气体信号分子硫化氢对阿尔茨海默病淀粉样前体蛋白代谢调控的研究

Endogenously generated hydrogen sulfide (H2S), a novel gaseous messenger and serves as an important neuromodulator in the central nervous system, may have multiple functions in brain and play important roles both in physiology and in disease. .In previous study, we had showed that the levels of H2S was severely decreased in the serum of Alzheimer's disease (AD) patients compared with the brains of the age matched normal individuals, and homocysteine obviously increased, and decreased the levels of phosphorylation of Akt and ERK1/2 in the downstream of the phosphatidylinositol 3-kinase(PI3-K) pathway. The β-site amyloid precursor protein cleaving enzyme 1(BACE-1) protein levels and β-amyloid peptides (Aβ) were significantly increased, and both decreased when given the sodium hydrosulfide (NaHS) or insulin or Insulin-like growth factor 1(IGF-1)..In this study, to explore the H2S modulated effect for APP metabolic pathway, active key target, we'll hold of the key pathogenesis of AD which abnormal metabolism of amyloid precursor protein(APP) and Aβ overly accumulate. Here, we'll investigate the levels of H2S in plasma, hippocampus and cortex with cultured neuron, normal SD rats and APPswe/PS1dE9 transgenic mice by sensitive sulphur electrode assay. To measure and analyze the Aβ,APP,BACE1,γ-secretase and mRNA with western blot and QPCR analysis. Then measure these markers again after exogenously applied H2S,CBS gene silencing and high expression. We hope to gain a new idea and methods in preventative and treatment for AD.

内源性气体信号分子硫化氢（H2S）参与了多种疾病的发病机制。我们前期实验显示AD患者血浆H2S含量降低,给予外源性H2S供体NaHS、胰岛素和胰岛素样生长因子后BACE1和Aβ表达均显著降低。本研究围绕AD淀粉样前体蛋白（APP）代谢异常致不溶性Aβ过度生成与沉积的核心发病机制,以体外培养的原代神经细胞，正常成年SD大鼠和APPswe/PS1dE9双转基因AD模型鼠为研究对象，进行体内、体外试验，用敏感硫电极法检测血液、海马和脑皮质H2S水平；荧光定量PCR/Western blot/ELISA技术等检测血液、海马神经元Aβ、APP、BACE1、γ-分泌酶等蛋白或mRNA表达、APP/Aβ代谢通路蛋白等表达水平；给予NaHS、CBS基因沉默和高表达等方法后检测前述各项指标的变化和学习记忆功能等。旨在探索和揭示内源性H2S对APP代谢的调控作用、机制和作用靶点,为AD的防治提供新的思路。

内源性硫化氢（H2S）参与了多种疾病的发病机制。我们前期实验显示AD患者血浆H2S含量降低。AD模型鼠血液中H2S含量显著降低，补充H2S供体后Aβ显著降低，认知行为功能显著改善。.本研究围绕AD淀粉样前体蛋白（APP）代谢异常致不溶性Aβ过度生成与沉积的核心发病机制进行了系统的体外和在体试验。结果显示：.1. 加入100μmol/L H2S供体NaHS时体外培养PC12细胞的存活率最高，500μmol/L以上剂量对细胞毒性大；10~200μmol/L各NaHS组的α分泌酶核心蛋白ADAM10和ADAM17蛋白表达水平上调，BACE1 和BACE1 mRNA表达均降低，Aβ40、Aβ42和sAPPβ蛋白表达降低，而sAPPα蛋白表达增高。.2. 加入5~20μmol/L 浓度NaHS时神经元存活率最高；30μmol/L以上NaHS组神经元活力降低、凋亡增加。低于30μmol/L浓度NaHS组，神经元中Aβ1-42、BACE-1 mRNA、PS1 mRNA水平显著降低（P<0.05）。添加PD98059和LY29002后检测BACE-1和PS1蛋白表达，仅PD98059组明显降低。提示H2S调节神经元和PC12细胞APP代谢可能通过PI3K/Akt通路。.3. APP/PS1转基因小鼠显示随年龄增长认知功能下降。6月龄时CSE、CBS、3MST酶活性降低，神经元中BACE1和PS1显著增加，Aβ蛋白过度表达。大脑皮质、海马及血浆中H2S水平逐渐降低。12月龄时严重下降(p<0.05)。3MST主要位于神经元线粒体中。给予NaHS后模型鼠视空间学习和记忆力改善、细胞外Aβ4\BACE1和PS1显著减少，ADAM17 显著增加。.4. APP/PS1转基因小鼠神经元和N2a细胞均见线粒体分裂融合异常。Drp1和Drp1mRNA、Fis1表达水平显着增加，Aβ40和Aβ42显著增高（P<0.01）。线粒体呼吸链复合物活性I~IV均降低。NaHS和AP39干预后呼吸链复合物活性明显增加，Drp1表达明显升高，Fis1蛋白水平显著降低，线粒体分裂明显减少，ATP产量增加，ROS水平，Aβ40和Aβ42显著降低。.科学意义：本研究揭示脑内H2S含量降低参与了AD发病，为开发防治AD的药物提供了实验依据，也为AD的线粒体损伤学说等研究提供了可用模型和方法，有较广泛的应用前景。