正交生物学方法对四君子汤与丝裂霉素C联用降低化疗毒性机制研究

This project intends to confirm the results of traditional non-targeted metabolomics on the integral of metabolites, proteins and genes levels by orthogonal biology analysis. And this integration project was used to solve the problem that the mechanism elucidating of chemotherapy-assisted Chinese medicine Sijunzi Decoction (SJZD) prevent chemotherapy drug mitomycin C (MMC)-induced toxicity. Based on the previous studies, 1H NMR and UPLC-MS /MS based non-targeted metabolomics and targeted metabolomics were used in this study to screen and confirm the biomarkers of MMC-induced toxicity and attenuation effect of SJZD. The metabolic pathways were deduced by KEGG, MetaboAnalyst and IPA, and were confirmed by stable isotope-assisted analysis. Finally, orthogonal biology analysis was used to confirm the key enzymes and genes involved in the pathways. ELISA, antimetabolites or immunohistochemistry was used on protein level and RNA interference or CRISPR-Cas technology were used on gene level. And then, comprehensive analysis on the mechanism of the toxicity attenuation effect was performed. This project provides a new way to confirm the results of non-targeted metabolomics and to optimize the combination of chemotherapeutic drugs with chemotherapy-assisted traditional Chinese medicine.

本项目拟通过正交生物学技术对传统非靶向代谢组学的研究结果进行代谢物、蛋白质和基因三个水平的综合确证，并利用该整合方案解决化疗辅助中药四君子汤（SJZD）与化疗药物丝裂霉素C（MMC）联用后降低MMC化疗毒性机制的问题。本课题组在前期研究基础上，拟首先采用基于1H NMR与UPLC-MS/MS非靶向代谢组学与靶向代谢组学研究，筛选并确证MMC引发毒性及SJZD减毒的生物标志物，然后利用KEGG、MetaboAnalyst及IPA等方法推断代谢通路并通过稳定同位素辅助分析方法确证代谢通路，最后采用正交生物学方法，通过ELISA、抗代谢物或免疫组织化学技术从蛋白质水平、利用RNA干扰或CRISPR-Cas技术从基因水平对代谢通路涉及关键酶及基因进行分析确证，进而综合分析SJZD联用方法对于MMC毒性的降低作用机制。本项目为代谢组学结果确证及优化化疗药物与化疗辅助中药的联合应用提供新的思路。

四君子汤（SJZD）作为一种效果良好的化疗辅助中药，与抗肿瘤药物丝裂霉素C（MMC）合用，可以有效降低MMC的毒副作用。本项目运用了结合非靶向与靶向代谢组学、稳定同位素标记、RT-PCR、 Western blot、MTT和ELISA等手段的正交生物学技术，从广泛定性到靶向定量两个层面构建MMC作用后大鼠体内整体的代谢特征及相关的异常代谢通路，监测SJZD联用给药前后内源性代谢物种类与含量变化，寻找生物标志物相关的代谢通路及其涉及的关键酶，对这些因素深层的相互关系进行构建和关联，并结合对免疫细胞及免疫分子的影响，综合分析MMC产生毒性及SJZD对于MMC的减毒作用机制。研究发现，天冬氨酸、异亮氨酸、琥珀酸、天冬酰胺和焦谷氨酸为MMC诱导免疫毒性的生物标志物， MMC诱导的免疫毒性主要与影响丙氨酸、天冬氨酸与谷氨酸代谢通路中ASNS催化反应相关；初步探讨出SJZD与MMC联用后，通过调节丙氨酸代谢和ASNS表达量，在正常细胞中可使肝细胞的糖异生和天冬酰胺合成维持在正常水平，有利于维护正常肝细胞的功能，在肝癌细胞中可能阻止肝癌细胞中DNA或RNA的形成，诱导肝癌细胞凋亡，抑制肝癌细胞的增殖、迁移、侵袭、克隆形成、自我更新等，有利于协同发挥抗肿瘤效应；SJZD对免疫细胞的生长及细胞因子（IL-2和IFN-γ）的分泌具有很好的促进作用，可以在一定程度减弱MMC对脾淋巴细胞的毒性以及对细胞分泌IL-2和IFN-γ的抑制作用，展现出了较好的免疫治疗前景。本研究为MMC及SJZD的临床应用提供科学依据，为化疗辅助中药的临床应用提供参考。