基于JAK/STAT及PI3K/Akt/mTOR信号通路研究贞术消积汤对肝癌细胞的干预作用及其机制

Zhenzhuxiaoji decoction is a classic prescription in the clinical treatment of liver cancer, which is composed of fructus ligustri lucidi, rthizoma zedoariae, spreading hedyotis herb, selfheal and licorice. In our preliminary in vivo experiments we found that the compound inhibited proliferation of hepatoma cell and induced cell apoptosis and autophagy. At the same time, signal transducer and activator of transcription (STAT3) and survivin gene expression were down-regulated. The decoction induced cell autophagy and apoptosis. Whether the autophagy and apoptosis are rivals or mates is the concern of this project. JAK/STAT and PI3K/Akt/mTOR signaling pathways which are key pathways in cell autophagy and apoptosis, will be taken as the breakthrough point. The possible mechanics will be clarified in cell autophagy and apoptosis after inhibiting the signal pathways. Experiments will conduct both in vivo and in vitro. Biological technologies methods including drug serum method, gene chip technology, flow cytometry, Elisa, real - time PCR and Western blot methods, will be adopted to explore the relationship between cell autophagy and apoptosis. Meanwhile, key factors of signaling pathways will be discovered in the process of cell apoptosis and autophagy. The achievement of this project will uncover the signal network mechanism of the decoction, which clarify the molecular mechanism of the TCM compound antitumor mechanism and to provide a scientific basis for clinical application.

贞术消积汤是临床经验方，由女贞子、莪术、白花蛇舌草、夏枯草和甘草组成，主治毒瘀互结，阴伤络阻，治法为滋阴解毒，活血通络。前期实验发现该复方抑制了肝癌细胞增殖、诱导细胞发生自噬和凋亡，信号转导和转录活化因子（STAT3）和生存素（Survivin）均表达下调。药物诱导细胞自噬和凋亡是拮抗作用，还是协同作用，是本项目重点探讨的问题。JAK/STAT和PI3K/Akt/mTOR信号通路是调控自噬和凋亡的关键性通路。本项目将以这两条信号通路为切入点，通过抑制信号通路，明确其参与凋亡、自噬的可能作用机制。拟通过体内、外实验，采用药物血清法、基因芯片、流式细胞术、Elisa、real-time PCR、Western blot等生物学技术方法，研究细胞自噬和凋亡的关系，筛选信号通路与凋亡、自噬相关的关键因子，以揭示该复方抗肿瘤的信号网络调控机制，为阐明该复方治疗肝癌分子机制和临床应用提供科学依据。

原发性肝癌是我国常见的消化道恶性肿瘤。贞术消积汤由女贞子、莪术、白花蛇舌草、夏枯草和甘草组成，主治毒瘀互结，阴伤络阻，治法为滋阴解毒，活血通络。本课题研究贞术消积汤治疗肝癌的分子机制。课题组开展了体内和体外实验，采用药物血清法、全基因芯片、流式细胞术、Elisa、real-time PCR、Western blot等生物学技术方法，从形态学、分子生物学等多方面进行研究。通过CCK8实验，确定含药血清最佳浓度是17.5%，时间48 h；倒置显微镜观察细胞，该复方致使其数量减少，形态变化，并发生脱落现象；Hoechst、AO和MDC染色，荧光显微镜下观察到细胞凋亡和自噬现象；电镜观察，细胞中出现大量自噬泡；该复方诱导细胞中活性氧（ROS）的产生；全基因组基因芯片分析，复方主要影响肿瘤相关基因，细胞凋亡和自噬及JAK/STAT信号通路相关基因；流式细胞术检测到药物诱导细胞凋亡；Elisa 实验检测到复方影响IL-2、IL-10等细胞因子含量；Real-time PCR、Western blot检测到复方抑制了JAK/STAT和PI3K/Akt/mTOR信号通路基因mRNA和蛋白的表达。课题组也开展了网络药理学研究，对预测的活性物质槲皮素和齐墩果酸抗肿瘤作用进行研究，发现药物在体内外对肿瘤均有抑制作用。实验结论：贞术消积汤通过影响JAK/STAT和PI3K/Akt/mTOR信号通路，诱导肝癌细胞自噬和凋亡。本复方养肝阴以扶正抑癌，且避免了祛邪药物伤正。女贞子作为君药补肾滋阴弥补了莪术的行气破血及白花蛇舌草的清热解毒而引起的伤阴作用，中药配伍理论具有一定的创新性。本课题为该复方的临床应用提供了实验依据。