基于PXR-UGT代谢网络研究炎性肠病-肿瘤演进中丹参酮IIA级联葡萄糖醛酸结合代谢与药效学调控机制

There is ample evidence indicating that the pregnant X receptor (PXR)- UDP-glucuronosyltransferases (UGTs) signaling pathway play an important role in colitis-associated colorectal carcinogenesis, whereby the activation of PXR could inhibit the NF-κB signaling pathways and the induction of UGT could lead to multi-drug resistance. Up to now no report is available concerning the dynamic expression of the PXR-UGT pathway.PXR could be activated by tanshinone IIa (TSA), an important lipophilic Danshen component possessing well proven anti-inflammation and anticancer effects. However, in-depth regulatory mechanism has not been explored. Our previous studies strongly indicated that the quinone reduction and the following glucuronidation play an important role in the intestinal elimination of TSA and the realization of its anti-tumor effects. Moreover, the expression of UGT could be induced by TSA. Based on this background, we hypothesize that the dynamic change of the PXR-UGT pathway during the colitis-colorectal carcinogenesis could influence the glucuronidation of TSA, thereby affecting its anti-tumor efficacy. The mechanism for the pharmacodynamic profile involves two aspects: Firstly,TSA could inhibit the NF-κB signaling pathways through activation of PXR during colitis and induce the expression of UGT to reduce the ROS generation from quinone reduction, collectively contributing to the protection of inflammatory bowel disease; Secondly, after tumor formation, the induction of UGT by TSA may promote its elimination and counteract the anti-tumor effect of ROS, thereby leading to drug-resistance and reduced anti-tumor efficacy of TSA. Colon biopsies both from patients and AOM-DSS treated mice are used to analyze the dynamic changes of PXR or UGT expression in different pathological stages of colitis-associated colorectal carcinogenesis and their influence on the glucuronidation of TSA could be investigated. Further studies, integrating small interfering RNA, reporter gene assay and glucuronidation metabolism analysis, will be performed to systematically validate our hypothesis and explore the underlying mechanism. This project will help to provide pharmacological basis for the use of TSA against colorectal cancer and guide future studies on researches integrating pharmacokinetics and pharmacodynamics methods.

文献研究表明PXR-UGT在肠炎癌症转化中起重要作用，激活PXR能抑制NF-κB，上调UGT能降低炎性损伤，肠癌中诱导UGT介导耐药；未见该病理进程中考察PXR/UGT动态变化的报道。丹参酮IIA(TSA)有PXR激动作用和确凿的抗炎抗肿瘤活性但深层药效机制未明。课题组研究表明级联葡萄糖醛酸代谢是TSA肠道代谢重要途径决定其药效，同时TSA能诱导UGT高表达。基于此提出假说：肠炎肿瘤转化中PXR/UGT的变化影响TSA级联代谢及药效；机制是TSA激活PXR抑制NF-κB通路同时上调UGT预防和治疗肠炎，肠癌发生后TSA激活PXR上调UGT介导耐药。研究采用人活检标本和小鼠模型明确肠炎癌症转化各病理阶段PXR/UGT变化及对TSA级联代谢的影响；应用siRNA和报告基因技术及代谢分析，从分子、细胞和整体水平结合药代药效多视角阐明TSA药效调控机制，为激活PXR中药的药效学机制研究提供新思路。

本研究采用AOM/DSS炎性肠病癌变小鼠，动态考察病理进程不同阶段小鼠病理进程中PXR、UGT表达情况。模型小鼠在4周、7周时PXR表达呈降低的趋势；12周时与癌旁组织比较肿瘤组织中的PXR表达显著升高；炎性肠病癌变对结肠组织Ugt1a9表达均无明显的影响。病理进程至4周时，TSA20mg/kg与PXR激动剂PCN均具有明显的抗肠道炎症作用；病理进程进展至7周和12周，TSA20mg/kg与PCN无显著抗炎性肠病癌变的作用。WB结果提示TSA抗炎作用与4周时其上调结肠组织PXR表达有关。进一步采用TNBS诱导慢性炎性肠病模型考察丹参IIA抗炎性肠病作用及调控机制。结果表明TNBS显著改善炎性肠病小鼠肠道炎症及病理评分；通过激活IKBα抑制NF-KB p65活性，同时上调PXR下游基因cyp3a11mRNA表达，提示丹参酮IIA激活PXR代谢通路抑制NF-κB 炎症通路对炎性肠病起治疗作用。丹参酮IIA抗炎性肠病药效确切，其在病理状态下药代动力学行为尚不清楚。口服给药后，虽然丹参酮IIA在正常和结肠炎小鼠中系统暴露低，但是在小鼠结肠组织中2h的组织暴露高达1015.5±411.9 ng/g，达到体外研究产生生物效应的组织浓度。模型小鼠中TSA AUC0-∞显著增高，MAT延长；进一步研究发现由于炎性肠病模型中UGT1A9表达上调，使得模型组小鼠结肠组织暴露显著降低，血浆中葡萄糖醛酸结合物在口服给药0.5，1，2h后显著升高；长期给予TSA能抑制炎性肠病模型中上调的UGT1A9，使模型小鼠结肠组织暴露回复正常水平。结果提示丹参酮IIA具有良好与治疗炎症性肠病相适应的药代动力学行为。由于TSA对PXR激活作用与其起主要代谢作用的亚型UGT1A9的表达不一致。因此主要考察UGT1A对丹参酮IIA在肿瘤细胞内累积和抗肿瘤药效的影响。结果表明HT29细胞中UGT1A高表达，而HCT116细胞中UGT1A不表达。HT29细胞的S9体系中丹参酮IIA发生明显的葡萄糖醛酸结合代谢，UGT1A的底物丙泊酚和siRNA能抑制其葡萄糖醛酸结合代谢。同时HT29细胞内TSA细胞内水平及细胞毒作用显著低于HT116细胞，相应地HT29细胞中应用UGT1A的底物丙泊酚和siRNA能增加其细胞内水平及其细胞毒作用，提示UGT1A折中解决丹参酮IIA肿瘤细胞内累积和抗肿瘤药效。