as well as suppressing cell apoptosis through the MAPKs signaling pathways.
4. The surface electrical properties and the microscopic structures of the peptide-oligosaccharide complex and its independent original molecules were performed by using zeta potential analyzer and Atomic force microscopy. Results indicated that the close combinations of oligosaccharide chains and peptide molecules mainly driven by the electrostatic interactions.
In conclusion, the surface electrical properties and the degree of polymerization (DP) of oligosaccharides have an influence to the different degree on the conformation of peptide-oligosaccharide complexes and protective effects against UV radiation. The present findings open up new avenues into the drug design of highly efficient peptide-oligosaccharide complexes and provide strong technical support for the high added value utilization of marine aquaculture industry.
1.复配物的制备与鉴定。低聚甘露糖酸、k-卡拉胶、琼脂糖,壳聚糖经降解制得不同聚合度的寡糖片段,与低分子量罗非鱼鱼皮胶原蛋白活性肽复配,通过HPLC和红外光谱分析二者间的相互作用,结果表明非共价键作用是复配物形成的内在驱动力,离子间缔合、分子间氢键以及疏水基间的相互作用均对二者的结合起到促进作用。
2.复配物抗氧化、抗紫外作用评价。于分子水平和细胞水平分别测定复配物对氧自由基的清除能力、细胞活力以及胞内抗氧化酶活力和脂质过氧化水平的影响.并将复配物与相应单一组分相比较,确定复配对其单一组分活性的提高作用。经筛选含羧酸根、具阴离子电荷性质、分子量1000左右的甘露糖醛酸与胶原蛋白活性肽复配产物的抗氧化、抗紫外作用最强。
3.复配物抗紫外辐照机理研究。同时建立低剂量和高剂量的紫外辐照细胞损伤模型。通过检测筛选所得复配物对胞内活性氧水平,胶原酶、Ⅰ型胶原蛋白含量,细胞凋亡以及MAPKs信号转导通路的影响,并结合FITC标记复配物的胞内定位,对复配物抗辐射机制进行研究。结果表明复配物能够定位于胞内线粒体,清除胞内氧自由基,提高抗氧化酶活性；通过抑制MAPKs信号转导途径激活,抑制辐照后的胶原降解、线粒体上游凋亡分子JNK,p38以及线粒体下游凋亡酶Caspase-3,9的表达,从而抑制光老化和凋亡的发生,降低光损伤程度,达到对皮肤细胞的保护作用。
4.复配物间作用方式及其微观结构分析。结合筛选得复配物及相应单一组分的Zeta-电势分析以及AFM对三者微观结构及状态变化的观察,为寡糖与肽间的离子间作用提供有力的数据支持及更为直观的证据。
以上研究提示,寡糖的电荷性质、聚合度以及连接与其上的极性基团,均会对其与鱼皮胶原蛋白活性肽间的作用方式产生不同程度的影响,引起复配物抗紫外活性的不同,其能够定位于线粒体,抑制皮肤细胞的光老化和凋亡,起到抗紫外辐照作用。本论文首次将不同电荷性质,不同聚合度的寡糖与胶原蛋白活性肽复配后的抗紫外活性进行比较,为海洋四大多糖以及海水养殖产业的高值化利用提供了有力的技术支撑,并为具有类似结构寡糖-肽复配物药物的设计与开发提供了新的线索和科学思路。
The 21st century is the age of ocean. Oceans are important natural resources for several peptides and oligosaccharides with potential antioxidant properties. Recently, there is a growing demand from the food, pharmaceutical and the cosmetics industry for the development of efficient antioxidants, especially from marine active extracts. The specific properties of each collagen peptide and marine oligosaccahride offer possibilities to produce complexes that can confer unique structural properties. The protein-polysaccharide complexes have been extensively studied and applied in tissue engineering. However, only few studies are available that relate to the improvement of protection agasint UV radiation of such complexes, in terms of their ROS scavenging capability. ROS are generated by UV radiation, resulting in photoaging, cancer and neurological degeneration. Thus, to satisfied with the possible economic and the treatment of disease, study on peptide-oligosaccharide complexes has both scientific significance and application values. In this study, we combined the different (isolated and purified) marine oligosaccharides with collagen peptides of the deep-sea fish skin, and evaluated their antioxidant activities and photoprotective effects, with the investigation of their protective mechanisms against UV-irradiated skin damage and the regular pattern of their cross-linking. The main ideas of this paper are listed below:
1. The peptide-oligosaccharide complexes were prepared by combining the different (isolated and purified) marine oligosaccharides with bioactive tilapia skin collagen peptides. The intermolecular interactions between the peptide and oligosaccharide have been investigated by Fourier transform infrared spectroscopy (FI-IR) and size exclusion chromatography (SE-HPLC). The results demonstrate that the intermolecular chain associations were formed between oligosaccharide chains and peptide molecules driven by the electrostatic, intermolecule hydrogen bond and hydrophobic interactions.
2. Chemical and cellular level assays were performed to measure the antioxidant activity and protection against UV radiation of some peptide-oligosaccharide complexes vis-a-vis the activity of its original or native molecules. Results indicated that the antioxidant activities and protective effects against UV radiation of all the complexes were stronger than that of their individual native peptides, especially for MA1000+CP and k-ca3000+CP.
3. Analysis of the protective mechanisms of peptide-oligosaccharide complexes was performed by FCM, ELISA, western blot and confocal. Results indicated that the complex targeted mitochondria after penetration of cell membrane, and suppressed intracellular ROS production as well as increased the antioxidant enzymes activities. The antioxidant properties of the complex can attenuate UV-induced skinphoto-damage by modulating MMP-1 and collagen I expression, as well as suppressing cell apoptosis through the MAPKs signaling pathways.
4. The surface electrical properties and the microscopic structures of the peptide-oligosaccharide complex and its independent original molecules were performed by using zeta potential analyzer and Atomic force microscopy. Results indicated that the close combinations of oligosaccharide chains and peptide molecules mainly driven by the electrostatic interactions.
In conclusion, the surface electrical properties and the degree of polymerization (DP) of oligosaccharides have an influence to the different degree on the conformation of peptide-oligosaccharide complexes and protective effects against UV radiation. The present findings open up new avenues into the drug design of highly efficient peptide-oligosaccharide complexes and provide strong technical support for the high added value utilization of marine aquaculture industry.
引文
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