Zhang Ying, Hong Zongchao, Yuan Zixin, Wang Tianshun, Wu Xingpan, Liu Bo, Ai Zhongzhu, Wu Hezhen, Yang Yanfang
Faculty of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, China.
Key Laboratory of Traditional Chinese Medicine Resources and Chemistry of Hubei Province, Hubei University of Chinese Medicine, Wuhan 430065, China.
ACS Omega. 2020 Dec 3;5(49):32123-32130. doi: 10.1021/acsomega.0c05227. eCollection 2020 Dec 15.
The main objective of this study was to investigate the antithrombotic and antiplatelet effect of the extract from (L.) Nees and understand the mechanisms by which it exerts its antithrombotic and antiplatelet mechanisms.
The antithrombotic effective parts (RPE) were isolated using D101 macroporous adsorption resin and potential active ingredients (JAC) were isolated using the preparative liquid-phase method. The lactate dehydrogenase kit was used to determine the toxicity of RPE and JAC to platelets. The antiadhesion effect of RPE and JAC on platelets was observed by fluorescence microscopy with rhodamine phalloidin. Antithrombotic efficacy of RPE and JAC in vivo was evaluated by establishing a rat tail thrombosis model. Contents of p-selectin, TXB, and 6-keto-PGF in rat serum were measured using an enzyme-linked immunosorbent (ELISA) assay, and the rat black tail rate was measured to prove the protective effect of RPE and JAC on the tail thrombus rat model. Western blot was used for detection of serum-related proteins in the tail thrombus rat model.
The results showed that RPE had antithrombotic and antiplatelet effects. RPE and JAC have no toxicity to platelets. In vitro experiments showed that RPE and JAC had antiadhesion effects on platelets. In vivo experiments showed that RPE significantly inhibited the increase of p-selectin and TXB and significantly increased the content of 6-keto-PGF in the serum of rats. Western blot results demonstrated that RPE and JDB significantly inhibited the phosphorylation of the MAPK protein family in the platelets of rats, and RPE also significantly inhibited the phosphorylation of β protein.
RPE has antithrombotic and antiplatelet activity in vivo and vitro. Its mechanism may be via preventing integrin αβ activation, which in turn leads to the inhibition of the phosphorylation of the MAPK family and further suppresses TXA, which leads to the antithrombotic and antiplatelet effects.
本研究的主要目的是研究（L.）Nees提取物的抗血栓和抗血小板作用，并了解其发挥抗血栓和抗血小板作用的机制。
采用D101大孔吸附树脂分离抗血栓有效部位（RPE），采用制备液相法分离潜在活性成分（JAC）。使用乳酸脱氢酶试剂盒测定RPE和JAC对血小板的毒性。用罗丹明鬼笔环肽荧光显微镜观察RPE和JAC对血小板的抗黏附作用。通过建立大鼠尾静脉血栓形成模型评估RPE和JAC在体内的抗血栓疗效。采用酶联免疫吸附（ELISA）法测定大鼠血清中p-选择素、TXB和6-酮-PGF的含量，并测定大鼠黑尾率以证明RPE和JAC对尾静脉血栓大鼠模型的保护作用。采用蛋白质免疫印迹法检测尾静脉血栓大鼠模型中的血清相关蛋白。
结果表明，RPE具有抗血栓和抗血小板作用。RPE和JAC对血小板无毒性。体外实验表明，RPE和JAC对血小板有抗黏附作用。体内实验表明，RPE显著抑制大鼠血清中p-选择素和TXB的升高，并显著提高6-酮-PGF的含量。蛋白质免疫印迹结果表明，RPE和JDB显著抑制大鼠血小板中MAPK蛋白家族的磷酸化，RPE还显著抑制β蛋白的磷酸化。
RPE在体内外均具有抗血栓和抗血小板活性。其机制可能是通过阻止整合素αβ活化，进而抑制MAPK家族的磷酸化，并进一步抑制TXA，从而产生抗血栓和抗血小板作用。
