【作者】 张超；

【导师】 石艳； 黄雅钦；

【作者基本信息】 北京化工大学 ， 化学， 2022， 博士

【摘要】 氧化应激是由于细胞内氧化还原失衡所造成的病理性状态,导致细胞、组织和器官的氧化损伤,已经被认为是多种慢性疾病的重要诱因。越来越多的研究表明,氧化应激会随着年龄的增长而逐渐增加,从而导致了年龄性相关疾病的发生。基于此,本研究设计制备了磷酸化胶原肽镁螯合物,并探究了其对氧化应激及其相关性骨质疏松症的疗效和作用机理。主要研究内容如下:（1）以牛骨胶原为原料,考察了碱性蛋白酶、木瓜蛋白酶、复合蛋白酶和菠萝蛋白酶及其复合对牛骨胶原催化水解产物的抗氧化活性,发现碱性蛋白酶和木瓜蛋白酶复合催化牛骨胶原水解可以得到高抗氧化活性的胶原肽。利用响应曲面优化方法对复合酶催化牛骨胶原水解反应条件进行了优化,得到制备高抗氧化活性胶原肽的最佳反应条件:反应温度为59.6℃,体系pH为7.7,总酶用量为8396 U/g,反应时间为120 min,底物浓度为0.25 g/m L,在此条件下制备得到的胶原肽表现出了良好的DPPH和ABTS自由基清除效果,IC₅₀值分别为4.99±0.17 mg/m L和2.14±0.10mg/m L。通过离子交换层析、反相高效液相色谱及液质联用技术对胶原肽进行了分离、纯化和鉴定,结合分子对接技术对鉴定得到的活性肽进行筛选,考察了不同活性肽与Keap1的结合能力。结果发现,GPAGPHyp GPIG、GPAGPIGPVG、IDGRPGPIGPA和ISGPHyp GPHyp GPA对Keap1表现出高亲和能力（Affinity≤-9.1 kcal/mol）,有望实现高抗氧化活性。合成的四种胶原肽的抗氧化活性测试进一步证明了IDGRPGPIGPA对DPPH和ABTS自由基有较高的清除能力,而GPAGPHyp GPIG能够通过促进细胞内抗氧化酶的激活及还原型谷胱甘肽水平的提升对H₂O₂诱导损伤细胞表现出较高的保护与修复效果。（2）基于牛骨胶原肽中丰富的甘氨酸和金属结合活性位点,成功地设计制备了高抗氧化活性的胶原肽镁离子螯合物,发现了提升胶原肽抗氧化活性的新途径。通过对比不同二价金属离子与牛骨胶原肽的结合能力及制备产物对自由基清除能力的影响发现,镁离子螯合能够显著提高牛骨胶原肽的抗氧化活性并具有较高的结合能力。胶原肽镁螯合物表现出显著提高的DPPH和ABTS自由基清除能力,IC₅₀值分别为2.05±0.11 mg/m L和1.56±0.09 mg/m L。通过对其结构进行表征发现,镁离子主要通过与牛骨胶原肽中的氨基氮和羰基氧结合,诱导并稳定其二级结构,导致牛骨胶原肽中β-转角结构的上升和无规卷曲含量的下降,促进更多活性氨基酸的暴露,从而提高了牛骨胶原肽的自由基清除能力。通过建立氧化损伤细胞模型,探究了胶原肽镁螯合物对氧化损伤细胞的保护与修复作用,证明了其能通过提高细胞内抗氧化酶活性和还原型谷胱甘肽含量对活性氧产生明显的清除作用,从而提高了细胞对氧化应激的抵抗能力。同时发现,镁离子螯合能显著提高胶原肽对细胞内促炎因子的抑制作用,从而达到有效的抗炎效果。（3）通过对胶原肽进行磷酸化修饰和镁离子螯合,成功地设计制备得到了磷酸化胶原肽镁螯合物。通过对比研究揭示了不同磷酸化试剂修饰对胶原肽磷酸化程度及镁离子螯合能力的影响,发现以三聚磷酸钠为磷酸化试剂时制备得到的磷酸化胶原肽镁螯合物显著提高了成骨细胞增殖、分化和矿化活性,并且能够对H₂O₂诱导引起的细胞内氧化应激起到明显抑制作用。探究了磷酸化胶原肽镁螯合物对去卵巢（OVX）小鼠骨质疏松的修复作用及其可能的作用机制。证明磷酸化胶原肽镁螯合物给药处理能够促进小鼠血清中抗氧化酶活性的提高,显著降低OVX小鼠血清中丙二醛水平,抑制小鼠体内氧化应激的形成。同时其能降低OVX小鼠体内肿瘤坏死因子-α（TNF-α）和白介素-6（IL-6）的生成,并通过提高转化生长因子-β（TGF-β）分泌水平发挥抗骨质疏松作用。Micro-CT结果表明,磷酸化胶原肽镁螯合物能够抑制OVX小鼠中BMD、BS、BV/TV、Tb.Th和Tb.N的下降和Tb.Sp的升高,组织学染色结果表明磷酸化胶原肽镁螯合物可以缓解OVX诱导的骨微结构的破坏。通过PCR array分析发现磷酸化胶原肽镁螯合物能够通过PI3K/Akt信号通路上调骨生成相关基因和成血管相关基因,从而有效实现骨组织结构的保持。更多还原

【Abstract】 Oxidative stress is a pathological state caused by the imbalance of intracellular redox,which leads to oxidative damage to cells,tissues,and organs.Therefore,oxidative stress has been considered as an important inducement for many chronic diseases.A growing number of studies have shown that oxidative stress gradually increases with age,leading to the development of age-related diseases.Hence,the phosphorylated collagen peptide-magnesium chelate was designed and prepared in this study,and its therapeutic effect on oxidative stress and osteoporosis as well as its possible mechanism were investigated.The main research contents are as follows:（1）The antioxidant activity of bovine bone collagen hydrolysates prepared by alcalase,papain,protemax,bromelain,and their combinations was investigated.The results showed that the bovine bone collagen hydrolysates prepared by alkaline coupled with papain exerted higher antioxidant activity.Response surface optimization methods were used to optimize the conditions of complex enzymatic hydrolysis,and the hydrolysis parameters were optimized as follows:temperature 59.6℃,pH 7.7,E/S 8396 U/g,time 120 min,and substrate concentration 0.25 g/m L.Under these parameters,the obtained collagen peptides exhibited desirable DPPH and ABTS radical scavenging activity,with IC₅₀ values of 4.99±0.17 mg/m L and 2.14±0.10 mg/m L,respectively.To explore the mechanism of antioxidant peptides,bovine bone collagen peptides were separated,purified,and identified by ion exchange chromatography,reversed-phase high performance liquid chromatography,and liquid chromatography-mass spectrometry.The identified active peptides were screened by molecular docking technology,and the binding ability of different active peptides to Keap1 was investigated.The results showed that the four active peptides,GPAGPHyp GPIG,GPAGPIGPVG,IDGRPGPIGPA,and ISGPHyp GPHyp GPA showed the highest affinity（Affinity≤-9.1 kcal/mol）,thus contributing to higher antioxidant capacity.The antioxidant activity test of the four synthesized peptides proved that IDGRPGPIGPA has a high scavenging capacity to DPPH and ABTS free radicals,while GPAGPHyp GPIG improves the resistance of cells to H₂O₂-induced oxidative damage through promoting the activation of intracellular antioxidant enzymes and the synthesis of reduced glutathione.（2）In view of the abundant glycine and metal binding active sites in bovine bone collagen peptides,we successfully designed and prepared the collagen peptide-magnesium chelate with high antioxidant activity,and developed a new method to enhance the antioxidant activity of collagen peptide.By comparing the binding capacity of different divalent metal ions with bovine bone collagen peptides and the effect of the prepared chelates on the free radical scavenging capacity,it was found that magnesium chelation could significantly improve the antioxidant activity of bovine bone collagen peptides and had a higher binding capacity.Magnesium chelation significantly improved the scavenging capacity of bovine bone collagen peptides to DPPH and ABTS radicals,with IC₅₀ values of 2.05±0.11 mg/m L and 1.56±0.09 mg/m L,respectively.The conformational structure of bovine bone collagen peptide-magnesium chelate was investigated,and the results indicated that magnesium ions can induce and stabilize the secondary structure of bovine bone collagen peptides via the binding with the amino nitrogen and carboxyl oxygen atoms,and leads to an increase ofβ-turn and a decrease of random coil,promoting the exposure of more active sites,thereby improving the free radical scavenging capacity of bovine bone collagen peptides.The protective and repair effects of collagen peptide-magnesium chelate on H₂O₂-induced oxidative damaged cells were investigated.The results demonstrated that collagen peptide-magnesium chelate exhibited a significant eliminating effect on intracellular ROS by activating the antioxidant enzymes and increasing the GSH levels,thus contributing to the resistance of cells to oxidative stress.Besides,collagen peptide-magnesium chelate can significantly improve the generation of proinflammatory factor TNF-αin lipopolysaccharide（LPS）-induced macrophages,thus achieving an effective anti-inflammatory effect.（3）Phosphorylated collagen peptide-magnesium chelate was successfully designed and prepared by phosphorylation modification and magnesium ion chelation.The influence of different phosphorylation reagents on the degree of phosphorylation and magnesium chelating capacity of collagen peptide was revealed and the results indicated that phosphorylated collagen peptide-magnesium chelate prepared with sodium tripolyphosphate（STPP）as the phosphorylation reagent could significantly improve the proliferation,differentiation,and mineralization of osteoblasts and inhibit the oxidative stress caused by H₂O₂.The preventive effect of phosphorylated collagen peptide-magnesium chelate on osteoporosis was investigated by an ovariectomy（OVX）-induced osteoporosis models.The results demonstrated that phosphorylated collagen peptide-magnesium chelate administration could promote the activation of antioxidant enzymes and reduce the generation of MDA in OVX mice,thus inhibiting the formation of oxidative stress in mice.phosphorylated collagen peptide-magnesium chelate administration also inhibited the generation of tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）,and promoted osteogenic activity by increasing the level of transforming growth factor-β（TGF-β）.Micro-CT results demonstrated that phosphorylated collagen peptide-magnesium chelate could inhibit the decrease of BMD,BS,BV/TV,Tb.Th,Tb.N and the increase of Tb.Sp.Histological staining also showed that phosphorylated collagen peptide-magnesium chelate could alleviate the destruction of bone microstructure induced by OVX.Further studies revealed that phosphorylated collagen peptide-magnesium chelate treatment coud up-regulate the expression of osteogenic and vasculogenic genes by stimulating the PI3K/Akt signaling pathway,thus effectively maintaining bone tissue structure.更多还原