藏药砂生槐子生物碱抗囊性包虫病的机理研究

Echinococcosis is one of zoonotic parasitic diseases which is seriously endangering the health of farmers and herdsmen and the livestock production, so it is badly constraining the development of local socio-economic. At present, the drug of treatment of echinococcosis, however, has side effects, poor absorption and drug-resistant, in addition, the germinal layer cells still proliferate and the granuloma still formates and continuously develops. The key to control development of echinococcosis is researching drugs that can both directly kill and inhibit the growth of germinal cells and prevent the formation of the granuloma.. Sophora moorcroftiana is a unique plant in Tibetan which is of great value in Chinese folk medicine for treatment of worms. The previous studies have shown that Sophora moorcroftiana alkaloids have insecticidal effects. Baseing on alkaloids, we study the mechanisms of insecticide and inhibiting germinal cells growth and granuloma formationa by detecting the cytotoxic effect of protoscolices and germinal cells and inducing apoptosis in vitro, establishing echinococcosis in mice model to observation apoptosis morphologic of treatment of cyst Organization (protoscolices, germinal cell) and using immunohistochemical technique to detect NF-κ b, Bcl-2, Bax, Fas, caspase-9, caspase-3 and IL-2 ,IL-4, IL-6, IFN-γ, TNF-α in the Granuloma expression levels comprared with the untreated control animals, and provide an important foundation information of further scientific development Sophora moorcroftiana .

包虫病是严重危害我国广大农牧民身体健康和畜牧业生产发展的人畜共患寄生虫病，其严重制约着当地社会经济的发展。目前治疗该病的药物毒副作用大，吸收差、易抗药，并且生发层细胞仍可增殖，肉芽肿继续形成和发展。研发既能直接杀虫、又能抑制生发层细胞增殖、阻止肉芽肿形成的药物是控制包虫病发展的关键。砂生槐是我国特有的民间治疗虫毒的一味藏药，发现其生物碱有杀虫的功效。本项目以生物碱为基础，进行体外原头蚴、生发层细胞的细胞毒效应及诱导细胞凋亡；建立小鼠棘球蚴病模型，观察治疗前后包囊组织（原头蚴、生发层细胞）的凋亡形态、免疫组化法检测NF-κB、Bcl-2、Bax 、Fas、caspase-9、caspase-3及肉芽肿中IL-2、4、6、IFN-γ、TNF-α的表达水平，研究其杀虫、抑制生发层细胞生长及对肉芽肿形成的作用机制，为进一步科学开发藏药砂生槐提供重要的基础资料。

砂生槐为青藏高原特有植物，是我国民间治疗虫病的一味藏药。本项目选择秀丽隐杆线虫和继发性小鼠棘球蚴病模型，针对藏药砂生槐子生物碱主要成分分析、体外及体内抗虫实验，采用HPLC、显微、亚显微及免疫因子检测等技术手段，探讨了藏药砂生槐子生物碱抗囊性包虫病的机理，已按计划完成研究任务。主要包括：（1）采用醇热回流提取、TLC定性鉴定，HPLC分析筛选获得砂生槐子生物碱有效的抗虫活性成分E2-a，为低极性的水溶性生物碱，其主要化学成分为苦参碱和槐果碱（68.5%）。（2）采用体外肝细胞模型，评价E2-a对HepG-2和Chang liver cell line细胞株的活力影响表明均有不同程度的抑制；利用健康BALB/c小鼠和继发性小鼠棘球蚴病模型，E2-a (100 mg/kg)分别灌胃实验，结果表明，E2-a处理组小鼠的肝肾功生化指标与未处理组比较均无差异，肝、肾脏组织切片未见病理学改变。（3）采用继发性小鼠棘球蚴病模型，通过显微及亚显微技术观察到E2-a可损伤囊泡生发层及角皮层，减少棘球蚴和泡球蚴囊泡组织湿重。采用流式细胞技术检测E2-a组小鼠脾脏T 细胞亚群、PD-1+T细胞亚群表明，可诱导CD4+T细胞增多、下调T细胞表面负调节分子PD-1的表达。采用ELISA夹心法检测各组IL-4和IFN-γ ，表明E2-a调节IFN-γ和IL-4，促进免疫功能。E2-a组小鼠脾细胞增殖能力无增高，采用继发性泡球蚴病小鼠模型也获得同样结果。E2-a能够影响秀丽隐杆线虫行为能力，降低生殖能力，缩短寿命，影响发育。（4）采用醇沉法提取砂生槐种子多糖，经Sevege法脱蛋白，氧化脱色素，气相色谱-质谱法联用分析组成成分。结果发现：砂生槐种子多糖含有葡萄糖，半乳糖和肌醇，其中葡萄糖的含量为35%。利用L4期秀丽隐杆线虫，测试砂生槐种子多糖对其寿命、行为、应激、生殖和抗菌能力的影响，结果表明砂生槐种子多糖可以延长秀丽隐杆线虫生命周期，并增强了秀丽隐杆线虫抗应激、抗衰老的能力，有效促进秀丽隐杆线虫排卵和延长线虫排卵时程，改善线虫的生殖能力，增强线虫抗细菌感染的能力。