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刘惠娜,李艳玲,曹旺,邓常清.药根碱靶向凝血因子Ⅻ发挥抗凝血作用的体外研究[J].湖南中医药大学学报,2021,41(1):27-33[点击复制] |
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| 药根碱靶向凝血因子Ⅻ发挥抗凝血作用的体外研究 |
| 刘惠娜,李艳玲,曹旺,邓常清 |
| (湖南中医药大学血管生物学实验室, 湖南 长沙 410208) |
| 摘要: |
| 目的 以凝血因子Ⅻ（coagulation factor Ⅻ，FⅫ）为靶点探讨药根碱抗血栓作用的机制。方法 通过Ledock软件将药根碱分子与内源性凝血途径接触激活阶段关键靶蛋白FⅫ、活化凝血因子FⅫ（actived coagulation factor Ⅻ，FⅫa）、凝血因子Ⅺ（coagulation factor Ⅺ，FⅪ）和活化凝血因子Ⅺ（actived coagulation factor Ⅺ，FⅪa）进行分子对接，以结合能低于-5 kcal/mol作为阈值判断药根碱与接触激活因子的结合活性；采用体外凝血实验法检测血浆活化部分凝血活酶时间（activated partial thromboplastin time，APTT）、血浆凝血酶原时间（plasma prothrombin time，PT）、凝血酶时间（plasma prothrombin time，TT）；乏因子血浆纠正试验检测FⅫ、FⅪ、凝血因子Ⅸ（coagulation factor Ⅸ，FⅨ）、凝血因子Ⅷ（coagulation factor Ⅷ，FⅧ）、凝血因子Ⅹ（coagulation factor Ⅹ，FⅩ）和凝血因子Ⅶ（coagulation factor Ⅶ，FⅦ）的活性的抑制作用；Western blot法检测药根碱对FⅫ蛋白激活的抑制作用。结果（1）分子对接实验表明，药根碱与FⅫa、FⅪa结合能均低于-5 kcal/mol，结合活性良好。（2）体外实验中，0.2~0.8 mg/mL药根碱均可显著延长APTT（P<0.01），但不延长PT和TT（P>0.05）。（3）药根碱各剂量均显著降低FⅫ活性（P<0.01），药根碱高剂量（0.8 mg/mL）可降低FⅪ活性（P<0.05），但对FⅨ、FⅧ、FⅩ及FⅦ活性无显著影响（P>0.05）。（4）药根碱各剂量均显著抑制FⅫ蛋白的激活（P<0.01）。结论 药根碱与FⅫa对接较好，显著延长APTT，抑制FⅫ蛋白激活，降低血浆FⅫ活性，提示其抗栓作用可能通过抑制FⅫ的活性而发挥。 |
| 关键词: 药根碱 内源性凝血途径 凝血因子Ⅻ 分子对接 |
| DOI：10.3969/j.issn.1674-070X.2021.01.006 |
| 投稿时间：2020-07-14 |
| 基金项目:国家自然科学基金项目（81904148）。 |
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| Anticoagulant Effect Study of Jatrorrhizine on the Target of Coagulation Factor XII In Vitro |
| LIU Huina,LI Yanling,CAO Wang,DENG Changqing |
| (Laboratory of Vascular Biology, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To investigate the anticoagulant mechanism of jatrorrhizine with coagulation factor Ⅻ(FⅫ) as the target. Methods Factor Ⅻ (FⅫ), actived coagulation factor Ⅻ (FⅫa), coagulation factor Ⅺ (FⅪ) and actived coagulation factor Ⅺ (FXIa), which are the key target proteins in the contact activation stage of endogenous coagulation pathway, were performed molecular docking with jatrorrhizine by the Ledock software, the binding activity between jatrorrhizine and contact activation factors was determined by the threshold that binding energy lower than -5 kcal/mol; the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) were detected by vitro coagulation test; the inhibitory effect of jatrorrhizine on the activities of FⅫ, FⅪ, coagulation factors Ⅸ (FⅨ), coagulation factors Ⅷ (FⅧ), coagulation factors Ⅹ (FⅩ) and coagulation factors Ⅶ (FⅦ) was detected by factor deficient plasma correction test; Western blot was used to detect the inhibitory effect of jatrorrhizine on activation of FⅫ protein. Results (1) The molecular docking experiments showed that the binding energies of jatrorrhizine with FⅫa and FXIa were lower than -5 kcal/mol, which indicated high binding activity. (2) Jatrorrhizine prolonged APTT at 0.2-0.8 mg/mL (P<0.01), but did not prolong PT and TT (P>0.05) in vitro experiment. (3) Various concentration of jatrorrhizine significantly decreased the activity of FⅫ (P<0.01), high concentration of jatrorrhizine (0.8 mg/mL) decreased the activity of FⅪ (P<0.05), but had no significant effect on the activities of FⅨ, FⅧ, FⅩ and FⅦ (P>0.05). (4) Various concentration of jatrorrhizine significantly inhibited the activity of FⅫ protein(P<0.01). Conclusion Jatrorrhizine has a good docking with FⅫa. It can significantly prolong APTT, inhibit activation of FⅫ protein and reduce the activity of plasma FⅫ. All suggested that antithrombotic effect of jatrorrhizine may be exerted by inhibiting the activity of FⅫ. |
| Key words: jatrorrhizine endogenous coagulation pathway coagulation factor Ⅻ molecular docking |
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