枫蓼肠胃康经IL-6/STAT3-PXR双向通路增效5-FU治疗结肠炎相关性结肠癌的作用及机制研究

5-FU is the preferred chemotherapeutic agents for colitis-associated colorectal cancer (CACC) treatment, however its chemotherapy effect usually decreases because of drug resistance caused by low chemotherapy efficiency. Activation of STAT3 pathway mediated by IL-6 could induce the conversion of colitis to colon cancer, and the reverse regulation of PXR on IL-6-STAT3 pathway could inhibit CACC. Previous pharmacodynamics research found that Feng-liao- chang-wei-kang could both function as an anti-gastroenteritis drug by inhibiting IL-6 and STAT3 expression, and also as a synergism drug of 5-FU by promoting early apoptosis of colon cancer cells. Previous pharmacokinetic study found that under the condition of colitis, Feng-liao- chang-wei-kang could induce PXR expression but inhibit P-gp expression. Therefore we propose that Feng-liao-chang-wei-kang can play a role of PD-PK related synergy with 5-FU via IL-6/STAT3- PXR bi-directional pathways. By using pharmacodynamics and pharmacokinetic study models including CACC model and different PXR，STAT3 expression cell models, this study intends to get the evidence of Feng-liao-chang-wei-kang’ s synergistic effect with 5-FU treatment on CACC, via bidirectional regulation on IL-6/STAT3-PXR pathways, which supplies data supports to find a safe and efficient adjuvant chemotherapy of CACC.

5-FU为治疗结肠炎相关性结肠癌（CACC）的首选化疗药，常由于化疗效率低下及易发生耐药而降低治疗效果。IL-6 介导的STAT3通路激活可以诱发CACC，核受体PXR可反向调控IL-6/STAT3通路而抑制CACC。我们前期研究发现FLCWK可抑制IL-6、STAT3表达，并能协同5-FU促结肠癌细胞凋亡；同时发现FLCWK虽可激活PXR转录调控，但却体现出抑制P-gp的作用。由此提出FLCWK可通过IL-6/STAT3-PXR双向通路增效5-FU，同时经IL-6/STAT3通路消除PXR对P-gp激活介导的5-FU耐药，发挥PD-PK协同增效作用。本课题拟借助CACC小鼠模型、PXR及STAT3不同表达水平的细胞模型进行药效学及药动学研究，获得枫蓼肠胃康经IL-6/STAT3-PXR 通路双向调控增效5-FU治疗CACC的证据，为发现安全有效的CACC化疗辅助药物提供数据支持。

本项目拟阐明枫蓼肠胃康（FLCWK）经IL-6/STAT3-PXR双向通路增效5-氟尿嘧啶（5-FU）治疗结肠炎相关性结肠癌的作用及机制。在体外研究方面通过FLCWK与5-FU联合处理结肠癌细胞后，MTT法检测细胞活力；流式细胞术检测细胞凋亡与周期分布；RT-PCR和Western-blot分别检测IL-6/STAT3通路相关蛋白及mRNA的表达情况。研究表明，FLCWK 能增效 5-FU 抑制 IL-6/ STAT3 信号通路的表达，有效抑制结肠癌细胞增殖并促进凋亡。本项目通过腹腔注射AOM、自由饮用DSS建立小鼠CACC模型，FLCWK与5-FU联合给药后，观察小鼠存活率、结肠长度、肿瘤数目；HE染色及免疫组化分别观察小鼠结肠的病理变化及Ki-67的表达情况；免疫荧光观察结肠组织STAT3相关通路蛋白的表达；ELISA检测小鼠血清中IL-6、IL-10、TNF-α炎症因子的表达情况。结果表明，FLCWK 显著增效 5-FU 抑制 CACC小鼠结肠肿瘤的数目，减轻炎症细胞浸润程度，抑制肿瘤细胞的异常增殖，提高小鼠存活率。FLCWK 联合5-FU 给药可有效降低 CACC 小鼠血清中 IL-6 和 TNF-α的表达，促进 IL-10的表达。FLCWK 联合 5-FU 能显著抑制结肠组织中 p-STAT3、IL-6、Bcl-2 以及 CyclinD1蛋白的表达。本项目通过皮下注射结肠癌细胞建立裸鼠皮下移植瘤模型，FLCWK与5-FU联合给药后，观察裸鼠肿瘤的生长情况；HE染色和免疫组化分别观察肿瘤组织的病理变化及Caspase3、p-STAT3及P-gp的表达； TUNEL显色法检测肿瘤组织的凋亡。研究表明，FLCWK能够增效5-FU抑制移植瘤的生长，促进移植瘤细胞凋亡，潜在作用机制是通过下调STAT3、P-gp蛋白表达从而发挥增效5-FU抗结肠癌的作用。我们的研究结果表明，FLCWK联合5-FU能显著抑制结肠癌细胞以及CACC小鼠IL-6/STAT3通路及通路相关蛋白及mRNA的表达，减轻CACC小鼠结肠内炎症细胞浸润，抑制肿瘤细胞的异常增殖，但基于PXR对结肠炎相关性结肠癌小鼠作用机制的研究尚在进行中，目前已建立PXR基因敲除小鼠模型进行深入研究。本课题的实施可为FLCWK作为5-FU的协同药物治疗结肠炎相关性结肠癌提供科学数据。