“UGT酶-外排转运蛋白”介导的肠道行为在黄芩抑制结肠癌中的作用及调控p53通路的分子机制

It has been proved that Scutellaria baicalensis Georgi. (SBG), one of Traditional Chinese medicines (TCMs) used for gastrointestinal treatments, and its corresponding biological flavonoids such as wogonin exhibit various pharmacological activities. However, our preliminary data showed that wogonin revealed the strongest anti-colon cancer effect compare to other non-intestinal tumors with respective inhibition rate of 55% and 0%. Our group has proved that flavonoids may subject to intestinal regional recycling, which resulted in their specific high concentration and long-term exposure in the intestine. Whether this intestinal regional recycling mediated by “UDP-glucuronosyltransferase -Efflux transporters” (UGTs-ETs) is associated with the preponderant efficacy in inhibiting colon cancer is not clear. Further mechanism study revealed that wogonin in SBG could directly promote phosphorylation and inhibit nuclear translocation of p53 protein, ultimately suppress colon cancer initiation. Nevertheless, the mechanism by which wogonin-regulates kinase to promote phosphorylation of p53 is unclear. At the same time, this regulating mechanism is related to the specifically intestinal behavior of wogonin has not been studied. Therefore, we hypothesized that the intestinal behavior of wogonin may directly regulate p53 signaling pathway to exert anti-tumor efficacy. Thus, in this proposal, molecular pharmacokinetics and biological methodologies were employed to find out 1) the correlation ship between “UGTs-ET” and specific colon cancer prevention efficacy of woginin and SBG; 2) the molecular mechanism of wogonin-regulates kinases activation of p53 phosphorylation; 3) determining the regulatory mechanisms of “UGTs-ET” and p53 signaling pathway of wogonin in colon cancer prevention. By studying UGTs-ET-p53 correlation of woginin and SBG, our study may provide novel strategy and experimental evidence for understanding the specific anti-cancer efficacy of TCMs.

中药黄芩及其黄酮类药效成分如汉黄芩素有多种药理作用。然而，我们预实验却发现，对结肠癌的抑制作用最优最强，抑瘤率达55%以上，而对其他非肠道肿瘤基本无效。我们前期证实，黄芩中的汉黄芩素，可在肠道形成局部循环，能够长时间、高浓度滞留在肠道。这种肠道调控行为与其抑制结肠癌是否相关，尚不清晰。进一步又发现，汉黄芩素可促进结肠癌细胞p53蛋白磷酸化，抑制其核转位。但激活p53磷酸化的机制尚不清晰，同时，该机制与汉黄芩素的肠道行为是否相关更未有研究。因此，本课题围绕“黄芩的黄酮类药效成分肠道行为在抑制结肠癌的作用和调控p53的分子机制”关键科学问题，采用已构建的“UGTs/ETs”基因工程鼠，阻断或增加肠道行为，以结肠癌为药效评判模型，研究肠道行为在黄芩抑制结肠癌的调控作用及机制。预期结果不仅能阐明黄芩抑制结肠癌的分子机理，更能发现和证实黄芩肠道特有的作用方式，为中药临床更合理应用及新药研发提供依据。

黄芩具有清热燥湿、泻火解毒的功效，黄酮类成分是其主要活性成分。本研究采用“UGTs/ETs” 细胞和基因工程鼠，深入研究外排转运蛋白调控黄芩水提物及黄酮类成分“肠肠循环”和“肠局部循环”，影响黄芩防治溃疡性结肠炎/结肠癌的作用机制。主要得到以下三方面的研究结论（1）黄芩素/黄芩苷，汉黄芩素/汉黄芩苷，千层纸素A（100:6:20:2:1:6）是黄芩水提物发挥防治结肠炎/结肠癌优势药效的物质基础；外排转运蛋白调控黄芩的肠道处置行为，使其高浓度（20倍，200倍 VS血液）、特异性富集于肠道（小肠、结肠）部位，发挥防治肠道疾病的优势药效；（2）黄芩中多个黄酮类成分均能和p53靶点蛋白相结合，以汉黄芩素结合能力最强；汉黄芩素剂量依赖地增加HCT-116细胞p53-ser376和ser315磷酸化水平，减少p53-ser15在细胞核中的定位，促进HCT-116细胞凋亡；而在HT-29细胞，汉黄芩素依赖地减少p53-ser376磷酸化水平，增加p53-ser15在细胞核中的定位，促进HT-29细胞自噬；（3）P-gp和BCRP是调控黄芩及其黄酮类成分，高浓度、长时间富集于肠道的主要外排转运蛋白；P-gp和BCRP敲除基因工程鼠，直接逆转黄芩防治溃疡性结肠炎/结肠癌的体内药效；黄芩及黄酮类成分通过调控NF-κB/IL-1β信号通路，发挥抑制溃疡性结肠炎的药效作用。以上研究结果为黄芩临床合理应用提供依据，课题拟发表SCI论文3篇，培养研究生2名，本科生3名。