【作者】 王洁；

【导师】 谭海宁；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2022， 硕士

【摘要】 骨质疏松症是以骨微结构破坏、骨密度和骨质量下降、骨脆性增加、极易骨折为病理特征的一种全身性疾病。骨质疏松症患者体内的氧化应激水平普遍高于正常人,改善骨质疏松或可通过改善氧化应激来实现。据报道,驴骨中的胶原蛋白具有改善骨质疏松大鼠和抗氧化的作用,本课题主要从氧化应激的角度探究驴骨胶原蛋白及其多肽对骨质疏松的改善作用。通过热水、碱和酶混合法提取驴骨中的胶原蛋白(donkey bone collagen,DC),DC在228 nm处有最高的紫外吸收峰,分子量在5 kDa和245 kDa之间呈弥散分布,分子间含有氢键,表现出一定的有序性。DC对1,1-二苯基-2-苦基肼自由基(DPPH)的清除率为17.89%,表现出一定的抗氧化活性。以H2O2建立成骨细胞氧化损伤模型,DC在100 μg/mL的浓度时,能够将细胞存活率提高27.31%,将细胞内ROS水平下调52.48%。此外,成骨细胞经50 μg/mL DC分化处理后,碱性磷酸酶表达量较模型组增加了 62.65%。由此可以说明,DC能够保护成骨细胞免受双氧水的损伤,显著降低细胞内过氧化物的水平,促进成骨细胞分化。以D-半乳糖联合低钙饲料喂养建立伴有氧化应激状态的小鼠骨质疏松模型,采集血清、骨骼和脏器探究各组药物对小鼠的干预情况。研究发现,小鼠经2.4 g/kg DC灌胃处理后,血清中超氧化物歧化酶含量升高19.17%,丙二醛水平降低38.95%,骨矿物质密度增加27.27%,骨小梁从稀疏变得致密,小鼠松质骨的异常结构被修复。该结果表明DC可以改善小鼠氧化应激状态、增加骨密度并改善骨微结构。为筛选口服DC产生的活性组分,本课题通过体外模拟胃肠消化降解DC,并对酶解产物进行分离纯化,最终得到抗氧化组分DCHcA3-α。通过LC-MS/MS对该组分进行测序,得到 ANLGPA、HYFL、PAEGPA、KLLHA、HVPNA、GGWFL 和 GWFK 共7个肽段。固相合成7个肽段,检测肽段对DPPH、超氧阴离子和(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)阳离子和羟自由基的清除活性,结果表明7种肽段均可以有效清除自由基,具有较好的抗氧化活性,其中肽ANLGPA的抗氧化活性较为全面。以H2O2建立成骨细胞氧化损伤模型,肽ANLGPA、GGWFL、GWFK可保护成骨细胞免受双氧水的损伤,显著降低细胞内过氧化物的水平,促进成骨细胞分化。综上,本研究为驴骨胶原蛋白对抗骨质疏松的药理作用及驴骨胶原蛋白来源抗氧化肽的应用奠定了实验基础。更多还原

【Abstract】 Osteoporosis is a systemic disease caused by various factors,characterized by the destruction of bone microstructure,decreased bone density and bone quality,increased bone fragility,and extreme susceptibility to fracture.The level of oxidative stress in patients with osteoporosis is generally higher than normal,and reducing the level of oxidative stress may improve osteoporosis.Related studies have shown that donkey bone collagen has the effect of improving osteoporosis in rats and antioxidants.This project mainly explores the impact of donkey bone collagen in improving osteoporosis from the perspective of oxidative stress.Donkey bone collagen(DC)was extracted by mixing hot water,alkali,and enzyme.We found that DC has the highest UV absorption peak at 228 nm,and the molecular weight is diffuse between 5 kDa and 245 kDa,containing hydrogen bonds between molecules and showing some orderliness.At a concentration of 500 μg/mL,DC showed some antioxidant activity with a scavenging rate of 17.89%against 1,1-diphenyl-2-picrylhydrazyl(DPPH).The model of oxidative damage to osteoblasts was established with H2O2.DC at the concentration of 100 μg/mL could increase the cell survival rate by 27.31%and reduce the intracellular ROS level by 52.48%.In addition,osteoblasts treated with 50 μg/mL DC differentiation showed a 62.65%increase in alkaline phosphatase expression compared to the model group.It can be shown that DC can protect osteoblasts from damage caused by hydrogen peroxide,significantly reduce the level of peroxides in cells,and promote osteoblastic differentiation.A mouse osteoporosis model with oxidative stress was established by feeding D-galactose with a low-calcium diet,and serum,bones,and organs were collected to explore the intervention of each group of drugs on mice.It was found that after gavage of 2.4 g/kg DC in mice,the content of superoxide dismutase in the serum increased by 19.17%,the content of malondialdehyde decreased by 38.95%,and the bone mineral density increased by 27.27%,the trabecular bone became dense from sparse,and the abnormal structure of the mouse cancellous bone was repaired.The result indicates that DC can reduce oxidative stress,increase bone density and improve bone microarchitecture in mice.In order to screen the active components produced by oral DC,DC was degraded by simulating gastrointestinal digestion in vitro.The enzymatic hydrolysis products were separated and purified,and finally,the antioxidant component DCHcA3-α was obtained.LCMS/MS determined that the composition included 7 peptides of ANLGPA,HYFL,PAEGPA,KLLHA,HVPNA,GGWFL,and GWFK.Solid-phase synthesis of 7 peptides was performed to detect the scavenging activities of the peptides on DPPH,superoxide anion,ABTS cations,and hydroxyl radicals to evaluate their antioxidant activity,the results showed that 7 peptides had good antioxidant activity and the peptide ANLGPA had a more comprehensive antioxidant activity.The oxidative damage model of osteoblasts was established with H2O2,and the three peptides of ANLGPA,GGWFL,and GWFK can protect osteoblasts from hydrogen peroxide damage,significantly reduce the level of intracellular peroxides,and promote the differentiation of osteoblasts.In conclusion,this study lays an experimental foundation for the pharmacological effect of donkey bone collagen against osteoporosis and the application of antioxidant peptides derived from collagen in donkey bone.更多还原