板栗壳原花青素诱导肿瘤细胞自噬性死亡及其机制研究

It is always a hotspot for tumor treatment research to look for efficiency and low toxicity antitumor drugs from plants. Autophagic death in tumor cells induced by active ingredient may provide novel strategies for the treatment of cancer treatment. Studies have proved that procyanidins is safe and nontoxic, which can promote the apoptosis of tumor cells. But the autophagic death in tumor cells induced by procyanidins and its mechanism remain unclear. Based on this fact, in view of the chemical composition, the mechanism, and the dose and time-dependent effect of procyanidins from chestnut shell (CSPCs) on the activity of autophagic death need further research. The composition and content of CSPCs will be determined, and then the autophagic death of human hepatoma HepG2 cell induced by CSPCs will be investigated in the project. This study mainly researches the effect of CSPCs on the express of three major effector molecule in phosphatidyl inositol 3-kinase/protein kinase B/mammalian target of Rapanycin, (P?3K/Akt/mTOR) pathway, Beclin 1 and associated Bcl-2 family members to the expression level of microtubule-associated protein 1 light chain 3 П (LC3П), attempts to reveal the molecular mechanism of tumor cell autophagic death induced by CSPCs, and provides a theoretical basis for the development of highly active plant ingredients from Chinese characteristics plant resources and agricultural processing by-products to improve the health of our country.

从植物中寻找高效低毒抗肿瘤作用的活性成分一直是世界抗肿瘤药物研究热点。活性成分诱导肿瘤自噬性死亡的研究是目前肿瘤治疗的新策略，业已证实原花青素是能促进多种肿瘤细胞凋亡的活性成分，且安全无毒，但其诱导肿瘤自噬性死亡活性及机制研究未见报道。基于此，本项目拟以板栗壳中原花青素(CSPCs)为对象，针对化学组成、剂量效应和时间效应对其诱导肿瘤细胞自噬性死亡活性不清楚，作用机制缺乏研究的现状，拟在确定CSPCs组成和含量后，以人肝癌HepG2为细胞模型，研究CSPCs诱导人肝癌细胞自噬性死亡作用。重点研究CSPCs对P?3K/Akt/mTOR通路的3个主要效应分子的表达、Beclin 1和与之相关的Bcl-2家族成员及其对LC3П表达量的影响，揭示CSPCs诱导肿瘤细胞自噬性死亡活性和分子机制，为深化我国特色植物资源和农产品加工副产物，开发高活性植物成分提高国民健康提供理论依据。

板栗壳占板栗重量8.9-13.5%，目前仍以燃烧和自然腐烂方式被废弃，仅少量被利用。而关于其化学成分报道甚少，更未涉及结构和活性研究。基于此，本项目确定了板栗壳抗肿瘤活性成分是以低聚体为主的原花青素类物质 (CSPCs)，进而研究其诱导人肝癌HepG2细胞自噬性死亡及其分子机制。结果如下：.（1）CSPCs分离与结构鉴定.以水为提取溶剂，大孔树脂富集、凝胶柱层析分离，采用UV、IR、HPLC、ESI-MS、NMR确定CSPCs是以低聚体为主的原花青素，含量90%以上。.（2）CSPCs诱导HepG2细胞凋亡及其分子机制.100-400μg/mL CSPCs能抑制HepG2细胞增殖，IC50为153.34μg/mL，300μg/mL的CSPCs作用4d抑制率达81.72%，凋亡率为11.25%，G0/G1期阻滞，ROS增加2.0倍，Bax/Bcl-2表达率增加3.2倍，进而激活caspase-3和-9诱导凋亡。.（3）CSPCs诱导HepG2细胞自噬及其分子机制.50-300μg/mL CSPCs作用HepG2细胞6-36h可诱导其自噬性死亡，增加自噬体和LC3-II量，GFP-LC3点状聚集增加5.9倍。CSPCs介导的自噬依赖于ROS产生。3-MA削弱CSPCs诱导的自噬，可下调PI3K、AKT和mTOR表达，上调Beclin1表达。.（4）CSPCs诱导HepG2细胞自噬和凋亡的相关性.自噬抑制剂3-MA和凋亡抑制剂Z-VAD能抑制CSPCs诱导细胞自噬性死亡。CSPCs下调PI3K/AKT/mTOR通路蛋白表达促进HepG2细胞自噬性死亡；LC3-II si-RNA干扰抑制LC3-II蛋白表达能有效减少caspase3表达；Bax、Bad和Bcl-2参与自噬和凋亡。CSPCs抑制PI3K/AKT/mTOR信号通路诱导细胞自噬，而促进凋亡。.（5）莲房原花青素（LSPCs）诱导HepG2细胞自噬及与凋亡相关性.25-100μg/mL LSPCs诱导HepG2细胞自噬，自噬体和LC3-II量均增加。LSPCs诱导HepG2细胞自噬与ROS有关，可被NAC阻断。3-MA 抑制CSPCs诱导细胞凋亡；Z-VAD抑制LSPCs诱导细胞自噬；Bax、Bad、Bcl-2参与ROS诱导的自噬和凋亡；PI3K/AKT/mTOR、ERK和JNK途径参与ROS诱导的自噬和凋亡。