核受体介导的CYP3A和外排转运蛋白在附子个体化用药中的作用及机制

Fuzi, the lateral roots of Aconitum Carmichaeli Debx, is a frequently used traditional Chinese medicine for more than two thousands of years. The major active/toxic components in Fuzi are aconitum alkaloids, mainly including diester-diterpene alkaloids (DDAs), monoester-diterpene alkaloids (MDAs) and aconines. It was reported that DDAs have the strongest effectivenesses in treating diseases such as rheumatics and inflammation. But DDAs also have the highest toxicity in the heart and nervous system with a very narrow safty window, which limits the safe usage of Fuzi in clinical therapeutics. Therefore, for greater effectiveness and minor toxicity, reasonably controlling the exposed concentration of DDAs in vivo it is very important for Fuzi clinical practices. However, individual differences lead to diversely absorption and elimination of DDAs. Therefore, the personalized practice of Fuzi, would be a necessary novel strategy for obtaining the less toxicity and greater effectiveness of Fuzi. .Our previous study showed that aconitine has an extremely low bioavailability (<7%), implying that the toxicity of Fuzi is ascribed to itself. We have revealed that coupling of CYP3A4/5 and efflux transporters (ETs), two key elements for the first line defense of the body in eliminating xenobiotics, controlled the exposed behavior of DDAs in vivo. DDAs were metabolized by CYP3A4/5 for their detoxicification, and excreted by ETs for their elimination. Moreover, we also found that DDAs up-regulated the gene and protein expressions of P-gp via transactivating PXR nuclear receptor (NR). Taken together, we have demonstrated that the bio-directional effects between DDAs and CYP3A/ETs/NRs could harmonize the blood concentration of DDAs in vivo. Therefore, our hypothesis in the proposal is that CYP3A/ETs/NRs would be major and important key elements for the personalized Fuzi practice because the diverse polymorphism of CYP3A/ETs/NRs could regulate the personal different exposure of DDAs in vivo. .Accordingly, in our proposed study, the diversity outbred (DO) mice that are cross-bred from 8 different mouse stains, and transfected cells will be employed for investigating the personalized characteristics of Fuzi in its toxicity and effectiveness. And gene polymorphism, genomic and epigenetic variations of CYP3A, ETs and NRs will be analyzed by personal genome machine. On the other hand, the mechanism of DDAs in regulating the transcriptional activations of CYP3A and ETs by NRs pathway will also be investigated using the cultured cell models. In conclusion, the newly generated findings from our proposed studies would shed light on the experimental principle for the personalized Fuzi clinical practice.

中药附子的毒性给临床带来风险。其毒效成分是双酯型乌头类生物碱（DDAs），安全窗窄。故附子个体化用药是临床安全使用之关键，但尚未研究。我们发现DDAs易被CYP3A4/5代谢和被外排转运蛋白（ETs）外排，使其生物利用度并不高（<7%）；又发现DDAs可干预核受体（NRs）而诱导ETs过表达而反制约其过度入血。由此，认为NRs介导的CYP3A/ETs与DDAs的反作用是DDAs适度入血的关键。CYP3A/ETs/NRs三元素的基因多态性和DDAs对它们基因的反调节，是附子个体化用药的重要机制。故课题采用基因多样性的多杂交系DO小鼠和细胞模型，运用基因组学等方法研究三元素基因多态性对DDAs入血的影响，并探讨DDAs对三元素基因表达的作用。以期为附子个体化用药提供依据，亦为后续开展附子临床个体化治疗提供实验基础。

项目背景：附子是中医临床常用的“回阳救逆”重要中药，用于“亡阳”危重患者，疗效显著。附子毒性大，治疗窗窄，临床疗效和毒性存在显著的个体差异，基于安全窗剂量下的个体化用药具有重要临床意义。乌头碱是附子的主要药效和毒性成分，药物代谢酶、外排转运蛋白以及调控它们的核受体的基因多态性及蛋白表达是影响药物药效和毒性敏感性的直接因素，是个体化用药的重要靶点与依据。DO小鼠（Diversity outbred mice）是目前可获得的最具遗传多样性（基因多样性）的小鼠品系，由 8 种祖源纯系小鼠（A/J，C57BL/6J，129S1/SvImJ，NOD/ShiLtJ，NZO/HiLtJ，CAST/EiJ，PWK/PhJ 和 WSB/EiJ）杂合而成，其基因组约有 45,000,000 个 SNPs 和基因结构变异位点，是理想的个体化用药研究的动物模型。主要研究内容：乌头碱毒性敏感和耐受DO小鼠的筛选；毒性敏感和耐受DO小鼠进行全外显子测序和生物信息学分析；RNA-seq筛选神经毒性敏感和耐受差异表达基因；RNA-seq筛选心脏毒性敏感和耐受差异表达基因；祖源小鼠毒性敏感和耐受小鼠筛选。重要结果：全外显子测序结果显示DO小鼠在SNP和Indel水平存在显著的个体差异；乌头碱毒性敏感组差异的基因主要富集在味觉传导，免疫系统，代谢相关的信号通路，耐受组差异的基因主要富集在嗅觉传导信号通路；敏感和耐受组乌头碱神经毒性差异表达基因主要富集在Neuroactive receptor ligand interaction; Adrenergic receptor signaling in cardiomyocytes; Calcium signaling pathway; cAMP signaling pathway等信号通路；敏感和耐受组乌头碱心脏毒性差异表达基因主要富集在PPAR signaling pathway; AMPK signaling pathway; Fatty acid metabolism signaling pathway；Insulin signaling pathway等信号通路。关键数据：找到毒性敏感和耐受小鼠差异的SNPs和Indels，找到毒性敏感和耐受差异基因参与的信号通路。

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