小分子抑制剂猫眼草黄素逆转P-gp介导的青蒿素多药耐药机制研究

Drug resistance for ART-based combination therapy has appeared under the current clinical treatment. No medicines could be used to cure malaria in the coming future if no effective actions are taken without delay. The mechanisms of ART resistance are very complex. Among them, multi-drug resistance mediated by P-gp is one of the pivotal mechanisms. Our obtained results showed that chrysosplenetin (CHR) can enhanced in vivo concentration and therefore anti-malarial activity of artemisinin (ART) by inhibiting P-gp protein and has the potency to be a reversal agent for ART resistance. But the mechanism is kept unclear up to now. This program investigated the significant difference for the expression of P-gp and its encoded protein MDR1 in small intestine between the mice infected by ART resistance P. falciparum and those by ART sensitive parasites with and without CHR in order to show the mechanism in the level of protein and gene expression. Secondly, cell membrane fluidity, ATP concentration and its activity were studied to confirm the mechanism in terms of P-gp function level at the same mice models. Finally, the impact of CHR on the transcriptional regulation of Pregnane X Receptor (PXR) and Constitutive Androstance Receptor (CAR), the key nuclear receptors on MDR1, was studied. Comprehensively, the reverting mechanism of CHR on ART resistance medicated by P-gp was elucidated in terms of protein and gene expression, P-gp function, and the transcription regulation levels. The results will lay the pivotal basis for finding the P-gp inhibitor with a clear mechanism in the future.

青蒿素（ART）为基础的抗疟疗法在现有的治疗方案下已产生抗药性，若不及时采取措施，疟疾治疗恐将无药可用。ART抗药性形成机制复杂，P-gp介导的多药耐药是关键机制之一。项目前期发现猫眼草黄素（CHR）通过抑制P-gp的外排功能，提高ART体内浓度和抗疟活性，有望成为ART抗性逆转剂，但作用机制不明。本项目通过建立ART抗性小鼠模型，考察CHR-ART合用前后抗性鼠和敏感鼠小肠P-gp及其编码蛋白MDR1的表达差异，明确CHR在蛋白和基因表达水平的作用机制；考察同一动物模型下，两药合用后小肠细胞膜流动性、ATP酶含量及活性差异，研究其对蛋白功能的影响；结合CHR对调控MDR1转录的关键核受体孕烷X受体（PXR）和组成型雄甾烷受体（CAR）的影响，从蛋白与基因表达、酶功能、核受体转录调控三个水平，深入研究CHR逆转P-gp介导的ART抗药性作用机制，为寻找机制明确的ART抗性逆转剂奠定基础。

项目在青蒿素敏感/抗性疟原虫感染小鼠模型上，研究了青蒿素单用、青蒿素-猫眼草黄素复配对小肠P-gp/Mdr1 mRNA表达水平、细胞膜流动性/ATP含量/ATP酶活性的影响，并采用双荧光素酶报告基因方法，在结肠腺癌细胞LS174T上考察了青蒿素-猫眼草黄素复配前后对PXR/CAR核受体调控Mdr1 mRNA转录表达差异，结果发现青蒿素显著上调健康及敏感、抗性疟原虫感染小鼠小肠P-gp/Mdr1 mRNA表达，而猫眼草黄素-青蒿素复配前后均能下调P-gp/Mdr1 mRNA水平，而乳腺癌多药耐药蛋白Bcrp蛋白表达和基因表达结果不一致，P-gp可能是猫眼草黄素逆转青蒿素耐药性的关键转运体；青蒿素对敏感株和抗性株感染小鼠小肠ATP含量存在显著差异，与抗性组相比，显著降低敏感组小鼠ATP含量，而猫眼草黄素单用及复配均显著提高敏感组ATP含量，降低抗性组ATP含量；与健康组相比，青蒿素显著下调敏感/抗性组小肠ABC家族依赖的ATP酶活性，猫眼草黄素单用和复配均显著上调抗性组ABC家族依赖的ATP酶活性。此外，青蒿素对PXR/CAR没有显著影响，这与文献结果并不一致，猫眼草黄素单用上调PXR/CAR转录表达水平，复配后PXR/CAR转录水平下调，表明猫眼草黄素是PXR的配体，鉴于CAR存在配体激活和非配体激活两种方式，故而猫眼草黄素是如何调控PXR/CAR转录调控Mdr1 mRNA表达还需进一步深入探讨。