青蒿内含增效组分协同促青蒿素抗疟活性的机制研究

Artemisinin-based combination therapies (ACTs) are now the most effective anti-malarial method. However, plasmodium falciparum has been currently reported to be resistant to artemisinin. It was found in our previous studies that synergistic components included in Artemisia annua L. (SCIAaL, including artemisinic acid, arteannuin B and scopoletin) themselves showed no significant anti-malarial activities, but they could significantly strengthen the anti-malarial activity of artemisinin. Therefore, it is speculated that the synergistic effects of SCIAaL on the anti-malarial activity of artemisinin might base on the following two synergistic mechanisms: 1. SCIAaL might inhibit the auto-induced metabolism of artemisinin, then reverse the decreased concentration of artemisinin in vivo; 2. SCIAaL might also block the bindings of intracellular transporters and artemisinin, inhibit the efflux of artemisinin from plasmodium, then increase the intracellular concentration of artemisinin, and finally decrease the resistance of plasmodium to artemisinin. In present study, the synergistic metabolic profiling of artemisinin and its SCIAaL will be investigated in-depth, and the molecular mechanism of artemisinin resistance in plasmodium falciparum will also be systematically studied. In view of the incomparable superiorities of traditional Chinese medicine and natural products in the field of drug resistance to the chemical medicines, the current research results will be helpful to the development of new anti-malarial strategy, provide new targets for the treatment of malaria, and ultimately provide the foundation for the development of new artemisinin-based anti-malarial compositions with low drug resistance.

基于青蒿素的联合疗法是当前对抗疟疾最有效的方式。然而，目前恶性疟原虫已对青蒿素产生耐药性。本课题组前期研究发现：青蒿酸、青蒿乙素、东莨菪内酯等青蒿内含增效组分（SCIAaL）本身并无明显的抗疟活性，却能协同显著增强青蒿素的抗疟活性。推测SCIAaL促青蒿素抗疟活性可能基于以下两种协同机制：1. SCIAaL可能抑制青蒿素的自身诱导代谢，从而逆转了自身诱导代谢所致的青蒿素体内浓度下降；2. SCIAaL可能竞争性阻断了疟原虫胞内转运子与青蒿素的结合，抑制了疟原虫对青蒿素的外排，增加了青蒿素的胞内浓度，从而降低了疟原虫对青蒿素的耐受性。本项目拟深入探讨青蒿素及SCIAaL的协同代谢谱，系统研究恶性疟原虫对青蒿素耐药性的分子机制。鉴于中药与天然产物在抗药性产生方面较化学药的优势，本研究结果将有助于发展新的截疟策略，为疟疾治疗提供新靶标，最终为开发低耐药的基于青蒿素的新型抗疟组合物奠定基础。

基于青蒿素的联合疗法是当前对抗疟疾最有效的方式。然而，目前恶性疟原虫已对青蒿素产生耐药性。本项目发现：“青蒿酸、青蒿乙素、东莨菪内酯”等青蒿内含增效组分（SCIAaL）本身并无明显的抗疟活性，却能协同增强青蒿素的抗疟活性。推测SCIAaL促青蒿素抗疟活性可能基于SCIAaL抑制青蒿素自身诱导代谢和抑制疟原虫对青蒿素的外排两种协同机制。本项目首先采用LC-ESI-MS/MS法对比了青蒿素及SCIAaL的大鼠药动学行为，发现SCIAaL组合可增加青蒿素的吸收（生物利用度增加1倍左右），并延缓其消除，表明SCIAaL可能存在针对青蒿素自身诱导代谢的抑制作用；其次，采用重组酶系孵育实验发现SCIAaL组合可明显抑制CYP 2C9、2B6、2C19和3A4酶，人和大鼠肝微粒体实验也表明SCIAaL组合可抑制青蒿素代谢的相关酶系，导致青蒿素代谢变慢。再次，应用青蒿素及青蒿素加SCIAaL处理疟原虫，发现采用青蒿素加SCIAaL处理后，青蒿素在疟原虫体内的浓度明显较高，而单用青蒿素处理后，青蒿素在疟原虫体内的浓度明显较低，证实SCIAaL可能对疟原虫外排青蒿素具有一定的抑制作用。应用RT-PCR法和Western blot法发现疟原虫PART8虫株可能通过增加药物的外排降低对青蒿素治疗的耐受性，PART8疟原虫中pbkelch13、pbggcs和pbmdr1基因的表达量显著上调，提示pbkelch13、pbggcs和pbmdr1基因的过度表达促进了疟原虫对青蒿素治疗的耐受性，而青蒿素的连续治疗可以诱导基因的表达，造成耐受性升高。SCIAaL组合能够延缓伯氏疟原虫对青蒿素连续治疗引起的药物耐受性的作用与耐药基因的表达相关。同时，pbggcs和pbkelch13基因在疟原虫中的过表达导致GSH水平升高，从而提高了疟原虫对青蒿素的耐受性。鉴于中药与天然产物在耐药性产生方面较化学药的优势，本研究结果有助于发展新的截疟策略，为疟疾治疗提供新靶标，最终为开发低耐药的基于青蒿素的新型抗疟组合物奠定基础。

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