不同制备工艺灵芝孢子粉抗血栓、改善心脏功能作用研究

Study on the Antithrombotic and Cardiac Function Improvement Effects of Ganoderma lucidum Spore Powder with Different Manufacturing Techniques

关键词:  血栓  心衰  斑马鱼  灵芝孢子粉

Key words:  thrombus  heart failure  zebrafish  Ganoderma lucidum spore powder

摘要:

目的 采用斑马鱼模型，比较研究破壁灵芝孢子粉（wall-broken Ganoderma lucidum spore powder，BGLSP）和去壁灵芝孢子粉（wall-removedGanoderma lucidum spore powder，RGLSP）对血栓形成预防作用及心脏功能改善作用。方法 首先采用紫外可见分光光度法分别测定BGLSP、RGLSP中总多糖和总三萜的含量。其次，在野生型AB品系斑马鱼中进行药物毒性试验，确定灵芝孢子粉的最大耐受浓度。然后，分别用苯肼、花生四烯酸以及普纳替尼诱发斑马鱼血栓模型，并用BGLSP、RGLSP及阳性药干预。建模完成后，用邻联茴香胺进行染色，并用NIS-Elements DTM图像处理软件进行图像分析，计算斑马鱼心脏红细胞染色强度及预防血栓作用。此外，用维拉帕米诱发斑马鱼心衰模型，测定斑马鱼心脏扩大面积、静脉淤血面积、心输出量以及血流速度，定量评价灵芝孢子粉对维拉帕米诱发的斑马鱼心衰的心脏功能改善作用。结果 RGLSP所含多糖和三萜类化合物的含量显著高于BGLSP （P<0.001），分别提高了11倍和5倍。苯肼、花生四烯酸和普纳替尼诱导后，斑马鱼心脏红细胞染色强度显著增高（P<0.01或P<0.001）。与模型对照组相比，BGLSP高剂量组显著抑制苯肼和普纳替尼诱发的斑马鱼血栓形成（P<0.05），并且能够显著提高心衰斑马鱼心脏扩大改善率和心输出量增加率（P<0.05或P<0.01）；RGLSP低、中剂量组对苯肼、花生四烯酸和普纳替尼诱发斑马鱼血栓形成，及对维拉帕米诱发斑马鱼心衰均具有显著保护作用（P<0.05或P<0.01或P<0.001），并且保护作用强于BGLSP。结论 BGLSP和RGLSP均具有显著的抗血栓及抗心衰作用，而RGLSP的药效作用更明显，可能与其较高的有效成分含量有关。

Abstract:

OBJECTIVE To investigate the antithrombotic and cardiac function improvement effects of wall-broken Ganoderma lucidum spore powder (BGLSP) and wall-removed Ganoderma lucidum spore powder (RGLSP) by zebrafish model. METHODS Firstly, the ultraviolet and visible spectrophotometry method was employed to determine the contents of total polysaccharide and triterpenoids in BGLSP and RGLSP. Secondly, the drug toxicity test was conducted in wild-type AB strains of zebrafish to determine the maximum tolerable concentration of BGLSP and RGLSP. Then, zebrafish thrombosis models were induced with phenylhydrazine, arachidonic acid, and ponatinib, respectively, and treated with various concentrations of BGLSP, RGLSP or positive drugs. After modeling, O-anisidine was used for staining, and NIS-Elements DTM image processing software was used for image analysis to calculate the staining intensity of zebrafish heart red blood cells and effect on preventing thrombosis. Moreover, the verapamil was used to establish a zebrafish heart failure model. The zebrafish heart enlargement area, venous congestion area, cardiac output, and blood flow velocity were measured to quantitatively evaluate the cardiac function improvement of verapamil-induced zebrafish heart failure. RESULTS The contents of polysaccharides and triterpenoids of RGLSP were significantly higher than those of BGLSP(P<0.001), with fold change of 11 and 5, respectively. The staining intensities of erythrocytes in the heart were significantly increased after induction by phenyl hydrazine, arachidonic acid, ponatinib or verapamil(P<0.01 or P<0.001), respectively. Compared with the model control group, high dose group of BGLSP could significantly inhibit phenylhydrazine and prnatinib-induced zebrafish thrombosis(P<0.05), and could improve the heart enlargement improvement rate, cardiac output increase rate of zebrafish with heart failure(P<0.05 or P<0.01); low and middle dose group of RGLSP showed protective effect on phenylhydrazine, arachidonic acid and prnatinib-induced zebrafish thrombosis, and verapamil-induced zebrafish heart failure(P<0.05 or P<0.01 or P<0.001), and the antithrombotic and anti-heart failure effect of RGLSP was better than that of the same dosage of BGLSP. CONCLUSION BGLSP and RGLSP have markedly protective effect on thrombosis and heart failure, the effect of RGLSP were better than BGLSP, which may be related to its higher content of active components.