嘎罗曼麦丸抗高原红细胞增多症的药效物质基础及作用机制研究

Pharmacodynamic Material Basis and Mechanism of Galuo manmai pill against High Altitude Polycythemia

中文关键词:嘎罗曼麦丸  高原红细胞增多症  HIF-1  谱效关系

keywords:Galuo Manmai Pill  High Altitude Polycythemia  HIF-1  spectrum-effect relationship

中文摘要:目的 分析嘎罗曼麦丸抗高原红细胞增多症的药效物质基础及作用机制。方法 采用低压氧环境控制系统联合腹腔注射氯化钴诱导建立高原红细胞增多症大鼠模型，同时各治疗组给予相应治疗药物，检测外周血象及血气，ELISA法检测血清中血红素（Heme）、促红细胞生成素（EPO）、缺氧诱导因子-1α（HIF-1α）、组织因子（TF）、组织因子途径抑制物（TFPI）、凝血酶-抗凝血酶复合物（TAT）含量，采用HE染色观察肺组织损伤，检测肾组织中HIF-1α/EPO信号通路的mRNA及蛋白表达，全面评价嘎罗曼麦丸对高原红细胞增多症的药效作用；收集嘎罗曼麦丸15批次，对其化学成分进行定性、定量分析，采用氯化钴诱导斑马鱼缺氧模型，检测不同批次样品对尾部红细胞的抑制率，采用灰色关联度联合斯皮尔曼秩相关分析谱-效数据，筛选嘎罗曼麦丸抗高原红细胞增多症的药效物质基础。结果 与对照组大鼠相比，模型组血红蛋白含量、红细胞数及红细胞压积显著升高；氧分压、钾离子及阴离子间隙显著升高，二氧化碳分压、总二氧化碳及碳酸氢根含量显著降低；血清Heme、EPO、HIF-1α、TF、TFPI、TAT水平显著升高；肺组织病理损伤明显；肾脏组织HIF-1α、EPO的mRNA及蛋白表达显著升高；与模型组大鼠相比，嘎罗曼麦丸给药组各指标显著改善；谱-效相关分析得出嘎罗曼麦丸中没食子酸、反式-草木犀苷、阿魏酸、新绿原酸、甘草素灰色关联度≥0.9且在斯皮尔曼秩相关分析中rs>0.5且差异具有统计学意义（P<0.05）。结论 嘎罗曼麦丸能够显著降低高原红细胞增多症模型大鼠血红蛋白含量及血液相关因子含量，改善血液流动性，减轻肺组织损伤，推测HIF-1α/EPO轴为其发挥药效作用的核心通路，没食子酸、反式-草木犀苷、阿魏酸、新绿原酸和甘草素为其防治高原红细胞增多症的关键物质基础。

Abstract:AIM Analyze the pharmacodynamic material basis and mechanism of Galuo manmai Pill against high altitude polycythemia (HAPC). Methods A rat model of HAPC was established by combined induction of a hypobaric oxygen environmental control system and intraperitoneal injection of cobalt chloride. Meanwhile, the corresponding therapeutic drugs were administered to each treatment group. Peripheral blood routine and blood gas indexes were detected, and the serum contents of Heme, Erythropoietin(EPO), Hypoxia-Inducible Factor 1α(HIF-1α), Tissue Factor (TF), Tissue Factor Pathway Inhibitor (TFPI) and Thrombin Antithrombin Complex(TAT）were determined by ELISA. HE staining was used to observe lung tissue injury, and the mRNA and protein expressions of HIF-1α/EPO signaling pathway in renal tissue were detected to comprehensively evaluate the pharmacodynamic effect of Galuo manmai Pill on HAPC. A total of 15 batches of Galuo manmai Pill were collected for qualitative and quantitative analysis of its chemical components. A cobalt chloride-induced zebrafish hypoxia model was established to detect the inhibition rate of tail erythrocytes by samples of different batches. The spectrum-effect data were analyzed by combining grey relational analysis with Spearman's rank correlation analysis to screen out the pharmacodynamic material basis of Galuo manmai Pill against HAPC. Results Compared with rats in the control group, the hemoglobin content, red blood cell count and hematocrit in the model group were significantly increased; the partial pressure of oxygen, potassium ion and anion gap were significantly elevated, while the partial pressure of carbon dioxide, total carbon dioxide and bicarbonate content were markedly decreased; the serum levels of Heme, EPO, HIF-1α, TF, TFPI and TAT were significantly raised; obvious pathological damage was observed in lung tissues; the mRNA and protein expressions of HIF-1α and EPO in renal tissues were significantly up-regulated. In contrast to the model group, all the above indicators were significantly improved in the Galuo manmai Pill treatment groups. Spectrum-effect correlation analysis revealed that gallic acid, trans-melilotoside, ferulic acid, neochlorogenic acid and liquiritigenin in Galuo manmai Pill had a grey correlation degree ≥0.9, with a Spearman’s rank correlation coefficient >0.5 and statistically significant differences (P<0.05). Conclusion Galuo manmai Pill could significantly reduce the hemoglobin content and the levels of blood-related factors in HAPC rats, improve blood fluidity and alleviate lung tissue injury. It is speculated that the HIF-1α/EPO axis serves as the core pathway for its pharmacodynamic effects, and gallic acid, trans-melilotoside, ferulic acid, neochlorogenic acid and liquiritigenin are the key material basis for its prevention and treatment of HAPC.