调节自噬的活性物质筛选及抗肿瘤和糖尿病心肌病的作用及机制

发布时间：2018-05-06 23:34

  本文选题：自噬 + 自噬流　； 参考：《北京协和医学院》2015年博士论文

【摘要】：自噬是细胞内的一种分解代谢过程,用于维持细胞内物质及能量的稳态。正常生理状态下,自噬常维持在基础水平,而在肿瘤、糖尿病、炎症及神经退行性疾病等多种病理状态下往往都伴随有自噬活性的改变。实验室前期研究也证实肿瘤、纤维化疾病及代谢综合症中均存在自噬活性的失调,我们认为,筛选不同活性的自噬调节剂应用于以上疾病模型具有治疗意义。因此,我们建立了自噬活性物质筛选系统,从具有结构及功能多样性的天然产物化合物库中筛选到多个自噬激动剂及自噬抑制剂,其中以XY1为代表的黄烷类化合物及以ZH33为代表的二氢黄酮类化合物能够明显激活自噬流。较强的自噬激活能够导致自噬性细胞死亡,而诱导肿瘤细胞发生自噬性细胞死亡是克服凋亡耐受的有效途径。自噬激动剂XY1及ZH33均具有抗肿瘤活性。XY1对小鼠黑色素瘤细胞B16-F10具有较强的抑制作用,而对小鼠AML-12正常肝细胞影响较小。进一步分子机制的研究发现,XY1可以选择性增加肿瘤细胞内ROS水平,对正常细胞无影响。利用NAC阻断细胞内ROS的产生及敲除Atg5抑制细胞自噬活性均可以减少XY1诱导的肿瘤细胞凋亡,说明XY1的抗肿瘤活性是自噬激活及ROS增加的共同作用结果。动物实验也表明,XY1可以抑制小鼠黑色素瘤的生长,激活肿瘤组织自噬,选择性诱导肿瘤组织ROS堆积。ZH33对人肝癌细胞Hepg2具有一定的抑制作用,分子机制研究结果显示ZH33也可以增加肿瘤细胞内ROS水平,诱导肿瘤细胞发生非凋亡依赖的自噬性细胞死亡。糖尿病是严重威胁人类健康的代谢紊乱疾病,而糖尿病心肌病作为糖尿病的主要并发症,是导致糖尿病患者死亡的首要原因。自噬作为一种物质及能量代谢过程与糖尿病的发生发展密切相关,目前对于自噬在2型糖尿病心肌病中的作用仍不明了。本研究利用高脂膳食合并STZ方法制备小鼠2型糖尿病心肌病模型,并从自噬流的角度发现2型糖尿病心肌病中自噬过度活化,表现为通过上游PI3K/AKT-mTOR及AMPK-mTOR信号通路激活自噬,自噬核心复合物PI3KC3、 Beclinl表达增加,自噬标志蛋白LC3-Ⅱ增加,自噬货车蛋白P62、NBR1及NIX下降。ERBB信号通路对于维持心肌细胞正常生长及损伤修复非常重要,我们发现伴随着心肌细胞自噬活化,ERBB家族蛋白降解增加导致ERBB信号通路受损。综上,利用自噬活性物质筛选系统我们筛选到了多种自噬激动剂及抑制剂,为自噬相关疾病的治疗提供了物质基础。一方面,我们通过对XY1及ZH33这两种自噬激动剂抗肿瘤作用及分子机制的研究,为自噬激动剂应用于抗肿瘤药物开发提供了理论依据。另一方面,我们通过阐明自噬激活促进糖尿病心肌病发生发展的分子机制,为自噬抑制剂应用于糖尿病心肌病治疗提供了新的理论依据。
[Abstract]:Autophagy is a catabolism process in cells, which is used to maintain the homeostasis of substance and energy in cells. In normal physiological state autophagy is often maintained at the basic level while autophagy is often accompanied by changes in autophagy in various pathological states such as tumor diabetes inflammation and neurodegenerative diseases. Previous laboratory studies have also confirmed that autophagy disorders exist in tumors, fibrotic diseases and metabolic syndrome. We believe that screening different active autophagy modulators for the above disease models has therapeutic significance. Therefore, we have established a screening system for autophagy active substances. A number of autophagy agonists and autophagy inhibitors have been screened from natural product compounds with structural and functional diversity. Among them, flavanides represented by XY1 and dihydroflavonoids represented by ZH33 could activate autophagy obviously. Strong autophagy activation can lead to autophagy cell death, and inducing autophagic cell death is an effective way to overcome apoptosis tolerance. Both autophagy agonists XY1 and ZH33 have anti-tumor activity. XY1 has a strong inhibitory effect on B16-F10 of mouse melanoma cells, but has little effect on normal hepatocytes of mice AML-12. Further molecular studies showed that XY1 could selectively increase the level of ROS in tumor cells, but had no effect on normal cells. Blocking the production of ROS and knockout of Atg5 by NAC can reduce the apoptosis of tumor cells induced by XY1, indicating that the anti-tumor activity of XY1 is the result of both autophagy activation and ROS increase. Animal experiments also showed that XY1 could inhibit the growth of murine melanoma, activate tumor autophagy, and selectively induce tumor tissue ROS accumulation. ZH33 could inhibit the Hepg2 of human hepatoma cells. The results of molecular mechanism showed that ZH33 could also increase the level of ROS in tumor cells and induce apoptosis dependent autophagy cell death. Diabetes mellitus (DM) is a metabolic disorder that threatens human health seriously. As a major complication of diabetes, diabetic cardiomyopathy (DM) is the leading cause of death in diabetic patients. Autophagy as a metabolic process of substance and energy is closely related to the occurrence and development of diabetes mellitus. The role of autophagy in type 2 diabetic cardiomyopathy is still unclear. In this study, the model of type 2 diabetic cardiomyopathy in mice was established by high fat diet combined with STZ method. From the point of view of autophagy, it was found that autophagy was activated by upstream PI3K/AKT-mTOR and AMPK-mTOR signaling pathway in type 2 diabetic cardiomyopathy. The expression of PI3KC3, Beclinl and LC3- 鈪，

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