中国科学院昆明动物研究所机构知识库

随着人们生活方式的改变和加快，肠炎、慢性疼痛和血栓的发病概率日益上升，成为困扰人们健康和影响生活质量的普遍问题。这些疾病发病因素多样而且容易反复发作，难以一次性根本治愈，然而现有的药物长期治疗存在广泛的个体差异性并具有各种显著的副作用。虽然引起这些疾病发生的原因是复杂的，但是饮食习惯是其中一个重要的因素。俗话说“病从口入”，随着生活水平的提高，健康的饮食习惯和“食疗”概念也逐渐深入人心，相关的膳食补充剂和保健品也层出不穷。探寻防范上述疾病的饮食因素，研究相关饮食因素对上述疾病的影响机理，针对上述疾病给予科学而有根据的饮食建议，发掘饮食中具有“低毒、高效”的药用和“食疗”分子具有十分重要的意义。 辣椒素和异黄酮是分别广泛存在于辣椒、胡椒等辛辣物质和各种豆类中的经常被人们大量食用的小分子化合物。本研究论文立足于生活实际，根据肠炎、慢性疼痛、血小板血栓的发病机理以及前人的临床和基础研究数据，我们发现饮食中的辣椒素非常有可能加重肠炎的发生，而异黄酮类化合物是目前市场中常见的一大类保健品，能够缓解疼痛症状，也能够预防血栓的形成。然而，尽管这些现象逐渐被人们生活中的常识所认可，但是这些化合物对其相关疾病的具体影响机理依旧不是很清楚。而且关于异黄酮化合物的作用靶点的研究也十分有限。 第一章的内容为本文研究的基础背景，分别介绍了肠道上皮通透性屏障和肠炎的关系；疼痛的信号传导和以钠离子通道为靶点的疼痛研究以及血栓的形成和抗血小板血栓的药物治疗。 在第二章中，我们发现肠炎因素可以诱导肠上皮细胞和肠道组织中辣椒素受体trpv1的表达，这暗示着trpv1可能和肠炎的发生发展相关联；随后，我们发现肠炎组织中erk激酶磷酸化水平显著升高，并且伴随着紧密连接关键蛋白occludin亚细胞分布的改变，这说明肠炎的发生是和erk通路的激活和肠道紧密连接的功能紊乱相关的。不仅如此，在肠道上皮细胞caco-2中，我们发现了辣椒素能够通过trpv1激活细胞内erk激酶信号通路，改变occludin亚细胞分布，进而改变细胞间紧密连接，破坏细胞旁通透性，从而有利于小分子和致病沙门氏菌穿透肠上皮细胞单分子层。同样的，动物实验结果也显示，辣椒素可以促进沙门氏菌诱导的小鼠肠炎，破坏小鼠肠道上皮结构，诱导炎性细胞向肠道粘膜浸润，显著降低疾病小鼠的体重并增加其死亡率。 在第三章中，我们发现在绿豆和葛根等“清凉、解毒、止痛”食物中广泛存在的3'-甲氧基大豆苷元能够强烈地抑制大鼠背根神经节电压门控钠离子电流，而背根神经节上的多种电压门控钠离子通道比如nav1.7、nav1.8、nav1.3等在背根神经节痛觉信号的传递中起到极其关键的作用。有意思的是，3'-甲氧基大豆苷元能够强烈并且有偏好性地抑制nav1.7、nav1.8、nav1.3电流，并显著地缓解由化学 (甲醛)、热 (红外辐射)、酸 (乙酸) 伤害诱导的小鼠疼痛反应。除此之外， 3'-甲氧基大豆苷元在小鼠致死性、体重和饮食、心率以及成瘾性方面没有任何副作用。通过对3'-甲氧基大豆苷元镇痛机理的研究，我们揭示了异黄酮化合物缓解疼痛的新机制，可以为慢性疼痛患者的饮食方式提供参考，也可以为开发高效、低副作用的异黄酮类镇痛药物提供借鉴。 在第四章的研究中，我们发现口服3',4',7-三羟异黄酮和3'-甲氧基大豆苷元能够显著地抑制角叉菜胶诱导的鼠尾血栓。我们随后的研究表明这种疗效的显著性可能和多种因素相关，这其中包括：3',4',7-三羟异黄酮和3'-甲氧基大豆苷元能够强烈地抑制胶原和adp诱导的血小板激活和聚集；同时它们也能抑制免疫细胞炎症因子的释放从而抑制持续的炎症；口服的异黄酮能够被迅速的吸收并在血液中维持较高含量很长时间等。不仅如此，相较于氯吡格雷能够引起血小板不可逆的功能丧失并增加出血风险，该异黄酮化合物可逆性地作用于血小板，并且不会增加出血风险。除此之外，其抗血小板聚集的机理可能是强烈的抑制pi3k/akt通路介导的血小板激活，并且抑制血小板脱颗粒和活性氧自由基产生等。虽然更为具体的分子机制探究还正在进行中，但是我们发现血小板功能重要的调节蛋白14-3-3可以与多种异黄酮化合物广泛地结合，而有意思的是，在血小板中，pi3k就是被14-3-3调节的重要的靶蛋白。我们希望能够以pi3k/akt和14-3-3为突破点，研究异黄酮作用于血小板的更为重要的机制。

Morbidity rate of enteritis, chronic pain and cardiovascular disease have been risen greatly nowadays and have significant impacts on patients’ health-related quality of life. Certain number of factors have been proved to promote the development of these diseases, such as genetic factors, diet, immunoregulatory defects and microbial exposure. These diseases are easy to recur and hard to be cured permanently. However, existing drugs for these diseases always have a wide range of individual differences and dose-limiting side-effects with a long-term medication. Diet factor is one of the factors that matter most for the development of these diseases mentioned above. With the development of the economy and the improvement of the human’s knowledge, “healthy-diet” concept has gradually become to a public recognition. Relevant dietary supplements and health care products become more and more popular. It’s important to find out effector molecules from diet that matters most for the development of these disease in order to give a reasonable recommendation on the prevention or treatment of these diseases. Capsaicin widely exists in hot and black pepper and natural isoflavones mostly exist in beans. Both of these compounds have been consumed all over the world for a long time. There are strong evidences that dietary factors such as capsaicin and isoflavone have been associated with the cause or prevention of enteritis, chronic pain and cardiovascular disease. The first chapter introduced the background of this thesis. This chapter contents: intestinal epithelial cells and its relationship with enteritis; pain signaling pathway and sodium channel targeted analgesic research; the form of thrombus and platelet- targeted medical research. In the second chapter, we found that bacterial inflammatory factors up-regulate TRPV1 expression both in intestinal epithelial cells and mouse intestinal. Besides, bacterial enteritis promotes the phosphorylation of ERK and disturbs the subcellular distribution of occludin. Capsaicin can activate ERK signaling pathway via TRPV1 activation, and thus change occludin distribution, disturb cell tight junction and elevate the paracellular permeability of intestinal epithelial cells. These facilitate the penetration of intestinal epithelial cells by bacteria and other compounds. Moreover, capsaicin promoted the enteritis triggered by salmonella, increased inflammatory cells infiltration, significantly reduced the weight and increase the mortality rate of enteritis mice. In the third chapter, we found that 3'-methoxydaidzein (3MOD) specifically and potently block voltage-gated sodium channels (VGSCs) in cultured rat dorsal root ganglion (DRG) neurons. Furthermore, 3MOD preferred to inhibit subtype Nav1.7, Nav1.8 and Nav1.3 with the estimated IC50 of 181, 397 and 505 nM, respectively. Interestingly, all these three sodium channel subtypes are considered to be potential targets for pain therapeutics. Thereafter, we proved that 3MOD is a potent analgesic in chemical, thermal and acid induced rodent pain models compared with morphine. Additionally, it had little side effects on heart rhythm, weight gain and drug addiction. For the first time, we identified that 3MOD, an isoflavone, could be a potent pain-relief molecule through inhibiting VGSC currents. This study reveals a new potential pain-killing mechanism of isoflavones which might be promising compounds or parent structures for the treatment of human chronic pain. In the last chapter, we introduced two isoflavonoids 3MOD and 3',4',7-trihydroxyisoflavone (THO), which can strongly inhibit platelet-aggregation evoked by ADP and collagen and thus exert remarkable antithrombotic activities induced by carrageenan. Subsequent research had shown that 3MOD and THO also inhibited the secretion of inflammatory factors. Oral-administrated 3MOD and THO absorbed quickly and had a high retention rate in the blood for a long time. Despite their excellent antithrombotic activities, 3MOD and THO will not increase the risk of bleeding in different animal models. What’s more, THO can inhibit PI3K agonist induced platelet-aggregation and the phosphorylation of AKT1 as well. Besides, isoflavone can bind to 14-3-3, which is an important regulator of PI3K in platelets.