CBS/H2S体系：帕金森病防治的新靶点

Hydrogen sulfide (H2S) has been proposed as a novel neuromodulator which plays critical roles in the central nervous system. Endogenous H2S is synthesized mainly by cystathionine-beta-synthase (CBS) in the brain. Through the establishment of PD cell model induced by MPP+ in primary cultured dopamine neurons and PD rat model induced by intraventricular point injection 6-hydroxydopamine (6-OHDA), this study is to observe the oxidative stress, the production of H2S and the activity of cystathionine-bet-synthase (CBS) in the primary cultured dopamine neurons or the Midbrain substantia nigra (SN) of affected side of PD rats. Tool agent such as CBS agonist specificity or inhibitors and Molecular biology techniques such as RNA interference (RNAi) or gene transfer techniques are applicated, which change the production of endogenous H2S by influencing the CBS activity or expression. To define the relationship of CBS / H2S system and Parkinson's disease, the effect of endogenous H2S on Parkinson's disease cell model and animal model is measured. This Study will illustrate the significance of CBS/H2S system on the prevention and treatment of Parkinson's disease. It will lay scientific experimental basis on increasing the content of H2S for PD control strategy or using CBS / H2S system for PD prevention and cure target in the future.

H2S是一种新的神经活性物质，在中枢神经系统中具有重要的调节作用。脑内H2S主要依赖于含硫氨基酸在胱硫醚-β-合酶代谢下生成。该课题拟通过建立MPP+ 损伤原代培养的多巴胺神经元的帕金森病（PD）细胞模型以及脑内注射6-OHDA 制备的PD大鼠模型，观察原代多巴胺神经元内或PD大鼠患侧中脑黒质组织的氧化应激损伤与H2S含量、CBS的活性及表达；应用 CBS的特异性激动剂或抑制剂等工具药以及RNA干涉（RNAi）或基因转染等分子生物学技术，通过改变CBS的活性或表达来改变内源性H2S的生成，探讨内源性H2S对帕金森病细胞模型和动物模型的影响，进一步揭示CBS/H2S体系与帕金森病的相互关系。该课题拟阐明CBS/H2S体系在防治帕金森病中的意义，为将来以增加脑内H2S含量为PD的防治策略，以CBS/H2S体系为PD的防治靶点奠定科学实验依据。

1. 用MPP+等损伤PC12细胞建立帕金森病的细胞模型，研究发现，MPP+降低PC12细胞的活性，促进PC12细胞的凋亡，抑制细胞线粒体膜电位，增加细胞ROS的产生；在抑制PC12细胞活性的同时，能降低细胞内源性H2S的生成，降低H2S合成酶CBS基因和蛋白的表达；CBS抑制剂AOAA能降低H2S的生成，加重MPP+对PC12细胞的毒性作用，进一步降低PC12线粒体膜电位水平、 促进细胞ROS的积累；而外源性H2S则能降低MPP+对PC12细胞的的毒性作用；2、合成针对CBS的siRNA并用慢病毒包装，转染PC12细胞；CBS基因沉默以后，CBS基因和蛋白表达明显降低；CBS基因沉默能明显降低内源性H2S的生成；CBS基因沉默后能进一步加重MPP+对PC12活性的抑制作用，促进PC12细胞的凋亡；CBS基因沉默后能进一步降低PC12细胞胞的线粒体膜电位，促进活性氧的积累；外源性H2S能够干预CBS基因沉默对细胞的作用。3、构建过表达CBS基因慢病毒表达载体，转染PC12细胞，并用嘌呤霉素筛选，构建稳定表达CBS的PC12细胞株；过表达CBS基因的PC12细胞其CBS基因和蛋白的表达明显升高；过表达CBS基因后能升高内源性H2S的生成；过表达CBS基因后能减轻MPP+对PC12活性的抑制作用，抑制PC12细胞的凋亡；过表达CBS基因后能改善MPP+降低PC12细胞的线粒体膜电位作用，减轻活性氧的积累。以上研究初步探讨了CBS/H2S体系与帕金森病模型的关系，CBS/H2S可能作为抗帕金森病的一个潜在靶点。