从Wnt/β-catenin通路探讨清热燥湿法修复化疗相关性腹泻肠道损伤的机理

Repair of intestinal damage was the key point and difficulty of the prevention and cure of Chemotherapy-induced diarrhea(CID). Wnt/β-catenin pathway is a key factor for affecting the repair of intestinal injury; Clear heat and dry dampness was a important and effective therapy for treating CID. The early clinical trials found that clear heat and dry dampness drug Xianglian pill was effective to prevent and to cure CID; based experiments also confirmed that Xianglian pill could relieve diarrhea and repair the intestinal injury in the CID mice; but its mechanism was unknown. Thus, we propose the hypothesis: Clear heat and dry dampness could regulate “Wnt/β-catenin pathway ”directly, and/or downregulate the antagonist proteins of Wnt to reduce the inhibition on Wnt/β-catenin pathway, to promote intestinal progenitor/stem cell proliferation and differentiation of intestinal mucosal cells to repair intestinal damage. In order to verify this hypothesis, we would build on a nude mouse model, colon cancer cells and nude intestine crypt intestine crypt model, to study and research above-mentioned questions from genes, proteins, cells, tissue and whole animal by using RT-PCR, Western blot, immunohistochemistry and so on. This project study will provide scientific basis for the intervention measures of traditional Chinese medicine in prevention to intestinal toxicity action of chemotherapy drugs.

修复肠道损伤是化疗相关性腹泻(CID) 防治的关键和难点；Wnt/β-catenin通路是影响肠道损伤修复的关键因素。清热燥湿法是中医防治CID的有效治法。课题组前期临床试验发现，清热燥湿法代表方剂香连丸防治CID具有可靠的疗效，基础实验也证实，香连丸可减轻CID小鼠腹泻程度、修复肠道损伤，但其作用机制尚不明确。为此，我们提出假说：香连丸通过上调Wnt激动蛋白而/和/或直接激活Wnt/β-catenin通路，和/或通过抑制Wnt拮抗蛋白、减少其对Wnt/β-catenin通路的抑制作用，促进肠道干/祖细胞增殖和向肠粘膜细胞分化，从而修复肠道损伤。为验证该假说，我们将采用小鼠结肠癌细胞、小肠隐窝上皮细胞、裸鼠CID模型，运用RT-PCR、Western blot、免疫组化等手段，从基因、蛋白、细胞及整体动物等多层次研究上述问题。本课题将为化疗药物肠道毒性反应中医药防治提供科学依据。

本研究采用腹腔注射伊立替康(CPT-11)法制备化疗相关性腹泻(CID)小鼠模型；从小鼠生存时间、大体状况(精神、活动状况及饮食)、腹泻程度、炎症因子表达、回肠粘膜病理损伤程度等方面证实香连丸具有减轻CID小鼠腹泻程度，改善小鼠大体状况，调节炎症因子表达，修复肠道损伤，延长小鼠生存时间的作用；香连丸修复CID肠道损伤的可能机理：①香连丸上调CID小鼠回肠组织中Wnt3、Fzd5、Lrp5和Pygo2的表达，激活Wnt/β-catenin信号通路，促进小肠干/祖细胞增殖，并向肠粘膜细胞如潘氏细胞、杯状细胞、肠内分泌细胞分化，从而替代损伤的小肠上皮细胞，修复肠道损伤；②香连丸上调Wnt/β-catenin信号通路激动蛋白R-spondin1的表达，激活Wnt/β-catenin信号通路，从而修复肠道损伤；③香连丸下调CID小鼠NF-κBp65表达，抑制NF-κB信号通路的活化，下调促炎细胞因子TNF-α、IFN-γ的表达，并上调抑炎细胞因子IL-4的表达，从而减轻肠道炎症反应，修复肠道损伤；④香连丸抑制CPT-11引起的小鼠小肠粘膜细胞的凋亡，促进CPT-11作用后小肠隐窝细胞的增殖。在完成国家自然科学基金任务书的前提下，我们还进行了以下实验：①观察香连丸活性成分黄连碱对CID小鼠Iκ Bα/NF-κB信号通路及炎症因子表达的影响；结果表明，黄连碱可下调NF-κBp65表达，上调IκBα表达，抑制NF-κB通路的活化，进而下调促炎细胞因子TNF-α、IL-6的表达，从而减轻CID小鼠肠道炎症反应，修复肠道损伤；②探讨了香连丸抑制人结肠癌细胞Caco-2细胞侵袭、转移的机理：香连丸可抑制Caco-2细胞NF-κB信号通路的活化，下调MMP-9的表达，降低MMP-9/TIMP-1的比值。. 本研究证实了清热燥湿代表方药香连丸对CID的治疗作用，并从调控Wnt/β-catenin信号通路及NF-κB通路方面，探讨了香连丸修复CID肠道损伤的机理；从而论证了CPT-11引起的CID从中医“湿”、“热”病机论治的科学性。本研究可为CID的治疗提供新的中医病机认识和治疗方法选择，并为抗肿瘤药物肠道毒性反应的中医药防治提供新的研究思路。