基于毒损肝络理论从线粒体途径研究健脾活血祛湿方调控AQP8、9对H22荷瘤小鼠、肝癌细胞凋亡的影响

“Toxin hurts liver collaterals”theory is an important theory to guide the prevention and treatment of liver disease of traditional Chinese medicineand.It suggests that the poison and blood stasis block in liver collaterals is the key to thedevelopmentoflivercancerpathogenesis..We make the Jianpihuoxuequshi prescription as part of treatment to liver cancer based upon the theory of “Toxin hurts liver collaterals”.Find it can significantly reduce the radiation and chemotherapy side effects, improve the quality of survival, but its mechanism is yet to be in-depth study. Liver cancer cell apoptosis related closely to occurrence and development of liver cancer, mitochondria pathway is an important way of promoting the cell apoptosis, but this way upstream factor is unclear. We have found this prescription can influnce the AQP8、9 expression and apoptosis of liver cancer cells through the recently research.This research based upon the theory of AQP8, 9 - mitochondrial apoptotic pathways activated - liver cancer cell apoptosis hypothesis, use the H22 tumor - burdened mice and H22 liver cancer cells in vito as the research object, the Jianpihuoxuequshi prescription to stimulate, by immunofluorescence and rt-pcr, WB, JC - 1 means in biology study the anti-tumor effect of this prescription and from mitochondria approach to study the prescription through regulating AQP8, 9 intervention mechanism of action of liver cancer cell apoptosis, for “Toxin hurts liver collaterals” instruction will provide a scientific basis for prevention and treatment of liver disease.

“毒损肝络”理论是指导中医药防治肝病的重要理论，认为毒瘀作祟、阻滞于肝络是肝癌发生发展的关键病机。课题组以此立论，组健脾活血祛湿方辅助治疗肝癌，能显著减少放化疗后副反应，提高患者生存质量，但其作用机制尚待深入研究。肝癌细胞凋亡与肝癌发生、发展关系密切，线粒体途径是细胞凋亡的重要途径，但此途径的上游因子尚不明确。前期研究发现该方能提高肝硬化腹水大鼠AQP8、9表达，减轻线粒体水肿，促进ATP合成，提高CYT-C表达，初步发现水通道蛋白与线粒体具有一定相关性。本项目以AQP8、9→线粒体凋亡通路激活→肝癌细胞凋亡假说立论，以荷H22肝癌移植瘤小鼠及体外培养的H22肝癌细胞为研究对象，健脾活血祛湿方为刺激方法，用免疫荧光、RT-PCR、WB、JC-1等生物学手段研究该方的抗肿瘤作用及从线粒体途径研究该方通过调控AQP8、9干预肝癌细胞凋亡的作用机制，为“毒损肝络”理论指导防治肝病提供科学依据。

肝癌是常见的恶性肿瘤，具有复发率高、转移率高、治愈率低的特点，死亡率仅次于胃癌和肺癌，是导致癌症相关死亡的第三大原因。“毒损肝络”是指导中医药防治肝病的重要理论，毒瘀作祟、阻滞于肝络，出现络虚、毒侵，而化毒为害则是肝病迁延和深化的关键所在。课题组以此立论，自拟健脾活血祛湿方辅助治疗肝癌，方由五爪龙、白术，田基黄、猪苓、白背叶根，土鳖虫、茜根，砂仁组成，全方化瘀利湿而不伤脾胃，共奏扶正解毒、健脾通络、祛湿之功。该方临床用于肝病防治效果显著。.促进肝癌细胞凋亡是目前防治肝癌的有效途径，线粒体途径是细胞凋亡的经典途径之一。前期研究发现该方能提高肝硬化腹水大鼠AQP8、9表达，减轻线粒体水肿，促进ATP合成，提高CYT-C表达，初步发现水通道蛋白与线粒体具有一定相关性，鉴于此，本项目以AQP8、9→线粒体凋亡通路激活→肝癌细胞凋亡假说立论，以荷H22肝癌移植瘤裸鼠及体外培养H22肝癌细胞为研究对象，以健脾活血祛湿方为干预手段，利用免疫荧光、RT-PCR、WB、JC-1等生物学检测方法，从线粒体细胞凋亡途径相关蛋白表达及AQP8、9表达变化方面，探究该方抗肿瘤的作用机制。.本研究表明健脾活血祛湿方可有效改善肝脏肿瘤病理形态，抑制肝癌细胞瘤生长，减小瘤体体积和质量，同时能起到改善肝功能作用，这可能是通过上调水通道蛋白 8、9，同时降低细胞线粒体膜电位、释放Cty-C、Bcl-2、Bax、Caspase-3等凋亡因子，促进肝癌细胞凋亡，提高肝肿瘤宿主的免疫应答（脾指数降低）造成的；同时离体实验结果也表明，健脾活血祛湿方干预后，H22肝癌细胞增殖抑制率呈剂量依赖性升高，降低细胞线粒体膜电位和抗凋亡因子Bcl-2水平，AQP8、9和AQP8、9mRNA以及促凋亡因子Bax的表达呈剂量依赖性升高，有效促进H22肝癌细胞的凋亡。课题研究为临床中医药治疗肝癌提供实验室依据，也为“毒损肝络”理论指导防治肝病提供科学依据，值得进一步推广。