基于mTOR信号通路调控缺氧微环境下细胞自噬的消癌解毒方抗肿瘤作用机制研究

Xiaoai Jiedu Prescription is an effective anti-cancer drug whose efficacy has been proved by a long period of clinical practice under the guidance of cancerous toxin theories proposed by Professor Zhou Zhongying, a great master of traditional Chinese medicine. Prior studies confirmed the anti-cancer effects of this drug and its benefits of enhancing efficacy and reducing toxicity when used in combination with chemotherapy. It was also shown that Xiaoai Jiedu Prescription was able to affect the mTOR signaling pathway and autophagy-related factors. The mTOR signaling pathway regulates autophagy of tumor cells in an anoxic microenvironment, which is an important influencing factor in tumor progression and sensitivity to chemotherapy. This may be the effective means for Xiaoai Jiedu Prescription to fight tumors and enhance efficacy and reduce toxicity in chemotherapy. . Based on previous research, this study looked at the mTOR signaling pathway-mediated autophagy. Through pharmacological and genetic intervention, animal carriers and cell research were used along with chemotherapeutic drugs to examine the mechanisms of Xiaoai Jiedu Prescription for fighting tumors and affecting the susceptibility of tumor cells to chemotherapy. This study would hopefully provide some new ideas in guiding the clinical use of Xiaoai Jiedu Prescription and tumor treatment with traditional Chinese medicine.

消癌解毒方是国医大师周仲瑛教授癌毒理论指导下，经长期临床实践证明的有效抗癌方。前期研究肯定了该方的抗肿瘤疗效以及临床配合化疗的增效减毒作用。我们前期工作发现消癌解毒方可以影响mTOR信号通路和细胞自噬相关因子，由于mTOR信号通路调控缺氧微环境下肿瘤细胞的自噬，是影响肿瘤发展和化疗敏感性的重要因素，这可能是消癌解毒方发挥抗肿瘤和对化疗增效减毒作用的有效途径。. 项目以前期研究为基础，从mTOR信号通路介导的细胞自噬入手，通过药理学及基因干预的方法，采用动物在体以及细胞研究相结合的方式，配合化疗药物，来探明消癌解毒方抗肿瘤和影响肿瘤细胞化疗效果的机制，进一步指导该方的临床应用，为中医药抗肿瘤研究提供新思路。

背景：立足前期研究，从细胞自噬角度，通过体内与体外实验，研究消癌解毒方（XJF）抗肿瘤作用机制。研究内容：1.体内实验①建立人肝癌SMMC-7721细胞裸鼠移植瘤模型，观测抑瘤率；HE染色法检测瘤体组织；免疫组化与western blot法检测自噬、凋亡相关蛋白表达情况。应用自噬化学抑制剂羟氯喹（HCQ）和化学诱导剂雷帕霉素（RAP），研究XJF对mTOR和自噬信号通路的调控作用。②对瘤体组织行miRNA测序分析，并筛选差异表达miRNA。2.体外实验①用人肝癌细胞株SMMC-7721进行研究。以透射电镜和GFP-RFP-LC3B双荧光蛋白检测自噬形成；结合HCQ、RAP和siRNA技术，以western blot法检测LC3、 beclin1等蛋白的表达。②检测XJF对正常人肝细胞的毒性作用。结果：1.体内实验①XJF能抑制SMMC-7721荷瘤裸鼠移植瘤的生长，促进LC3II、Beclin1和mTOR信号通路关键蛋白的表达，并呈剂量依赖。XJF联合RAP能提高抑瘤率；TUNEL法显示瘤体凋亡率明显增加；Western Blot法显示Bax、cleaved-caspase3表达增加，Bcl-2蛋白降低。而XJF联合HCQ则表现出相反结果。②移植瘤miRNA测序分析，共筛选出185个差异表达miRNA和263条肿瘤相关信号通路。2.体外实验①XJF含药血清能剂量依赖性抑制SMMC-7721细胞活性，诱导细胞内自噬体和自噬溶酶体的累积，促进LC3II和Beclin1蛋白表达，抑制p62蛋白表达；联合HCQ及si-Beclin1能够反调控上述蛋白。XJF含药血清能抑制p-AKT、p-mTOR、p-p70s6K、p-p4EBP1蛋白表达，联用si-AKT后，LC3蛋白表达明显增加。XJF含药血清能增加Bax、cleaved-caspase3表达，降低Bcl-2表达，而HCQ和si-Beclin1能逆转此作用。②XJF含药血清体外对人正常肝细胞LO2活性未见明显影响。科学意义：本研究发现，XJF能通过诱导SMMC-7721细胞自噬发挥抗肿瘤作用，增强RAP的抗肿瘤效果。并确定该方作用该通路的关键因子，筛选相关差异表达的miRNA。为该方的抗癌机理研究，提供新思路，为临床治疗提供实验依据，进一步丰富“癌毒”理论的内涵。