青蒿素及其衍生物抗鸡球虫的分子机理

It has been found that nitric oxide (NO) generated by bacterial nitric oxide synthase (NOS) mediated the resistance of bacteria to a broad spectrum of antibiotics, and this finding provides a new sight for the clinical therapy relying on antibiotics. Recently, it has been demonstrated that artemisinin and its derivatives could increase the effectiveness of antibiotics antimicrobial therapy by inhibiting the NOS in bacteria. Over the past few decades, many studies suggested that artemisinin based drugs possess an effective anticoccidiosis, however, the specific underlying mechanism is still not clear at present. Considering the mitochondria was shown as one of the important direct targets for antimalarial effect of artemisinin, and now we detected the NO production from freshly isolated unsporulated Eimeria tenella oocysts, plus another fact that E.tenella in various stages contains large amount of mitochondria, so we hypothesize mitochondria in Eimeria spp may be the potential target of artemisinin-based drugs..We will illustrate the effects of artimisinin and its derivatives on sporozoite invasion, NOS expression, S-nitrothiols contents in mitochondrial memberane and mitochondrial permeability transition pore (mPTP) in this project, using cell culture and animal examination. Moreover, the artemisinin or its derivatives will combine with the specific inhibitors (mPTP or ANT) to detect the cascade reaction induced by mitochondrial damage in oocysts or sporozoites, and then confirmed the potential roles of NO and mitochondria pathway on activating the damaging reactions against Eimeria sporozoites or merozoites induced by artimisinin or its derivatives. Based on the series of studies, the potential targets of artemisinins against Eimeria spp will be analysed and elucidated. Meanwhile, the synergistic effects of combining artemisinin and its derivatives with other anticoccidiostats on the expression of inducible NOS, NF-κB、Bcl and c-FLIP proteins, and the location or exprerssion of the key apoptosis association signal moleculars, such as caspase-3, caspase-9 and apoptosis inducing factor (AIF) in host cells will be determined by modern molecular biological techniques. Therefore, the indirect mechanism of artemsinin and its derivatives decreasing the proliferation of Eimeria merozoites or macrogametes via promoting the cellular apoptosis of infected host cells will be clarified. .To explore the previous important events and their relationships，this project will contribute significantly to the theoretical basis for the discovery of new anticoccidial targets and reducing the drug resistence development of Eimeria spp through reasonable usage of artemisinin and its derivatives.

细菌通过自身一氧化氮合酶（NOS）释放NO介导耐药性产生途径的发现为依赖抗生素的治疗提供了新思路，青蒿素类药物能抑制NOS而增强抗生素疗效。青蒿素类药物具有明确的抗球虫作用但机理不明，鉴于线粒体是青蒿素发挥抗疟活性的重要靶位而球虫各阶段虫体均富含线粒体且能产生自身NO。本项目拟通过细胞培养和动物试验来研究青蒿素类药物对虫体NOS、入侵活力、线粒体膜S-亚硝基化以及mPTP开放等关键指标的分析，结合mPTP、ANT等特异抑制剂的使用，阐明药物致虫体线粒体损伤的信号级联反应，确定NO-线粒体途径在青蒿素类药物抗球虫活性中的潜在作用。同时联合抗球虫药，研究感染细胞NOS，抗凋亡因子NF-κB、Bcl和c-FLIP以及凋亡关键信号分子的表达与定位，明确青蒿素类药物通过促进感染细胞凋亡而抑制虫体繁殖的间接机制及增效作用。本研究将为抗球虫药物新靶点的发现以及指导合理用药而降低耐药性发生提供理论依据。

前期研究表明ART处理柔嫩艾美耳球虫（E. tenella）感染鸡可抑制球虫卵囊壁的形成，并抑制卵囊的孢子生殖。本项目的研究表明ART比其衍生物青蒿琥酯（ARS）和蒿甲醚（ARM）具有更好的抗球虫作用，且毒副作用小。在体外试验中，ARS溶解度高，故可用于体外细胞培养试验，ARS处理细胞和子孢子2-12h均可显著降低E. tenella子孢子对DF-1细胞的入侵率，使入侵率下降60%左右，凋亡抑制剂米酵菌酸对ARS的影响不明显，说明青蒿琥酯抑制入侵不是通过凋亡途径，而是改变了细胞膜上受体或虫体膜抗原的结构。体内试验中ART处理E. tenella感染鸡可使第二代裂殖子数量明显减少，使部分子孢子发生细胞核固缩和染色质异常；裂殖子线粒体膜电位下降(47%)而使虫体凋亡比率增加了61.9%，同时ART处理可显著增加感染细胞促凋亡因子（Bax，caspase-3）的表达而减少抗凋亡因子（Bcl-2）的表达，这有利于感染细胞启动凋亡过程而减少虫体繁殖，最终减轻感染程度。以上数据提示ART可从多个环节发挥抗球虫作用。ART单独使用的抗球虫效果难以达到传统抗球虫的疗效，与柳氮磺吡啶、二甲氧苄胺嘧啶以及癸氧喹酯联合应用可显示增效作用，有必要进一步优化剂量和配比，为临床使用提供参考。