基于PI3K-Akt-mTOR信号通路介导的自噬性死亡探讨培土生金法干预NSCLC顺铂耐药的分子机制

Autophagy is an important reason for the tumor drug resistance, so inducing cell autophagic death and/or apoptosis is an effective method to relieve tumor drug resistance. PI3K-Akt-mTOR signaling pathway is the focal point which affects apoptosis, autophagy and tumor drug resistance. Traditional Chinese Medicine (TCM) holds that “insufficiency of healthy-Qi” is the principal reason of cisplatin resistance of lung cancer. Early research has shown that the method of strengthening the lung by way of reinforcing the spleen can effectively intervene in cisplatin resistance of non-small cell lung cancer (NSCLC) through inhibiting PI3K-Akt signaling pathway and intervening mTOR expression. Regarding the PI3K-Akt-mTOR signaling pathway as the entry point, with the model of cisplatin resistance of lung orthotopic transplantation tumor models and A549/DDP cells as the study object, by techniques of animal In-Vivo Imaging System, RNAi, transmission electron microscopy and immunoblotting, the project study that the method of strengthening the lung by way of reinforcing the spleen effects intervening cisplatin-resistant cells of NSCLC on inner relation and the feedback mechanism of gene and protein expression of signal related proteins and substrate. So the subject explores the mechanism of the method of strengthening the lung by way of reinforcing the spleen intervening cisplatin resistance of NSCLC by inducing cell autophagic death on PI3K-Akt-mTOR signaling intervening, which further proves PI3K-Akt-mTOR singal transduction regulation is feasible as a new target which improves cisplatin resistance of NSCLC. And it will provide the referable experimental method and theoretical basis for filtering Chinese medicine to improve cisplatin resistance.

细胞自噬是肿瘤耐药的重要原因，诱导细胞自噬性死亡和/或凋亡是逆转肿瘤耐药的有效手段，PI3K-Akt-mTOR信号通路是细胞凋亡、自噬与肿瘤耐药调控的汇聚点。中医认为正气虚损是肺癌顺铂耐药发生的主要原因，前期研究发现培土生金法分别通过抑制PI3K-Akt信号通路、调控耐药相关基因mTOR表达等方式有效干预肺癌顺铂耐药。本项目以PI3K-Akt-mTOR为切入点，以顺铂耐药肺癌原位移植瘤动物模型和A549/DDP细胞为研究对象，运用小动物活体成像系统、RNAi、透射电镜、免疫印迹等技术，分别在组织、细胞、蛋白及基因水平上，观察培土生金法对肺癌顺铂耐药及自噬信号通路相关蛋白及其底物的调控，阐明培土生金法干预PI3K-Akt-mTOR信号通路诱导细胞自噬性死亡逆转NSCLC顺铂耐药的机制，为寻找中西医结合治疗肺癌的有效干预靶点提供实验依据，同时为丰富肺癌耐药病机与治法理论提供新的思路与方法。

肺癌属“肺积”、“痞癖”、“咳嗽”、“咯血”等范畴，为本虚标实之证，其病机特点是全身虚为本，局部瘀痰毒互结成积为标，正虚即是肺癌发生的内因，也是肺癌传变的重要因素；正气不足又以后天脾胃虚弱为基础，土不生金为关键，故治疗肺癌多运用培土生金法。.本研究分别利用补中益气汤（BZYQT）及参芪扶正注射液（SFI）为培土生金法代表方药，协同顺铂共同干预肺癌细胞，观察培土生金方药逆转非小细胞肺癌顺铂耐药的分子机制。.1. 以人肺腺癌顺铂耐药细胞（A549/DDP）为模式细胞，构建裸鼠皮下转移瘤动物模型，培土生金方药联合顺铂具有抑制非小细胞肺癌皮下转移瘤生长的作用。细胞水平上分别应用不同浓度SFI及BZYQT联合顺铂干预A549/DDP细胞，可逆转A549/DDP细胞顺铂耐药的作用。.2. 不同浓度SFI联合顺铂干预A549/DDP细胞，可明显将细胞阻滞于G2/M期，并检测到P53及P21蛋白表达上调；激活caspase 3及PARP活性片段的表达，下调Bcl-2蛋白、上调Bax蛋白的表达，且凋亡抑制剂z-VAD-fmk预干预，可部分抑制培土生金联合顺铂诱导的细胞死亡；诱导细胞发生自噬性死亡，且自噬抑制剂3-MA预干预，可部分抑制BZYQD 联合顺铂诱导的细胞死亡；增加细胞中ROS含量，抑制剂（NAC）几乎完全抑制BZYQD联合顺铂所引起的A549/DDP细胞死亡；上调顺铂耐药细胞A549/DDP线粒体融合蛋白（Mfn2）的表达，并增加细胞中活性氧ROS含量、降低线粒体膜电位、抑制剂NAC几乎完全抑制了SFI与顺铂协同作用所引起的A549/DDP细胞Mfn2的升高、逆转了SFI与顺铂协同作用所引起的细胞周期阻滞及细胞凋亡。.此外，该项目还支持了中药缓解化疗不良反应分子机制的研究，结果显示：化疗前给予补中益气汤干预可有效改善5-氟尿嘧啶所致的小鼠腹泻症状、降低小肠炎症反应、抑制黏膜细胞凋亡；此外，还可增加肾组织抗氧化相关酶（超氧化物歧化酶、过氧化氢酶等）活性、抑制化疗药物诱导的肾小球细胞凋亡，改善肾脏功能；中药肉苁蓉通过调节卵巢组织ATP合成酶活性、调控线粒体融合蛋白表达、抑制化疗药物引起的卵巢颗粒细胞凋亡，从而保护卵巢，降低化疗药物引发的卵巢早衰现象。