谷胱甘肽调控微囊藻毒素生物毒性的作用机制与可行性研究

Evaluation and regulation on the biological toxicity effect of Microcystins (MCs) was an important guarantee of drinking water safety. This research focused on the regulatory mechanism and regulation effects of glutathione (GSH) on the biological toxicity of MCs. By investigating the regulatory processes of GSH on protein phosphatase activity (inhibited by MCs) and oxidative stress effect (stimulated by MCs), the biological toxicity of MCs was effectively controlled by GSH. Firstly, the regulating effect of MCs and MC-GSH was investigate in combination with enzyme activity experiment and electrospray mass spectrometry/molecular simulation technology, and the molecular mechanism of MC-GSH transformation to regulate the inhibition toxicity of MCs on protein phosphatases was clarified. Then the enzyme activity of protein phosphatases in liver cells treated with MCs, MC-GSH, GSH+MCs was compared, and the regulation effect of MC-GSH pathway on the toxicity of MCs targeted to protein phosphatases was verified. Meanwhile, the regulatory mechanisms on intracellular oxidative stress effect by GSH directly inhibit pathway and MC-GSH/protein phosphatases pathway were also explored. Finally, our research achievements on GSH regulation at molecular and cellular levels was used to intervene the toxicity effect of MCs contaminated animals, and the feasibility of GSH regulation on toxic MCs was discussed in-depth.

微囊藻毒素(MCs)生物毒性效应的评价与调控是饮用水安全的重要保障。本课题拟以谷胱甘肽(GSH)调控MCs生物毒性的研究为契入点,在深入考察GSH调节MCs抑制蛋白磷酸酶（PPs）活性和抑制氧化应激效应作用机制的基础上实现对MCs毒理过程的有效调控:首先结合酶活性实验与电喷雾质谱/分子模拟技术考察MC-GSH转化对MCs抑制PPs致毒过程的调节效果,阐明GSH调控MCs抑制PPs毒性的分子机制;然后对比MCs、MC-GSH、GSH+MCs作用下肝细胞内PPs酶活性的变化规律,验证MC-GSH转化途径调控MCs抑制PPs毒性的有效性,并探明MC-GSH调节PPs活性途径和GSH直接抑制途径对胞内氧化应激效应的调控机制;最后将分子、细胞水平上关于GSH调控MCs生物毒性作用机制的成果用于MCs染毒动物生物毒性效应的干预研究，深入探讨通过外源性GSH调控MCs生物毒性的可行性。

与MCs的源消减调控策略和针对MCs迁移过程的调控策略相比，关于MCs生物毒性作用及其调控机制的研究值得关注。本课题以GSH调控MCs生物毒性作用的研究为契入点,在考察GSH抑制氧化应激效应和调节MCs抑制PPs活性效应具体机制的基础上实现对MCs毒理过程的有效调控。研究通过模拟GSH(biothiols)与MCs的亲电反应生成MC-GSH(MC-biothiols)结合产物并借鉴MCs的制备工艺实现MC-GSH(MC-biothiols)的纯化制备，以满足后续分子、细胞、水平的MCs毒性评价与调控研究。在产物色谱制备的基础上,对比MCs、MC-GSH对PP1酶活性直接抑制作用差异，明确MC-GSHs转化途径对MCs抑制PP1致毒过程的调控效果，为后续细胞水平的毒性实验提供依据；同时借助分子模拟技术考察MCs、MC-GSH与PP1相互作用的活性位点、作用模式以及作用强度差异，深入阐明转化产物MC-GSH调控MCs抑制PP1毒性的分子作用机制。在分子水平研究的基础上，通过考察MCLR、GSH+MCLR、MCLR-GSH作用下肝细胞PPs酶活性的变化趋势，验证GSH转化途径调控MCLR抑制PPs毒性的有效性；同时明确GSH直接抑制途径和MC-GSH调节PPs活性途径对肝细胞氧化应激效应调控的贡献程度和机制。最后将分子、细胞毒性实验关于GSH调控MCs生物毒性作用机制的研究成果应用于biothiols调控MCLR毒性的效果研究，探讨通过合理补充外源性biothiols进一步调控MCs生物毒性的可行性。本研究有利于削弱MCs的生物毒性和环境风险，同时完善了对MCs调控策略的认知，因而具有重要的理论和实际应用价值。