参苓白术散对脾虚湿困型溃疡性结肠炎大鼠Toll样受体2/NF-κB/COX-2信号通路的调控机制研究

In recent years, Shenling Baizhu powder has shown good clinical effects on ulcerative colitis. Our study shown that Shenling Baizhu powder could improve the level of inflammatory factor in serum, and increase the activity of SOD and decrease the level of MDA. But the mechanism of the effects was lack of further study. This research supply TLR2/NF-κB/COX-2 as the cutting point, to observe the expression of protein and mRNA of IKKβ, IκBɑ, TLR2, Myd88, NF-κB p65, COX-2 and damage factor of downstream, to investigate the intervention effect of Shenling Baizhu powder on TLR2/NF-κB/COX-2 signal pathway in ulcerative colitis rats with syndrome of dampness stagnancy due to spleen deficiency. To preliminarily verify the relationship between therapeutically effects of Shenling Baizhu powder and TLR2/NF-κB/COX-2 signal pathway using the antagonist and agonist of TLR2. To reveal the therapeutically mechanism of Shenling Baizhu powder on TLR2/NF-κB/COX-2 signal pathway in ulcerative colitis rats with syndrome of dampness stagnancy due to spleen deficiency from molecular biological level, and to provide the experimental basis for improve the clinical efficacy.

近年来，临床应用参苓白术散加减治疗慢性溃疡性结肠炎（UC）取得了较好疗效。我们前期的研究发现，参苓白术散能显著改善脾虚湿困型UC大鼠血清炎症因子的水平，提高大鼠结肠组织SOD活性，降低MDA水平，但是对于其作用机制仍缺乏深入的研究。本项目拟以TLR2/NF-κB/COX-2信号通路为切入点，观察参苓白术散对脾虚湿困型UC大鼠IKKβ、IκBɑ、TLR2、Myd88、NF-κB p65、COX-2蛋白及mRNA表达和下游致损伤因子的影响，探讨参苓白术散对脾虚湿困型UC大鼠TLR2/NF-κB/COX-2信号通路的干预作用；并通过应用TLR2拮抗剂和激动剂，初步探明参苓白术散对脾虚湿困型UC的治疗作用与TLR2/NF-κB/COX-2信号通路变化之间的关系，旨在从分子生物学水平揭示参苓白术散对脾虚湿困型UC大鼠的治疗机制，为提高临床疗效提供实验依据。

溃疡性结肠炎（UC）是一种病因尚不明确的慢性非特异性炎性肠病，其发病机制较为复杂，已知免疫、遗传及环境等多种因素参与了UC的发病过程。近年来临床研究已经证实，参苓白术散加减治疗慢性UC疗效确切。项目组前期研究发现，参苓白术散能显著改善脾虚湿困型UC大鼠血清炎症因子的水平，提高大鼠结肠组织SOD活性，降低MDA水平，但是对于其作用机制仍缺乏系统深入的研究。本项目以TLR2/NF-κB/COX-2信号通路为切入点，观察参苓白术散对脾虚湿困型UC大鼠IKKβ、IκBɑ、TLR2、Myd88、NF-κB p65、COX-2蛋白及mRNA表达和下游致损伤因子的影响，探讨参苓白术散对脾虚湿困型UC大鼠TLR2/NF-κB/COX-2信号通路的干预作用；并通过应用TLR2拮抗剂和激动剂，初步探明参苓白术散对脾虚湿困型UC的治疗作用与TLR2/NF-κB/COX-2信号通路变化之间的关系。结果显示，大鼠NF-κB p65蛋白表达及IL-6、IL-8、TNF-α水平明显高于正常组，参苓白术散组大鼠结肠组织NF-κB p65蛋白表达及IL-6、IL-8、TNF-α水平较模型组显著降低，其间差异有统计学意义；脾虚湿困型UC大鼠结肠组织ERK、p38MAPK、IKKβ蛋白表达明显增强，IκB蛋白表达明显降低；参苓白术散治疗可显著降低ERK、p38MAPK、IKKβ蛋白的表达水平，增强IκB蛋白表达；脾虚湿困型UC大鼠结肠组织TLR2、Myd88、COX-2 mRNA表达呈高表达，参苓白术散可明显下调脾虚湿困型UC大鼠结肠组织TLR2、Myd88、COX-2 mRNA表达，TLR2可能通过正调节Myd88、COX-2的mRNA表达水平来参与参苓白术散的抗炎效应；TLR2 拮抗剂组大鼠结肠组织NF-kB p65、TLR2、Myd88、COX-2 蛋白表达均显著降低，TLR2激动剂组大鼠结肠组织NF-kB p65、TLR2、Myd88、COX-2 蛋白表达均显著升高。提示参苓白术散治疗脾虚湿困型UC的可能机制是通过抑制结肠组织NF-κB的表达和NF-κB通路的激活，从而调控IL-6、IL-8、TNF-α等炎症因子的释放，减轻肠黏膜损伤；TLR2参与了参苓白术散改善脾虚湿困型UC大鼠肠粘膜损伤机制，TLR2/NF-κB/COX-2 信号通路在参苓白术散治疗脾虚湿困型UC 大鼠效应中发挥重要的作用。