四神丸调控Nemo及NLK/TCF/LET信号泛素化预防慢性溃疡性结肠炎复发的作用机制研究

In our previous study, all results had shown that the effect of SiShen Wan (SSW) effectively treated ulcerative colitis (UC) was realized by regulating P38MAPK signal pathway. In the clinical of UC, SSW effectively prevented the relapse of chronic ulcerative colitis. Many studies have hinted that Nemo and ubiquitination of NLK/TCF/LET signal play a key role in attack of chronic UC and activation of P38MAPK signal. We will verify the effectiveness of SSW on preventing the relapse of chronic UC by the model of chronic and relapsed ulcerative colitis, while effect of SSW on regulating ubiquitination of Nemo and NLK/TCF/LET signal which regulated activation of NF-κB signal and P38MAPK signal, negatively regulated Wnt/β-catenin were observed in the study. Furthermore, the probable mechanisms of SSW treated UC, prevented the relapse of UC were disclosed, and to explore possible mechanism of Chinese medicine effectively prevented ulcerative colitis and provide a scientific reference for research and development of new drugs in Chinese herb prevented the relapse of chronic ulcerative colitis.

我们以前的研究，表明四神丸有效治疗溃疡性结肠炎的作用途径可能和P38MAPK信号途径有关，根据四神丸临床可有效预防慢性溃疡性结肠炎复发，且Nemo及NLK/TCF/LET信号泛素化在慢性溃疡性结肠炎发作及其在P38MAPK信号活化过程中发挥了关键性作用，我们拟通过复制慢性复发型溃疡性结肠炎大鼠模型，在验证四神丸预防其复发的有效性基础上，从调控Nemo及NLK/TCF/LET信号泛素化水平的角度，观察四神丸对Nemo及NLK/TCF/LET信号泛素化的核心指标，以及与NF-κB信号活化、P38MAPK信号活化及负性调控Wnt/β-catenin信号的关联性指标进行观察，探索四神丸预防慢性溃疡性结肠炎的可能作用机制。进一步揭示四神丸治疗溃疡性结肠炎并预防其复发的可能作用靶点，并借此探索中医药有效预防溃疡性结肠炎复发的可能作用机制，为探索预防溃疡性结肠炎复发的中药新药研发提供科学的参考。

Nemo/NLK信号及其泛素化水平是NF-κB信号通路被激活的关键性因素之一。近年来发现Nemo及（NLK）/TCF/LET信号泛素化水平在慢性溃疡性结肠炎发作过程中发挥了关键性作用。溃疡性结肠炎治疗的难点在于高复发率。四神丸预防溃疡性结肠炎复发的作用机制不明确。在本实验中采用病理形态学等方法评价四神丸预防慢性溃疡性结肠炎复发的有效性。采用WB、流式细胞术、RT-PCR等方法，对Nemo及NLK/TCF/LET信号泛素化、NF-κB信号活化、负性调控Wnt/β-catenin信号进行检测，发现结肠重量指数、肉眼下及镜下病理损伤评分明显下降，病理形态学分析下的结肠粘膜损伤缓解、抗氧化损伤、降低 IL-1β,IL-17表达，升高IL-4表达等证据表明四神丸可有效缓解和防治TNBS诱导的实验性结肠炎发病与复发。同时四神丸还可有效抑制NF-κB p65和NLK活性，抑制NEMO泛素化水平，ATP水平明显升高，Wnt/β-catenin信号被抑制，NARF, TCF 及下游蛋白c-Myc, COX-2, cyclin D1, survivin, STAT 3、ZIPK明显被抑制，而LEF泛素化水平及 β-TrCP表达明显升高，结肠上皮细胞凋亡率明显下降，表明四神丸防治实验性结肠炎复发可能是通过抑制NEMO/NLK信号途径进而抑制NF-κB活性和Wnt/β-catenin信号来实现的，这揭示了四神丸治疗溃疡性结肠炎并预防其复发的可能作用靶点，为探索中医药有效预防溃疡性结肠炎复发的可能作用机制和中药新药研究提供科学的科学参考。