Hsp90抑制肌内磷脂氧化的作用机制研究

Lipid oxidation could easily lead to rancidity and flavor deterioration, a problem often confronted during meat processing and storage. The highly unsaturated phospholipids are major targets for lipid oxidation, and have been given much attention in the regulation of lipid oxidation. Applicant’s previous study demonstrated that heat shock protein 90 (Hsp90) could form a stable complex with phospholipids and prevent enzymatic hydrolysis of phospholipids; it was further found that the complex was capable of resisting the external oxidative attack. To thoroughly clarify the role of Hsp90 on phospholipids oxidation and its underlying mechanism, in this project Hsp90 will be extracted and purified from duck muscle, and the binding characteristics of Hsp90 to phospholipid will be analyzed, subsequently the in vitro liposomal oxidation system will be established which is induced by radical, non-radical, and lipoxygenase respectively, and the fluorescence resonance energy transfer spectra (FRET) , HPLC-MS/MS and Western Blot will be applied to evaluate Hsp90 self-oxidation and shielding effect of Hsp90 on phospholipid, so as to explore the approach and mechanism of the inhibitory effect of Hsp90-phospholipid complex on phospholipid oxidation; On this basis, the duck muscle storage experiment will be carried out with the addition of Hsp90 to verify the inhibitory effect of Hsp90 on phospholipid oxidation. The fulfillment of this project will provide new ideas on the control of meat flavor and quality, and provide the theoretical basis to develop natural antioxidant for meat products.

脂肪氧化易引起酸败和风味劣变，是肉品加工和贮藏中经常面对的难题。其中高度不饱和性的磷脂是脂肪氧化主要底物，成为脂肪氧化调控的重点对象。申请者前期实验证实，热休克蛋白90（Hsp90）可与磷脂结合成稳定的绑定体，并抑制磷脂的酶解；进而又发现，该绑定体具有直接抵御外界氧化攻击的能力。为更全面阐明Hsp90抑制磷脂氧化的内在机制，本项目采用鸭肉中提取纯化的Hsp90，解析其与磷脂绑定的特点，并分别建立以自由基、非自由基、脂氧合酶等介导的体外氧化模拟体系，运用荧光共振能量转移光谱（FRET）、HPLC-MS/MS、Western Blot等手段，分析Hsp90-磷脂绑定体中Hsp90自氧化及屏蔽磷脂氧化的效价，探究Hsp90抑制磷脂氧化的途径和规律；在此基础上，开展鸭肉中添加Hsp90的贮藏试验，验证其对磷脂氧化的抑制效果。本项目成果将对肉品风味控制提供新思路，并为天然抗氧化剂开发提供理论支撑。

构建热休克蛋白90（Hsp90）的原核表达体系，对Hsp90进行分离纯化；采用表面等离子体共振（SPR）分析Hsp90与不同磷脂相互作用特性和影响因素，根据磷脂相变和流动性分析Hsp90对膜磷脂状态的影响；建立以过氧化氢自由基介导的氧化模拟体系，根据磷脂脂质体的氧化情况和形态，分析Hsp90对磷脂氧化的抑制作用；将表达Hsp90的原核细胞进行氧化，观察在细胞中Hsp90对细胞膜磷脂的保护作用；明确Hsp90与肉品品质的相关性。结果表明，Hsp90可与磷脂稳定结合，胆固醇和NaCl对相互作用有较大影响；Hsp90可降低膜磷脂的流动性，提高磷脂的相变温度；Hsp90能抑制过氧化氢介导的脂质体氧化，在原核细胞中防止细胞膜的氧化损伤；在鸡肉中Hsp90含量与保水性显著相关，高滴水损失组的Hsp90含量最低,推测Hsp90可保护细胞膜的完整性，防止水分流失。本项目成果将对天然抗氧化剂开发和肉品风味形成机理研究提供新思路。