【作者】 杨光；

【导师】 唐煌；

【作者基本信息】 广西师范大学 ， 生物与医药， 2023， 硕士

【摘要】 铜离子与Aβ蛋白形成可溶性的Aβ寡聚体（oAβ）,破坏细胞内的钙平衡,损伤细胞的线粒体,加速促炎因子的分泌,降低神经胶质细胞的吞噬能力,导致活性氧（ROS）的升高,细胞极化向M1型转变,最终导致神经元细胞凋亡。因此调节Cu²⁺介导的oAβ的解离是未来抗阿尔茨海默病药物研究的一个新热点。氮杂蒽醌类生物碱本身就具有广泛的生物活性,近期课题组合成了多种氮杂蒽醌类生物碱的衍生物,其中1,6-二氮杂蒽醌衍生物具有较好的抗胆碱酯酶活性。本论文选择其中毒性较小,对Cu²⁺的螯合能力较强的化合物6作为研究对象,研究其调节Cu²⁺-Aβ复合物介导的神经毒性、细胞极化和氧化应激的活性与机制。其主要研究内容包括:1.通过测定Aβ42转基因秀丽隐杆线虫（CL2120）的瘫痪率和APP/PS1双转基因小鼠的行为能力来评价化合物对Cu²⁺-Aβ复合物介导的神经毒性的抑制能力;2.通过蛋白免疫印迹实验来检测化合物和Cu²⁺干预后转基因线虫和转基因小鼠体内oAβ的表达;并使用透射电镜在体外观察不同条件下Aβ42的形态变化;3.利用DCFH-DA染色测定化合物对Cu²⁺介导后转基因线虫体内ROS水平的影响;4.利用ELISA试剂盒测定化合物对Cu²⁺-Aβ复合物刺激的BV2细胞产生NO,TNF-α,IL-1β,IL-6等促炎因子和TGF-β等抗炎因子的影响;利用PE标记的抗体通过流式细胞仪测定化合物对Cu²⁺-Aβ复合物刺激的BV2细胞产生的细胞极化标志物CD206和CD86的影响;5.利用FITC和PI染色和流式细胞仪测试化合物对Cu²⁺-Aβ复合物介导的BV2细胞凋亡的抑制能力和吞噬能力的影响,并通过WB实验测定BV2细胞的髓系细胞触发受体2（TREM-2）的表达水平变化。实验结果表明:化合物6能通过抑制Cu²⁺-Aβ复合物的形成来降低Aβ42转基因线虫的瘫痪率,恢复转基因小鼠的学习能力并且抑制Aβ42转基因线虫体内ROS的分泌;而且效果明显优于先导化合物5。同时化合物能够通过调节极化蛋白的表达,抑制Cu²⁺-Aβ复合物介导的胶质细胞分泌NO,TNF-α,IL-1β,IL-6等促炎因子,促进TGF-β等抗炎因子的分泌;最后,化合物6能够通过促进TREM-2蛋白的表达来增强胶质细胞的吞噬能力,使胶质细胞由表型M1向M2转变。总之,通过对先导化合物5进行结构修饰,引入含氮侧链基团,极大增强了衍生物抑制Cu²⁺-Aβ复合物的形成能力,同时衍生物还表现出更强的缓解Cu²⁺-Aβ复合物介导的毒性、氧化应激和加速胶质细胞极化的活性。更多还原

【Abstract】 The amyloidβ-protein（Aβ）form soluble Aβoligomers（oAβ）with copper ions which disrupts the calcium balance in cells,damage the mitochondria of cells,accelerates the secretion of proinflammatory factors,and reduces the phagocytic capacity of glia cells,leading to the increase of reactive oxygen species（ROS）,cell polarization to M1 type transformation,and eventually leading to neuronal cell apoptosis.Therefore,regulating Cu²⁺-mediated dissociation of oAβis a new hotspot for future anti-Alzheimer’s drug studies.Azanthraquinone alkaloids have a wide range of biological activities.Recently,our team has synthesized a variety of derivatives of azanthraquinone alkaloids,among which 1,6-diazazanthraquinone derivatives have good anticholinesterase activity.In this paper,compound 6 with less toxicity and strong chelating ability for Cu²⁺was selected to study the activity and mechanism of regulating Cu²⁺-Aβcomplex mediated neurotoxicity,cell polarization and oxidative stress.The main research contents include:1.The ability of the compound to inhibit the Cu²⁺-Aβcomplex mediated neurotoxicity was evaluated by determining the paralysis rate of Aβ42 transgenic C.elegans（CL2120）and the behavior ability of APP/PS1 double transgenic mice;2.Protein immunoblotting experiments were performed to examine oAβ expression in transgenic nematodes and transgenic mice after compound and Cu²⁺intervention.The morphological changes of Aβ42 under different conditions were observed using transmission electron microscopy in vitro.3.The effect of compound on ROS levels in transgenic nematodes after intervening with Cu²⁺was determined by DCFH-DA staining.4.The effects of the compound on the production of proinflammatory factors（NO,TNF-α,IL-1βand IL-6）,and anti-inflammatory factors（TGF-β）in Cu²⁺-Aβ complex mediated BV2 cells were determined by the ELISA kit.The effects of the compound on the production of the cell polarization markers CD206 and CD86 in Cu²⁺-Aβcomplex mediated BV2 cells were investigated by flow cytometry using PE-labeled antibodies.5.The effects of the compound on the inhibitory capacity of apoptosis and phagocytic capacity in Cu²⁺-Aβcomplex mediated BV2 cells were tested by FITC and PI staining and flow cytometry.Changes in the expression levels of triggering receptor expressed on myeloid cells-2（TREM-2）in BV2 cells were also determined by WB experiments.The experimental results showed that compound 6 can reduce the paralysis rate of Aβ42 transgenic nematodes,restore the learning ability of transgenic mice and inhibit the secretion of ROS in the body of Aβ42 transgenic nematodes by inhibiting the formation of Cu²⁺-Aβcomplex.Moreover,the effects of compound 6 were significantly better than that of the lead compound 5.Meanwhile,the compound could inhibit the secretion of pro-inflammatory factors（NO,TNF-α,IL-1βand IL-6）and promote the secretion of anti-inflammatory factors（TGF-β）mediated by Cu²⁺-Aβcomplex by regulating the expression of polarized proteins in glial cells.Finally,compound 6 was able to enhance glial phagocytosis by promoting TREM-2 protein expression.It altered glial cells from the phenotype M1 to M2.In conclusion,the structural modification of the lead compound 5 greatly enhanced the ability of the derivative to inhibit the formation of the Cu²⁺-Aβcomplex.Meanwhile,the derivative also showed a stronger activity to alleviate Cu²⁺-Aβcomplex-mediated toxicity,oxidative stress and accelerate glial cells polarization.更多还原