基于代谢酶调控的当归补血汤在吉西他滨治疗非小细胞肺癌中的减毒增效机制研究

Chemotherapy, a major treatment for non-small cell lung cancer (NSCLC), with median survival time (MST) of 8 months, leads to obvious side effect like myelosuppression and nephrotoxicity by using first-line drugs as gemcitabine. When gemcitabine was accompanied with Danggui buxue tang, the patients’ immunity improved and side effects alleviated, thus enhancing tolerance and prolonging survival time. Herb-drug interaction is ubiquitous，and the important proteins P-gp and GST-Pi could be regulated by the main active components in Astragalus membranaceus and Angelica sinensis, respectively. Yet it still remains unknown whether there is a herb-drug interaction, and how the interaction works between Danggui buxue tang and gemcitabine. Therefore, this project will compare pharmacokinetics of the major active components of gemcitabine and Danggui buxue tang with these two drugs administrated alone or combined, by hyphenated techniques on tumor-bearing mice and lung cancer cells, to study the influences on the physiological disposition. Comparing the expression and the activity of metabolic enzymes (dCK, CDA, GST-Pi) and transporters (hENT, hCNT, P-gp) on tissues, A549 cells and A549/Gem cells in different administration groups, to study the regulation mechanism, as well as analyzing the drug efficacy, toxicity and cytokine, will clarify the mechanism of efficacy enhancement and toxicity reduction of herb-drug combination. This work will not only provide scientific evidence for the clinical efficiency, but also guide rational usage of herb-drug combination, which facilitate further studies for anti-tumor mechanism of herb-drug combination.

化疗是治疗非小细胞肺癌的主要手段，但即使应用一线药物吉西他滨，患者的中位生存期也仅8个月，且伴随明显的骨髓抑制和肾毒性等副作用。当归补血汤联合吉西他滨治疗，可显著提高患者免疫功能、缓解毒副作用，延长生存时间。药物相互作用普遍存在，当归补血汤中黄芪和当归的主要活性成分分别对吉西他滨耐药相关蛋白有调节作用，但当归补血汤和吉西他滨是否存在相互作用、减毒增效机制如何发挥均未见报道。本项目拟从动物和细胞两水平，采用联用技术分析两药联用前后，主要活性成分在体内、外的药代动力学过程，研究药物相互作用；通过测定两药联用前、后，荷瘤小鼠各组织、肺癌细胞和耐药细胞中吉西他滨转运体和代谢酶的表达及活力，研究当归补血汤对吉西他滨代谢酶/转运体调控机制和逆转耐药机制，并结合药效、毒性评价和细胞因子分析，最终揭示当归补血汤与吉西他滨的增效减毒机制，为临床合理用药提供科学依据，并为其他中西药联用抗肿瘤机制研究提供思路。

肺癌是目前全世界发病量与死亡病例最多的癌症，非小细胞型肺癌约占肺癌总数的85%。吉西他滨作为第三代细胞毒性化疗药，广泛应用于肺癌、胰腺癌等癌症的临床全身性化疗中。然而，吉西他滨的化疗耐药性问题日趋严重，对肿瘤的转移和复发黔驴技穷。近年来，临床研究发现，传统中药与化疗药联合治疗肿瘤，可显著提高治疗响应率，减轻毒副反应，提高患者耐受性，延长生存期。但是，目前中西药联用治疗肿瘤的作用机制相关研究仍然极度匮乏。在本研究中，我们首先优化了当归补血汤的提取方法，之后，本研究采用高效液相色谱-质谱联用（HPLC-MS/MS）技术，比较了吉西他滨联用当归补血汤后大鼠外周血单个核细胞中吉西他滨代谢物的药代动力学行为，发现吉西他滨联用当归补血汤可显著增加外周血单个核细胞中吉西他滨三磷酸盐代谢产物的含量。在药效学实验中联用组的抗肿瘤效果更加显著，因此，对肿瘤组织中吉西他滨关键代谢酶以及其上游、下游蛋白进行了研究，比较了中西药联用前后荷瘤小鼠的血象及免疫指标，结果表明当归补血汤不仅调控脱氧胞苷激酶及其相关蛋白而且可改善吉西他滨造成的免疫及造血系统损伤。在体内动物实验中发现当归补血汤可有效减轻吉西他滨引起的骨髓抑制。首先，分析了器官指数，外周血细胞和造血相关细胞因子，当归补血汤可显著改善吉西他滨诱导的骨髓抑制小鼠的造血功能。之后，发现当归补血汤可通过增加细胞计数，加速细胞周期过程和上调细胞周期蛋白D1、细胞周期蛋白依赖性激酶来促进骨髓细胞的增殖。本研究从药代动力学和药效学两个方面，评价了当归补血汤联用吉西他滨的体内相互作用及其作用机制，阐明了临床中西药联合用药的合理性，为临床当归补血汤的合理用药提供了科学依据。