基于IFN-γ-JAK/STAT1-IFITM3通路探讨黄芩汤治疗溃疡性结肠炎湿热证的机制

Abnormal level of IFN-γ is closely related to the pathogenesis of UC. It participates the pathogenesis of UC by destroying the intestinal mucosal barrier, preventing migration of intestinal epithelial cells and repairing mucosal injury, however, there are no researches to investigate the relation between IFN-γ-JAK/STAT1 pathway and the pathogenesis of UC; Scutellaria Decoction plays a role via this pathway, but there are no further researches to explore the mechanism of downstream gene IFITM3 induced by IFN-γ-JAK/STAT1 pathway in the pathogenesis of UC. This study plans to investigate the mechanism of Scutellaria Decoction in the treatment of UC with dampness heat syndrome via IFN-γ-JAK/STAT1-IFITM3 pathway. Screens effective dose of Scutellaria Decoction, using rats model of UC with dampness heat syndrome and normal colonic epithelium cells HT-29, and detects the expression of mRNA and protein of key genes in this pathway and cell proliferation﹑cell apoptosis and so on, reveals the mechanism of participating the pathogenies of UC with dampness heat syndrome, and clarifies Scutellaria Decoction cures UC with dampness heat syndrome effectively via this pathway. It is of great significance to elaborate the scientific connotation of prescription to cure UC damp-heat syndrome by the way of multi-target and multi-channel, and to provide the research foundation for clinical innovation drug research and development.

IFN-γ水平的异常与UC湿热证发病关系密切。目前研究发现，它通过破坏肠黏膜屏障、阻止肠上皮细胞移行和黏膜损伤修复等参与UC发病，但并未通过激活JAK/STAT1通路探讨其与UC发病的关系；黄芩汤通过该通路发挥作用，但并未进一步探讨IFN-γ-JAK/STAT1通路诱导的下游基因IFITM3在UC发病中的作用。本课题拟研究黄芩汤通过IFN-γ-JAK/STAT1-IFITM3通路治疗UC湿热证的作用机制。筛选黄芩汤有效剂量，在UC湿热证大鼠模型及HT-29结肠上皮细胞模型上，通过检测该信号通路上、下游关键基因的mRNA、蛋白表达及细胞增殖、凋亡等指标，揭示IFN-γ-JAK/STAT1-IFITM3通路参与UC湿热证发病的机制，阐明黄芩汤通过该通路有效治疗UC湿热证。对阐述清热燥湿方剂多靶点、多途径治疗UC湿热证的科学内涵有重要意义，为临床创新药物研发提供研究基础。

研究背景：UC湿热证患者血清促炎因子IFNγ高表达，且与疾病活动度相关，它通过破坏肠黏膜屏障、阻止肠上皮细胞移行和黏膜损伤修复等参与UC发病；JAK/STAT通路参与炎症性肠病的发病，UC患者或者大鼠的JAK2、STAT1、IFITM3的mRNA或蛋白表达都增高，IFITM3可能通过上调ETS-1的表达参与UC的发病；黄芩汤能够通过抑制JAK/STAT通路、降低促炎因子的表达水平、防止肠粘膜上皮细胞凋亡、增强抗氧化活性等方法，有效地治疗UC。本课题通过构建UC湿热证小鼠模型及体外HT-29细胞炎症模型，探讨验证IFNγ-JAK/STAT1-IFITM3通路可能是黄芩汤治疗UC湿热证的作用机制之一。研究内容：采用“湿热环境+自由饮用DSS水”的复合方法造模，观察UC湿热证小鼠一般状态、结肠组织形态学及超微结构，检测血清细胞因子含量及IFNγ-JAK/STAT1-IFITM3通路关键基因蛋白的表达，分析治疗前后小鼠肠道微生物的变化；采用HT-29细胞炎症模型，观察黄芩汤含药血清对炎症因子的影响，检测关键基因蛋白的表达及细胞凋亡情况。研究结果：1.黄芩汤能有效缓解UC湿热证小鼠腹泻、便血的程度，改善结肠粘膜的炎症，促进肠上皮细胞的修复；2.黄芩汤能显著降低血清IFNγ、TNF-α、IL-8、IL-17的含量，抑制结肠组织IFN-γ、IFITM3、JAK1、JAK2、ETS-1等蛋白的高表达，提升BCL-2的蛋白表达；3.黄芩汤治疗后使UC湿热证小鼠肠道菌群发生改变，可能发挥治疗作用的关键菌群有Parabacteroides_goldsteinii、Parabcteroides、Tannerellaceae、Bacteroides_pectinophilus_group、Desulfovibrio_fairfieldensis；4、黄芩汤含药血清能明显促进HT-29细胞的增殖活力；5、黄芩汤含药血清能显著降低HT-29细胞培养上清促炎因子IFN-γ和TNF-α的表达水平；6、采用IFN-γ复合LPS诱导HT-29细胞炎症模型成功，但该模型没有明显的细胞凋亡。科学意义：本项目主要从体内外两方面验证了IFNγ-JAK/STAT1-IFITM3通路是治疗UC湿热证的作用机制之一，对阐述黄芩汤多靶点、多途径治疗UC湿热证的科学内涵有重要意义，为临床创新药物研发提供研究基础。