基于RNA-Seq转录组技术探讨豨莶草炮制减毒机理

According to the traditional Chinese medicine theory, processing with rice wine can reduce the toxicity of Siegesbeckiae Herba (SH). SH is commonly used for treating chronic diseases, while, its chronic toxicity and mechanisms underlying the toxicity-reducing effect of processing are still unknown. Our preliminary studies showed that processing with rice wine reduced the chronic pulmonary toxicities of SH in SD rats. RNA-sequencing (RNA-seq) analyses of the lung tissues showed that SH but not processed-SH (PSH) significantly up-regulated c-Jun N-terminal kinases 2 (JNK2). It was reported that JNK2 activation plays a causative role in lung injury. We hypothesize that 1) activation of JNK2 signaling is one of the mechanisms responsible for SH-induced pulmonary toxicities; 2) inhibition of JNK2 signaling contributes to the toxicity-reducing effect of processing. To address these hypotheses, we propose to 1) determine the dose- and time-dependent effects of SH on JNK2 signaling in vitro and in vivo; 2) determine the contribution of JNK2 signaling in the effects of SH on inflammatory response and fibroblast proliferation; and 3) explore the upstream mediator of JNK2 in SH-treated cells. Outcomes of this study can help us to optimize the manufacturing procedure for PSH by specifically inhibiting the JNK2 activating property of the herb; and to reduce SH’s pulmonary toxicities in the clinic by combining other herbs that are able to suppress the effect of SH on JNK2. This study will provide a model for exploring mechanisms of processing for reducing toxicities of toxic herbs with multiple components and multi-target natures.

根据中医药理论，酒制可降低豨莶草毒性。豨莶草常用于治疗慢性病，而其长期毒性及炮制减毒机理还不清楚。我们前期的大鼠长期毒性研究表明，酒制能降低豨莶草的肺毒性。大鼠肺组织RNA-Seq转录组分析发现，豨莶草生品明显上调JNK2而炮制品无影响。据报道，JNK2通路的激活与肺毒性相关。基于此，我们提出以下工作假说：1）豨莶草激活JNK2信号通路是其肺毒性的机制之一；2）抑制JNK2信号通路与豨莶草炮制减毒有关。我们将研究：1）豨莶草是否在体内、外剂量及时间依赖性地调控JNK2信号通路；2）JNK2信号通路在豨莶草影响炎症反应及成纤维细胞增殖中的贡献；3）豨莶草影响JNK2通路的上游调控因子。研究结果将有助我们针对性地抑制JNK2通路来优化豨莶草炮制工艺；并提示我们，可通过临床配伍，抑制豨莶草激活JNK2通路而降低该药毒性。同时该研究也为探讨多成分、多靶点的有毒中药的炮制减毒机理提供借鉴。

豨莶草为临床常用中药，具有祛风湿、利关节等功效，临床上常用于治疗类风湿性关节炎等慢性疾病，但其具有小毒。根据传统中医药理论，酒炙可降低其毒性。为了探讨豨莶草的长期毒性及酒炙减毒的机理，本课题建立了体内模型（大鼠长期毒性模型）、体外模型（小鼠巨噬细胞RAW264.7、大鼠肺泡巨噬细胞NR8383、肺成纤维细胞IMR90模型以及RAW264.7-原代成纤维细胞共培养模型）等，试图从体内、外角度深入探讨豨莶草的毒性机理及酒炙减毒的科学内涵。研究结果发现豨莶草生品激活JNK2信号通路诱导炎症反应是其引起肺毒性的可能原因，而酒炙通过抑制JNK2信号通路的激活而降低豨莶草的毒性。此外，通过血清代谢组学分析，我们发现了32个显著改变的代谢物，主要包括氨基酸、脂类、碳氢化合物等。相比较空白组而言，SO组中有14个显著改变的代谢物在PSO组中并没有明显差异。更重要的是，我们还发现豨莶草生品诱导了氧化应激反应，主要通过影响精氨酸的生物合成和甘油磷脂代谢途径；而酒炙后主要通过影响组氨酸代谢，增强自由基的清除能力，从而达到降低毒性的目的。综上，激活JNK2通路引起的炎症反应以及诱导氧化应激反应可能是豨莶草引起肺毒性的主要原因，而酒炙通过抑制JNK2信号通路的激活以及清除自由基来降低豨莶草的毒性。该项目研究结果有助于未来我们针对性地优化豨莶草的炮制工艺；同时，可通过临床配伍，抑制豨莶草激活JNK2通路和氧化应激反应从而降低该药的毒性，为临床安全合理用豨莶草提供研究基础和科学依据。