桑黄吡喃酮类成分调控Hedgehog信号通路的抗结肠癌分子机制研究

The incidence and mortality of colon carcinoma have been ranked the forefront of cancer. Phellinus igniarius, a medicinal mushroom, because of its anti-tumor activity shows a good prospect. Hedgehog (Hh) gene family is an important signal molecule in the endoderm, abnormal activation of which often leads the occurrence of tumor. Our previous studies have shown that a pyrones component called Inoscavin A, which separated from Phellinus igniarius, has an anti-colon carcinoma activity, and it can inhibit Hh signaling pathway. Through molecular docking we discovered that Inoscavin A could form stable complexes with Smo which is the key receptor of Hh pathway, speculating that Smo is the target of Inoscavin A. Based on these, the project intends to use HT-29-Luc cell lines to construct orthotopic transplantation tumor as animal model in vivo, and use HT-29 cell lines and Smo siRNA and pCMV-Smo-Flag HT-29 cell lines in vitro, via small animal living imaging technique, immunofluorescence, cell chip technology and so on, to observe the anti-colon carcinoma activity of Inoscavin A, and to reveal the anti-colon carcinoma molecular mechanism of Inoscavin A based on direct inhibiting Smo receptor to block Hh signaling pathway. These results will lay the foundation of the anti-colon carcinoma application of Phellinus igniarius in clinic, and provide reference for further study of anti-tumor mechanism of Phellinus igniarius.

结肠癌的发病率和死亡率已位居恶性肿瘤前列。药用真菌桑黄因其抗肿瘤活性展现出良好的应用前景。Hedgehog(Hh)基因家族是重要的内胚层信号分子，其异常激活常常导致肿瘤的发生。课题组前期研究表明桑黄吡喃酮类成分Inoscavin A具有抗结肠癌活性，可抑制Hh信号通路，分子对接显示Inoscavin A与Hh通路关键受体Smo可形成稳定的复合物，推测Smo为Inoscavin A作用靶点。基于此，本项目体内拟采用HT-29-Luc细胞系构建原位移植瘤动物模型，体外拟采用HT-29细胞系及Smo siRNA和pCMV-Smo-Flag HT-29细胞系，通过小动物活体成像、免疫荧光和细胞芯片等技术，观测Inoscavin A抗结肠癌活性，揭示Inoscavin A直接抑制Smo受体阻断Hh信号通路的抗结肠癌分子机制。研究结果将为桑黄抗结肠癌临床应用奠定基础，为探讨桑黄抗肿瘤作用机制提供参考。

Hedgehog信号通路的异常激活与结肠癌发生密切相关，可干扰下游Caspase介导的细胞凋亡途径。桑黄吡喃酮类成分Inoscavin A具有抗结肠癌活性，本项目通过体内外实验，结合组织病理学、免疫荧光、质粒转染、细胞生物学和分子生物学等方法，对Inoscavin A抑制Hedgehog信号通路诱导细胞凋亡的抗结肠癌分子机制进行了研究。首先，构建HT-29细胞裸鼠移植瘤动物模型，通过瘤体大小测量和组织病理学观察证实Inoscavin A对HT-29细胞裸鼠移植瘤具有抑制作用，平均抑制率23.4%；其次，通过免疫荧光、RT-PCR和Western blot方法，证实Inoscavin A可抑制肿瘤组织中Shh、Ptch、Smo和Gli1的表达，阻断Hedgehog信号通路。之后，采用Smo Double Nickase 质粒和Smo CRISPR Activation 质粒构建Smo沉默和过表达HT-29细胞系，通过平板克隆法、划痕法和Transwell法证实Smo沉默可增强Inoscavin A对HT-29细胞增殖、迁移和侵袭能力的抑制作用，而Smo过表达则可削弱该作用；同时，通过线粒体膜电位检测、TUNEL凋亡检测和流式细胞技术证实Smo沉默可增强Inoscavin A 诱导HT-29细胞凋亡作用，而Smo过表达则可削弱该作用；随后，通过RT-PCR和Western blot方法证实Smo沉默可增强Inoscavin A 对Hedgehog信号通路的阻断作用，而Smo过表达则可削弱该作用，提示Smo是Inoscavin A的作用靶点，揭示Inoscavin A可直接抑制Smo阻断Hedgehog信号通路诱导细胞凋亡来发挥抗结肠癌作用。除项目规定外，课题组还通过高分辨质谱和分子生物学技术对Inoscavin A主要富集部位桑黄二氯甲烷提取部位（P. igniarius DCM）进行化学成分和抗结肠癌机理研究，明确P. igniarius DCM主要由20种化学成分组成，并通过体外细胞实验证实P. igniarius DCM抗结肠癌作用是通过调控线粒体凋亡途径（上调Bax/Bcl-2比率，促进Caspase3/9切割）诱导细胞凋亡来实现的。这些研究结果初步明确了桑黄抗结肠癌的药效物质和作用机理，可为桑黄抗结肠癌临床应用奠定基础，为深入探讨桑黄抗肿瘤作用机制提供参考。