苦马豆素抗牛病毒性腹泻病毒的研究

Study on Inhibitory Effect of the Swainsonine from Alkaloid of Astragalus strictus Grah. Ex Bend on Bovine Viral Diarrhea Virus

【目的】疯草是中国西部影响草地生态和畜牧业健康发展的毒草之一。主要有豆科棘豆属和黄芪属有毒植物等,在冬季牧草严重缺乏的时节,牛羊等牲畜被迫采食后可引起中毒和生产性能下降,甚至死亡,每年给我国草地生态和畜牧业造成的直接经济损失达几十亿元,是危害最严重的毒草。目前,我国西部天然草地动物因疯草中毒死亡所造成的经济损失仍持续剧增。苦马豆素被认为是引起动物疯草中毒的主要毒性成分。由于疯草分布广泛,资源丰富,如何改善草地生态,合理开发利用疯草成为研究的课题和方向。近几十年来,随着对疯草研究的深入和扩展,人们发现苦马豆素不仅具有良好的抗肿瘤效果,还可作为免疫调节剂、抗HIV及扩散抑制剂、抗病毒和细胞保护剂等药物使用。目前,国内在对苦马豆素抗肿瘤、调节机体免疫方面研究较热,但是,对苦马豆素在抗病毒活性、作用机理方面的研究尚无相关报道。为了探讨茎直黄芪中生物碱苦马豆素(swainsonine,SW)抗牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的作用机制,明确其体外抗病毒作用效果,为抗BVDV的药物筛选提供参考依据。【方法】利用细胞培养技术,采用CPE观察法和MTT比色法相结合的方法测定不同浓度SW对牛肾原代细胞(madin-darby bovine kidney cells,MDBK)的毒性作用,确定药物的安全浓度和TD50,并分别采用先加药后感染病毒、先感染病毒后加药、药毒作用2 h后加入、感染病毒同时给药后再加药四种作用方式,检测不同浓度SW对BVDV入侵的阻断作用、复制的抑制作用、直接杀伤作用和综合作用,并计算不同作用方式下的治疗指数。【结果】结果显示:SW浓度小于0.256μg·mL-1范围内对MBDK细胞无毒性作用,在大于0.512μg·mL-1浓度下MBDK细胞表现出一定程度的病变,TD50为2.512μg·mL-1,按Reed-Muench氏法计算BVDV TCID50为10-4.7/0.1mL;在体外随苦马豆素质量浓度的增加,其抗BVDV作用具有很好的量效关系,且与作用方式有关,比较四种作用方式下的治疗指数1.05、2.47、2.08和3.21,说明SW对BVDV的综合作用(65.29%,P<0.01)和复制(65.05%,P<0.01)的抑制作用较好,亦有一定的直接杀伤作用,但对其入侵的阻断作用不佳。【结论】SW在体外有良好抗BVDV活性作用,并推测苦马豆素(SW)抗病毒的主要效应可能是SW能进入细胞内抑制BVDV的复制增殖以及直接灭活游离BVDV而发挥作用。

[Objective] Locoweed is one of the poisonous weeds in western China that influences the healthy development of grassland and animal husbandry. Locoweeds mainly belong to Astragalus and Oxytropis genera. As a serious lack of forage in winter season, livestock such as cattle and sheep are forced to feed them. Locoweed can decrease the production performance, toxicity and death of animals, and the direct economic loss of the grassland ecological and animal husbandry is about billions of dollars, which is the most serious poisonous weed. At present, the economic losses of animal death caused by locoweed poisoning continues to soar. Swainsonine is the main toxic component and believed to be the cause of animal locoweed poisoning. Locoweed is widely distributed and is rich in resources, and how to improve the grassland ecology and use of locoweed is becoming the topic of research subject and direction. In recent years, with the study and expansion of locoweed, people have found that swainsonine not only has a good anti-tumor effect, also it can be used as an immunomodulator, anti HIV and anti proliferation inhibitor, anti viral and cell protective agent and other drugs. At present, the domestic research on swainsonine anti-tumor and regulation of immunity is hot, However, no research reports are found in antiviral activity and mechanism of action of swainsonine up to the present. This experiment was designed to investigate the anti-virus mechanism of the swainsonine from alkaloid of Astragalus on bovine viral diarrhea virus （BVDV）, and to make clear of the antiviral effect in vitro, it will provide references for the screening of anti BVDV drugs. [Method] Using cell culture techniques, and combined with CPE observation method and MTT colorimetric method, different concentrations SW toxicity with bovine kidney primary cells （Madin-Darby Bovine Kidney Cells, MDBK） were detected, the concentration of the drug’s safety and TD50 was determined. They were measured by four administration routes of virus before swainsonine, virus after swainsonine, virus-and-swainsonine after 2 hours, and virus-and-swainsonine after swainsonine, which are the effect of different concentrations of SW against blocking effect on BVDV invasion, replicatting inhibition, directly killing effect and combining effects, respectively. The treatment indexes under different interaction modes were also calculated.[Result]It was found that the TCID50 of BVDV-MDV is 10-4.7. SW was not toxic to MDBK when its concentration was below 0.256μg＆#183;mL-1, and TD50 is 2.512μg＆#183;mL-1. Under the way of virus after SW, IC50 is 2.399μg＆#183;mL-1, TI is 1.05. Under the way of SW after virus, IC50 is 1.018μg＆#183;mL-1, TI is 2.47. Under the way of 2 h after mixed virus with SW, IC50 is 1.205 μg＆#183;mL-1, TI is 2.08. Under the way of synthetic action, IC50 is 0.782 μg＆#183;mL-1,TI is 3.21. After the TI of different modes of action was compared, it showed that the comprehensive effect on BVDV of SW （65.29%,P＆lt;0.01） and the inhibition effect of SW on the reproduction of BVDV （65.05%, P＆lt;0.01） were better, and SW had a certain direct killing effect on BVDV, but the blocking effect of SW on the invasion of BVDV was poor.[Conclusion]The effect of swainsonine （SW）-anti-BVDV activity in vitro was better, and the authors infered that the antiviral mechanism of SW might be that it could enter into the cells and inhibit the reproduction of BVDV, or it could play a role by killing the uncombined BVDV.