【作者】 王燕妮；

【导师】 赵全义； 贺殿；

【作者基本信息】 兰州大学 ， 药学， 2022， 硕士

【摘要】 胆汁酸（BAs）是经由肝脏中胆固醇合成的一种内源性的甾体化合物,主要存在于哺乳动物的胆汁中。BAs参与多种生理过程,作为信号分子调节细胞内葡萄糖、脂质和能量的代谢,参与免疫反应等。因其种类繁多、具有两亲性以及强大的生物学功能,吸引着许多国内外学者尝试以BAs为先导化合物进行新药研究。一些胆汁酸及其衍生物已作为临床上治疗胆汁淤积性肝病的药物,众多研究表明胆汁酸衍生物具有抗肿瘤、抗炎等多种药理活性,这提示我们以胆汁酸为先导化合物的结构修饰具有巨大潜力。基于新药设计策略,本课题尝试从胆汁酸骨架出发,将一些活性小分子或其类似物与胆酸、鹅去氧胆酸、熊去氧胆酸、石胆酸的24位羧基反应得到了未见文献报道的系列胆汁酸衍生物,通过¹H NMR、¹³C NMR和MS进行表征,并对其进行了体外抗肿瘤和抗炎活性研究。研究结果表明,20个胆汁酸衍生物对5株人结肠癌细胞系HCT116、HT-29、RKO、LOVO、SW480、人胃癌细胞系MGC-803、人肺癌细胞系A549以及2株人肝癌细胞系Hu H7、SK都有显著的细胞增殖抑制活性,尤其对结肠癌细胞抑制活性最优,据此,选用对结肠癌细胞HCT116抑制活性较好的胆汁酸衍生物4、5、6、7、15、16(IC₅₀在21.32-28.90μM之间)探索了该类胆汁酸衍生物的抗肿瘤机制。首先,细胞克隆形成实验表明六个胆汁酸衍生物均可以有效抑制HCT116细胞集落的形成,Transwell实验显示六个胆汁酸衍生物能够抑制HCT116细胞的迁移和侵袭过程;其次,通过姬姆萨染色、DAPI染色以及流式细胞术,发现六个胆汁酸衍生物均可以诱导HCT116细胞在晚期发生凋亡,并将HCT116细胞的有丝分裂过程阻滞在细胞周期的G₂/M期;第三,六个化合物还可以通过降低HCT116细胞的线粒体膜电位、提高细胞内ROS水平、降低抗凋亡蛋白Bcl-2以及增加活化的凋亡蛋白Cleaved Caspase 3的表达来诱导HCT116细胞凋亡。另外一方面,通过脂多糖（LPS）刺激的RAW264.7细胞炎症模型筛选了这20个胆汁酸衍生物的抗炎活性,研究表明含3,5-二酚羟基结构的化合物10和18可以有效抑制RAW264.7细胞中促炎介质NO、TNF-α的表达,具有较强的抗炎活性。综上,本课题的研究为胆汁酸衍生物候选药物的发现提供了一些研究基础和实验依据。更多还原

【Abstract】 Bile acids（BAs）are endogenous steroids synthesized from cholesterol in liver,which mainly exists in bile of mammals.BAs are involved in a variety of physiological processes,as signaling molecules to regulate the metabolism of glucose,lipids and energy in cells,and to participate in immune responses.Many domestic and foreign researchers have been attracted to try to use BAs as lead compounds to design and synthesize new drugs due to their wide range of amphipathic and powerful biological functions.Some bile acids and their derivatives have been used as clinical drugs to treat cholestatic liver disease.Numerous studies have shown that bile acids derivatives have various pharmacological activities such as antitumor,anti-inflammatory.It is suggested that structural modifications in which bile acids are lead compounds have great potential.Based on the drug design strategy,this project attempts to start from the bile acid skeleton,some active small molecules or their analogues react with the 24-carboxyl group of cholic acid,chenodeoxycholic acid,ursodeoxycholic acid,and lithocholic acid to obtain a series of bile acid derivatives.These derivatives have not been reported in literature,and were characterized by ¹H NMR,¹³C NMR and MS.Their antitumor and anti-inflammatory activities were studied in vitro.The results showed that twenty bile acid derivatives had significant inhibitory activity against five human colon cancer cell lines HCT116,HT-29,RKO,LOVO,SW480,human gastric cancer cell lines MGC-803,human lung cancer cell lines A549,and two human liver cancer cell lines Hu H7,SK,especially for colon cancer cell lines.Accordingly,bile acid derivatives 4,5,6,7,15,16(IC₅₀ ranged from 21.32μM to 28.90μM)with better inhibitory activity on colon cancer cell lines HCT116 were selected to explore the antitumor mechanism of these bile acid derivatives.Firstly,the plate clony formation assay showed that the six bile acid derivatives could effectively inhibit the formation of HCT116 cells colonies.Transwell experiments showed that the six bile acid derivatives inhibited the migration and invasion process of HCT116 cells.Secondly,by giemsa staining,DAPI staining and flow cytometry,it was found that six bile acid derivatives could induce the late process of apoptosis in HCT116 cells and block the mitotic process of HCT116 cells in G₂/M phase of the cell cycle.Thirdly,six compounds could also induce HCT116 cells apoptosis by reducing the mitochondrial membrane potential of HCT116 cells,increasing the level of intracellular ROS,reducing the anti-apoptotic protein Bcl-2 and increasing the expression of the activated apoptotic protein Cleaved Caspase 3.On the other hand,the anti-inflammatory activities of 20 bile acid derivatives were screened by lipopolysaccharide（LPS）-stimulated RAW264.7 cells inflammation model.Compounds 10 and 18 with 3,5-diphenol hydroxyl structure were found to effectively inhibit the expression of pro-inflammatory mediators NO and TNF-αin RAW264.7cells,indicating strong anti-inflammatory activity.In conclusion,the research of this project provides some research basis and experimental basis for the discovery of bile acid derivatives as candidate drugs.更多还原