解毒颗粒调控MyD88/Snail信号轴介导的EMT抑制肝癌转移的作用及分子机制

Metastasis is the leading cause of death in patients with hepatocellular carcinoma (HCC) and lack of effective treatment. Jiedu granule is the preparation developed under the guidance of TCM "qingrejiedu" anticancer therapeutic principle. The clinical application shows that it has exact effect on the recurrence and metastasis of HCC. Our previous work also suggested that Jiedu granule could inhibit the migration and invasion of hepatocellular carcinoma cells in vitro, but the related molecular mechanism remains unclear. Epithelial-mesenchymal transition( EMT）is one of the key biological processes in HCC metastasis which can promote the occurrence of metastasis and is one of the possible targets for clinical treatment. Therefore, it has important biological significance. MyD88/Snail signal axis is an important regulatory pathway of HCC metastasis, recent studies have shown that it can regulate the occurrence of EMT and promote the invasion of hepatocellular carcinoma cells. Our preliminary experimental data show that Jiedu granule can influence the expression of MyD88 and Snail, as well as EMT molecular markers such as E-cadherin and Vimentin of HCC cells. Based on the above results, we plan to observe the anti-metastasis effect and further analyze the regulatory mechanism on EMT mediated by MyD88/Snail signal axis. We intend to explore the detail molecular mechanism by small animal in vivo imaging technology, invasion assay and other assays both in vitro and in vivo. Our research could be contribute to clarify the anti-metastasis mechanism of Jiedu granule, offer valuable reference for clinical treatment of HCC metastasis and further provide experimental evidence for related anticancer therapeutic principle of TCM.

肝癌的转移是临床治疗的难点。解毒颗粒是本科室在中医“清热解毒”抗癌理论指导下研发的抗肿瘤制剂，临床实践表明其具有良好的抗肝癌复发转移作用，课题组前期工作也提示解毒颗粒能够在体外抑制肝癌细胞的迁移和侵袭，但具体机制尚不清楚。上皮间质转化（EMT）是肝癌转移过程中的关键环节之一，能够促进肝癌细胞的侵袭，是临床治疗的潜在靶点，具有重要的生物学意义。近年研究显示，MyD88/Snail信号轴是肝癌转移的重要调控通路，能够通过调节EMT发挥促转移作用。我们的预实验结果发现解毒颗粒能够影响肝癌细胞EMT分子标志物及MyD88、Snail分子的表达。基于以上结果，本项目拟从MyD88/Snail信号轴介导的EMT调控出发，利用小动物活体成像、侵袭实验等方法在体内外明确解毒颗粒抗肝癌转移效应，进一步解析相关的作用途径和分子机制。本研究有助于明确解毒颗粒的抗转移机理，并为中医相关抗癌理论提供实验依据。

肝癌的转移是临床治疗的难点。解毒颗粒是在中医“清热解毒”理论指导下研发的抗肿瘤制剂，课题组前期临床实践和预实验均提示其具有良好的抗肝癌转移作用，但相关机制仍不清楚。上皮间质转化（EMT）是肿瘤转移过程中的重要环节，也是临床治疗的潜在目标。本项目以MyD88/Snail信号轴介导的EMT调控为切入点，探讨解毒颗粒抗肝癌转移效应和机制。结果显示，解毒颗粒能够抑制多种肝癌细胞的迁移、侵袭及EMT的发生，可以上调肝癌细胞中E-cadherin分子的表达，同时下调N-cadherin、Vimentin等分子的表达。信号通路分析发现解毒颗粒能够抑制NF-κB的转录活性、IKBɑ的磷酸化以及ERK1/2的磷酸化，并能够抑制肿瘤转移调节分子Rac1的水平但促进抑癌分子Rb的表达。进一步的机制研究提示，解毒颗粒能够抑制MyD88的表达，并下调EMT上游转录因子Snail的水平，但对Twist等调控因子无明显影响。功能实验结果表明，在MHCC97H细胞中过表达MyD88分子后，能够部分逆转解毒颗粒对肝癌细胞迁移、侵袭的抑制效应，并能改变解毒颗粒对EMT相关分子标志的调控状态。过表达MyD88还能够部分逆转解毒颗粒对EMT上游转录因子Snail水平的下调以及对NF-κB、ERK1/2等信号通路的抑制，同时改变解毒颗粒对Rac1表达的调节作用。此外，解毒颗粒还能够抑制TNFɑ、IL-6等炎性因子的表达及VEGF、MMP-2等肿瘤转移相关因子的分泌，过表达MyD88能够部分逆转解毒颗粒对上述因子表达和分泌的抑制作用。这些结果明确了解毒颗粒的抗肝癌转移效应和对EMT的调节功能，提示MyD88/Snail信号轴可能是其关键作用靶点之一。以上结果有助于进一步揭示解毒颗粒抗肝癌作用的分子机制，为其临床应用提供实验依据，并可以丰富肝癌的中西医结合治疗思路及可能的治疗靶点，具有较好的科学意义和价值。