PI3K-Akt/mTOR通路调控P-β-catenin介导溃疡性结肠炎相关癌变及健脾清热活血方药干预研究

Previous research has proved that Jian Pi Qing Re Huo Xue (JPQRHX) decoction exerts an effect on preventing the onset of ulcerative colitis associated carcinogenesis (UCAC). We consider the effect target of JPQRHX decoction maybe relate with interfering Wnt signal pathway and regulating phosphorylation of β-catenin.Meanwhile,there is a sign that phosphorylation of β-catenin and nuclear abnoramal translocation involve in multi-signal pathway.Except the canonical Wnt/β-catenin pathway, We put forward a hypothesis that excessive activation of PI3K/Akt-mTOR after EGF combined with EGFR plays an important role in the onset of UCAC under inflammation.That signal pathway we have mentioned above which cooperative with Wnt signal pathway induce excessive phosphorylation of β-catenin which may lead to downstream target gene excessive transcription.We will compare with the changes of EGF/EGFR-PI3K/Akt-mTOR signal pathway in UCAC after intervention with LY294002 and JPQRHX decoction respectively in vivo and in vitro.In order to prove that the JPQRHX decoction is characterized by multi-pathway and multi-target in mediating PI3K-Akt/mTOR pathway.Those results may provide new idea and experimental basis for preventing UCAC.

前期证实健脾清热活血方药具有防治溃结相关癌变（UCAC）效应，其靶点可能与干预经典Wnt通路调控β-catenin磷酸化（P-β-catenin）相关。但是项目组发现β-catenin过度磷酸化及核内异常转位是涉及多信号复杂过程，除外经典Wnt通路，PI3K-Akt/mTOR通路在P-β-catenin作用不容忽视，炎症状态下PI3K-Akt/mTOR通路在EGF与EGFR结合刺激下过度活化，协同Wnt通路，介导P-β-catenin，下游靶基因过度转录，致肠上皮细胞异常增殖、癌变。项目拟从在体实验和离体实验应用LY294002阻断PI3K-Akt/mTOR通路以及健脾清热活血方干预，应用分子生物学技术对比UCAC治疗前后相关指标变化，证实健脾清热活血方防治UCAC可能与介导PI3K-Akt/mTOR通路协同Wnt通路调控P-β-catenin相关，研究结果将为中医药防治UCAC提供新思路

目的：从 PI3K-Akt/mTOR通路介导β-catenin磷酸化核内转位参与溃结癌变角度，探讨健脾清热活血方防治溃结癌变机制。方法：⑴在体实验：Balb/c小鼠随机分为正常组、模型组、西药组、治疗组（低、中、高剂量）及LY294002组。除正常组，其余均采用DMH/DSS法制备溃结癌变模型后，分组干预。采用现代生物技术检测结肠黏膜形态、超微结构、PI3K-Akt/mTOR通路相关指标及p-β-catenin核内转位。⑵离体实验：①应用高效液相色谱法测定健脾清热活血含药血清组份，建立质控。②SW480细胞分空白组、治疗组、LY294002组，分组干预。应用现代生物技术检测细胞增殖、凋亡、周期、PI3K-Akt/mTOR通路中指标及β-catenin核内转位。结果：⑴模型组见结肠黏膜上皮脱落、形成溃疡，腺体萎缩、部分呈浸润癌；电镜下微绒毛稀疏甚至脱落消失，粗面内质网扩张及大量细胞坏死。治疗组见结肠黏膜损伤程度较模型组改善，仍有不典型增生,未见癌变；电镜见上皮细胞排列尚整齐，核染色质边集，凋亡小体形成，局灶可见坏死。与模型组比较，治疗组EGFR、P85、P-Akt、P-β-catenin基因表达下调，各组间EGF、P110、mTOR基因表达比较无差异。与模型组比较，治疗组EGFR、P85、CDK1、P-β-catenin蛋白表达下降；而EGF、P110、P -Akt、mTOR蛋白表达无差异。正常p-β-catenin呈胞膜表达为主，而溃结相关癌变存在不同程度p-β-catenin核内异常转位和膜表达缺失。⑵含药血清以三七皂苷R1、人参皂苷Rg1、甘草酸铵为主。治疗组增殖抑制率分别2.55%、4.90%及16.30%、细胞凋亡率上升，与空白组比较有差异。与空白组比较，治疗组EGF、EGFR、P110、P-AKT、mTOR、CDK1基因表达下降，P85、P-β-Catenin基因表达无差异；与空白组比较，治疗组EGFR、P85及CDK1蛋白表达下调；而EGF、P110、mTOR、P-β-Catenin、P-Akt蛋白表达无差异。结论：健脾清热活血方药防治溃结癌变，与下调EGFR、P85、P-Akt、P-β-catenin基因，调控P-β-catenin核内转位，减少CDK1蛋白表达，抑制细胞增殖，诱导细胞凋亡相关。