基于HIF-1a/NF-kB信号通路的藏药麻花秦艽抗高原缺氧物质基础及作用机制研究

Altitude sickness seriously affects people's health, and Hypoxia is the main factor to induce acute or chronic altitude sickness, therefore looking for effective plateau hypoxia drugs has become one of the highlights in agro-scientific research of the altitude sickness. Tibentan Gentiana straminea is a characteristic plant of Qinghai-tibet plateau in China and it has a glorious history as the medicine. The research group found thatalcohol extracted from Tibentan Gentiana straminea shows obvious anti-hypoxia and anti-oxidation effects in the previous research. On this basis, we hope to study the anti-hypoxia effect and the possible mechanism to find out the effective component of Tibentan Gentiana straminea on anoxia by continuing this research. We identify anti-hypoxia ingredients extracted from Tibentan Gentiana straminea and find out the possible mechanism of its anti-hypoxia actions by simulating plateau hypoxia mice model and hypoxic cells in vitro at the same time. we study anti-hypoxia actions of Tibentan Gentiana straminea in the hypoxia mice models and explore it's possible mechanism from 3 aspects of morphologic, oxidative stress and inflammatory factor of pharmacological and biochemical indexes. We also investigate the anti-hypoxia effects of Tibentan Gentianas traminea from molecular biological detection. Western-blot and RT-PCR are used to study expression of HIF-1 and NF- kB mRNA in two cell lines and the protein expression is also examined. The effects of the overall animal model, cells, molecules, proteins levels reveal the hypoxia HIF-1a/NF-kB inflammatory key signaling pathways of regulation and control function of Tibentan Gentiana straminea. The experimental results provide theoretical references and drug targets for the Tibentan Gentiana straminea used in the prevention and control of clinical plateau hypoxia disease.

高原病严重影响人们的身体健康，高原低压缺氧是诱发急慢性高原病的主要因素，寻找有效抗高原缺氧药物成为高原病的研究热点之一。麻花秦艽是我国青藏高原特色植物，是藏医药中重要的常用药材。课题组前期发现麻花秦艽醇提物具有抗缺氧作用，本研究在此基础上，拟对其抗高原缺氧物质基础及作用做进一步研究。通过模拟高原缺氧小鼠模型和体外缺氧细胞模型筛选抗缺氧活性成分，明确其药效物质基础，同时结合对模拟高原缺氧小鼠缺氧组织的形态、氧化应激反应、炎症因子等多方面药理和生化指标的影响，从整体动物模型、细胞、分子、蛋白等多方位研究麻花秦艽抗缺氧作用并揭示其抗氧化应激及炎性机制，以及对HIF-1a及NF-kB mRNA、蛋白的表达，分析其对缺氧细胞保护的HIF-1a/NF-kB的炎性关键信号通路的调控作用。为麻花秦艽用于治疗高原缺氧疾病的开发奠定科学依据，为临床高原缺氧疾病新的防治策略提供理论依据与药物靶点。

麻花秦艽(Gentiana straminea Maxim) 为龙胆科(Gentianaceae)龙胆属多年生草本植物，藏医称“解吉嘎保”，是我国常用藏药材资源之一。本课题组在前期研究发现麻花秦艽具有抗缺氧新用途，本项目在此基础上探讨麻花秦艽对急性高原缺氧损伤的保护作用并揭示其可能机制及物质基础。.本项目首先采用常压缺氧和低氧高原缺氧试验验证西藏麻花秦艽抗缺氧作用，并通过常压缺氧动物模型和PC12细胞建立物理缺氧和化学缺氧模型，从体内外筛选了麻花秦艽抗缺氧作用的部位，并对该部位进行了分离、纯化和结构解析，再分别从细胞水平和整体动物水平对麻花秦艽的抗缺氧作用及其机制进行研究。.筛选实验结果证实西藏麻花秦艽具有抗缺氧和抗氧化作用，其活性部位主要是乙酸乙酯部位，通过HPLC初步验证了乙酸乙酯和正丁醇两个部位均含有环烯醚萜类化合物，并建立了4种环烯醚萜苷类化合物含量测定的HPLC方法，运用色谱及高分辨率质谱手段对麻花秦艽抗缺氧有效部位的成分进行分析鉴定，确定了乙酸乙酯部位含有龙胆二糖、马钱苷酸等20种化合物成分。.动物和细胞试验结果表明，麻花秦艽乙酸乙酯部位抗缺氧作用是多靶点的，具可升高SOD、CAT、GSH-PX活力，降低MDA、H2O2、NO、LD、LDH水平，对DPPH、•OH、•O等自由基均有一定清除能力，可升高ATP和Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性，降低炎症因子IL-6、IL-1β、TNF-α含量，降低Hif-1a、NF-KB mRNA及蛋白表达水平，升高Bcl-2以及Bcl-2/Bax水平，通过提高抗氧化应激能力、调节能量代谢、抗炎、抗凋亡等多方面发挥抗缺氧作用，初步揭示可能通过NF-KB/HIF-1α调控其抗缺氧作用。.本课题将为麻花秦艽用于治疗高原缺氧疾病的开发奠定科学依据，为临床高原缺氧疾病新的防治策略提供理论依据与药物靶点，为开发藏药麻花秦艽的新用途提供思路。