COX-2介导的TLR4/NF-κB信号通路在溃疡性结肠炎相关异型增生及癌变中的作用

Enteric chronic inflammatory is associated with dysplasia and carcinogenesis of epithelial cells in the intestinal mucosa, and the colorectal cancer risk of chronic nonspecific ulcerative colitis (UC) patients is ten times higher than the general population. Previous studies have demonstrated that COX-2 mediated oxidative stress injury plays a significant role in the development of UC associated dysplasia and carcinogenesis, but the exact mechanism remains unclear. Our previous experimental results have proved that "dietary iron supplementation+DSS distilled water feed" can aggravate intestinal mucosa barrier injury of mice, and increase the incidence of DSS-induced colitis associated dysplasia and carcinogenesis. Therefore, in the present project, this mice model and human colorectal cancer cell lines are treated with antagonist/agonist as well as cell transfection. Expression of COX-2 and its downstream genes, such as PGE2, TLR4, TAK-1, NF-κB, STAT3, are detected. Meanwhile, the effects of TLR4/NF-κB signaling pathway on proliferation, apoptosis, invasion, migration of cells and wounding/repairing ability, permeability of intestinal mucosa are observed. The aims are to explore the role and mechanism of COX-2 mediated TLR4/NF-κB signaling pathway from levels of tissue, cell and molecular on UC associated dysplasia and carcinogenesis, and to further provide experimental evidence for clinical prevention and therapy of UC associated cancer.

肠道慢性炎症与肠黏膜上皮细胞的异型增生及癌变密切相关，其中慢性非特异性溃疡性结肠炎（UC）发生结直肠癌的风险是一般人群的10倍。已有研究表明COX-2介导的氧化应激损伤在UC相关异型增生及癌变形成过程中具有重要作用，但具体机制尚未明了。我们前期实验结果表明"高铁饮食+DSS蒸馏水饲养"能够加重小鼠肠黏膜屏障损伤，增加DSS诱导的结肠炎相关异型增生及癌变发生率。因此，本课题拟利用拮抗剂/激动剂和细胞转染等方法分别干预上述动物模型和人结肠腺癌细胞株，检测COX-2及其下游基因PGE2、TLR4、TAK-1、NF-κB、STAT3的表达，并观察TLR4/NF-κB信号通路对细胞增殖、凋亡、侵袭、迁移和肠黏膜损伤/修复能力、通透性的影响，旨在从组织、细胞和分子水平探讨COX-2介导的TLR4/NF-κB信号通路在UC相关异型增生及癌变中的作用及其机制，从而为UC相关癌的临床防治提供实验依据。

肠道慢性炎症与肠黏膜上皮细胞的异型增生及癌变密切相关，其中慢性非特异性溃疡性结肠炎（UC）发生结直肠癌的风险是一般人群的10 倍。已有研究表明COX-2 介导的氧化应激损伤在UC 相关异型增生及癌变形成过程中具有重要作用，但具体机制尚未明了。我们前期实验结果表明“高铁饮食+DSS 蒸馏水饲养”能够加重小鼠肠黏膜屏障损伤，增加DSS 诱导的结肠炎相关异型增生及癌变发生率。。因此，本课题拟利用拮抗剂/激动剂和细胞转染等方法分别干预上述动物模型和人结肠腺癌细胞株，检测COX-2 及其下游基因PGE2、TLR4、TAK-1、NF-κB、STAT3 的表达，并观察TLR4/NF-κB 信号通路对细胞增殖、凋亡、侵袭、迁移和肠黏膜损伤/修复能力、通透性的影响。.建立nimesulide、LPS、三叶青黄酮干预小鼠模型。ELISA方法检测nimesulide干预组和三叶青黄酮干预组血清COX-2、PGE2水平显著降低（P<0.05），LPS干预组血清COX-2、PGE2水平显著增高（P<0.05）。Western blot法检测nimesulide干预组和三叶青黄酮干预组TLR4、TAK-1蛋白表达显著降低（P<0.05），LPS干预组TLR4、TAK-1蛋白表达显著增高（P<0.05）。RT-qPCR检测nimesulide干预组和三叶青黄酮干预组NF-κB p65、STAT3 mRNA水平显著增高，，LPS干预组NF-κB p65、STAT3 mRNA水平显著增高（P<0.05）。免疫组化结果显示，nimesulide干预组和三叶青黄酮干预组TLR4、NF-κB p65在结肠黏膜上皮细胞和固有层表达的OD值较模型组均显著降低（P<0.05），LPS干预组TLR4、NF-κB p65在结肠黏膜上皮细胞和固有层表达的OD值较模型组显著增高（P<0.05）。. HT-29细胞实验证实nimesulide干预组PGE2、NF-κB、STAT3表达较正常对照组明显下降；三叶青黄酮干预组COX-2、PGE2、AKT表达较正常对照组明显下降；LPS干预组PGE2、NF-κB、STAT3表达较正常对照组明显增高。MTT结果显示三叶青黄酮能显著抑制结肠癌细胞株HT-29的增殖能力，并且呈剂量依赖性。.本课题为UC 相关癌的临床防治提供实验依据。