基于肝癌小鼠肾上腺皮质功能异常研究SGK1通路在因实致虚病机中的作用及中药拆方干预

Syndrome differentiation and treatment are used broadly in China to prevent and treat primary liver cancer (PLC). On the basis of the unified understanding of the pathogenesis of PLC, which is deficiency resulted from excess syndrome and the deficiency and excess appearing together. And the mTORC2/GR-SGK1 signaling pathway gene expression are abnormal in tumor and adrenal tissue. The hypothesis is proposed that excess syndrome maybe caused by the mTORC2/GR-SGK1 signaling pathway genes are activated in tumor and deficiency syndrome maybe resulted from adrenal decompensation. Therefore, we prepare to screen the high expression SGK1 cell among HepG2, SMMC7721 and Huh-7 hepatocellular carcinoma cells line. Then, SGK1 knock-out stable expression hepatocellular carcinoma cell line will be establised by RNAi. After that, mTOR inhibitor rapamycin and Glucocorticoid and different Chinese medicine prescription will be treated the SGK1 knock-out hepatocellular carcinoma cells, and cell proliferation and apoptosis will be detected by MTT and flow cytometry, SGK1 target genes such as FOXO3, FasL and p27 will be tested by real-time quantitative PCR (qPCR), mTORC2, SGK1, p-SGK1 and GR are tested by western blot. Additionally, the serum of liver cancer mice will be pretreated adrenocortical cells H295R, then different Chinese medicine prescriptions are added the cells. And H295R cell proliferation will be detected, cortisol also be tested by ELISA and some enzymes such as StAR, CYP11A1, CYP21A2 and CYP11B1 will be arrayed by real-time quantitative PCR. It is to study the secretions interaction of between tumor and adrenal cells in vitro. Further, BALB/C-nu/nu mcie will be injected with SGK1 over-expression or SGK1 knockout hepatocellular carcinoma cells. After about 7 days, different Chinese medicine prescriptions will be used to treat for some weeks. And plasma corticosterone are tested by ELISA, tumor cell proliferation and apoptosis will be detected, the mTORC2/GR-SGK1 signaling pathway in the tumor are monitored by qPCR and western blot. Adrenal tissue will be observed by transmission electron microscope and some enzymes will be tested by qPCR. It is incredible value for the study, as the molecular mechanism of deficiency resulted from excess syndrome in liver cancer mice will be clear, and the adrenal dysfunction maybe a key role. It is also help to enrich and develop the theory of deficiency and excess syndromes appearing together in tumor.

辨证论治在我国肝癌防治中具有广泛的社会需求。基于对肝癌病机"因实致虚、虚实夹杂"的基本共识，及课题组发现肝癌小鼠肿瘤与肾上腺存在SGK1相关信号通路基因表达异常，提出了该通路激活导致肝癌恶性增殖继发肾上腺皮质机能衰退是因实致虚病机的假说。基于此，本研究拟先筛选高表达SGK1肝癌细胞，通过RNA干扰建立SGK1基因沉默的稳定表达肝癌细胞株，采用糖皮质激素与不同治法方药及其拆方干预SGK1高表达与沉默的肝癌细胞，并观察肝癌小鼠血清对肾上腺皮质细胞功能的影响及中药的作用，探索激素对肝癌细胞增殖的作用、及肿瘤对肾上腺皮质功能的影响。再通过不同表达状态SGK1肝癌移植瘤裸鼠实验，研究不同治法方药及其拆方对肝癌增殖与凋亡的影响，以及对肾上腺皮质功能的保护作用。从方证相应的角度揭示SGK1调控肝癌细胞增殖继发肾上腺皮质功能异常是因实致虚病机的部分物质基础，有助于丰富和发展肿瘤虚实夹杂证理论的科学内涵。

肝癌发病隐匿、癌细胞增殖快、易复发、预后差。中医认为“因实致虚、虚实夹杂”是其病机，实证以癌细胞恶性增殖为主，虚证以神经内分泌免疫功能低下为主。中医药治疗优势不在于消除肿瘤，而是调节免疫内分泌系统功能为主。本研究着眼点肾上腺皮质功能，探索了肝癌小鼠虚实夹杂病机及中药调控作用。一是围绕肝癌全方及其相应的清热解毒、行气活血、健脾益气3个治法拆方对SMMC7721人肝癌细胞mTORC2/GR-SGK1信号通路的调节研究，同步观察了温补肾阳中药主要成分对此信号通路的调控作用；二是从肿瘤组织对肾上腺皮质激素合成的影响探索肝癌全方及其拆方对皮质功能的调节作用；三是肝癌小鼠因实致虚病机的论证以及肝癌全方及其拆方对裸鼠肿瘤mTORC2/GR-SGK1信号通路及其内分泌功能的调节作用，同时拓展了控食所致虚证对小鼠肿瘤生长的影响、并延伸了氢化可的松诱导的药源性虚证小鼠证候物质基础的研究。.结果发现：1）尽管SGK1在SMMC7721细胞高表达，但是SGK1并非肝癌细胞增殖所必需。2）与肝癌全方比较，行气活血拆方抑制作用最强，健脾益气拆方抑制作用弱；以抑制SGK1为主要靶点；并抑制SGK1磷酸化蛋白与CyclinE1蛋白表达，将SMMC7721细胞阻滞在G2/M期。3）肝癌全方及不同治法拆方对肝癌裸鼠肿瘤生长抑制不明显，但可促进肿瘤细胞凋亡发生；并促进肾上腺类固醇激素合成酶转录以合成皮质激素去适应肿瘤慢性应激。4）H22肝癌小鼠血清或肿瘤组织匀浆液模拟肿瘤慢性长期应激可损耗肾上腺皮质功能，肝癌全方及其拆方通过增强Star、Cyp11a1、Cyp21a1与Cyp11b1基因表达，提振类固醇激素向糖皮质激素转化，以适应肿瘤慢性应激。5）淫羊藿苷、蛇床子素、补骨脂素均抑制SMMC7721细胞增殖及诱导凋亡。6）持续性控食可引发肾上腺皮质功能紊乱致虚证，加速肿瘤小鼠不良预后。7）氢化可的松诱导的药源性证候小鼠以虚证为主，定位于肾与脾藏象。.本项目论证了肝癌虚实夹杂的复杂病机学说，阐明了肝癌全方及其不同治法拆方调控肿瘤虚实病机的差异性，丰富了中晚期肝癌攻补兼施、重在补益为主的科学内涵。