基于miRNAs对T细胞免疫平衡调控探讨痛泻要方治疗肝郁脾虚型溃疡性结肠炎的机制

Ulcerative colitis (UC), as s chronic non-specific inflammatory disease, is a syndrome of deficiency in origin and excess in superficiality and belongs to the category of diarrhea. MiRNAs that are closely related to the mechanism of diarrhea were screened out by the applicant at earlier stage, and it was found that miRNAs could regulate T cell-mediated Th1/Th2 and Th17/Treg inflammatory balance and participate in mediating the mechanism of diarrhea as an intermediate link. In addition, previous literature researches have confirmed that stagnation of liver qi and spleen deficiency is a common syndrome of this disease and tong xie yao fang is the treatment guidance, which can regulate the ratio values of Th1/Th2 and Th17/Treg and regulate the expressions of miRNAs related to diarrhea; however, no analysis of its therapeutic mechanism and which miRNAs are directly associated with the regulation of T cell-mediated inflammatory balance was conducted. On the basis of existing researches, this study intends to apply TNBS combined with diet binding method to construct the model of stagnation of liver qi and spleen deficiency, and miRNAs chip and transfection technology to screen out differentiated miRNAs, verify their regulatory effects on the inflammatory balance of Th1/Th2 and Th17/Treg, and elaborate the intervention mechanism of tong xie yao fang, providing basic research support for further functional analysis.

溃疡性结肠炎（UC）为慢性非特异性炎症性疾病，属本虚标实之证，归属泄泻范畴。申请人前期筛选出与泄泻机制密切相关的miRNAs并发现其作为中间环节参与T细胞介导Th1/Th2、Th17/Treg免疫平衡的调节；前期研究证实肝郁脾虚证是本病常见证候且痛泻要方是指导用方，其能够通过调节T细胞免疫平衡及与泄泻相关的miRNAs的表达参与UC的治疗，然并未分析其作用机制与具体哪些靶向miRNAs相关，以及这些靶向miRNAs是否对T细胞介导免疫平衡具有直接调节作用。本研究拟在已有研究基础上，采用TNBS配合饮食束缚法建立肝郁脾虚型UC大鼠模型，通过miRNAs芯片筛选找到靶向miRNAs并使用转染技术验证其对Th1/Th2、Th17/Treg免疫平衡的调节作用，以此阐述痛泻要方干预治疗机制，为下一步功能分析提供基础研究支持。

溃疡性结肠炎（UC）为慢性非特异性炎症性疾病，属本虚标实之证，归属泄泻范畴。申请人前期筛选出与泄泻机制密切相关的miRNAs并发现其作为中间环节参与T细胞介导Th1/Th2、Th17/Treg免疫平衡的调节，为了证实痛泻要方治疗机制与miRNAs在T细胞分化中的作用相关，本项目采用TNBS配合饮食束缚法建立肝郁脾虚型UC大鼠模型，通过miRNAs芯片筛选找到靶向miRNAs并使用转染技术验证其对Th1/Th2、Th17/Treg免疫平衡的调节作用。重要研究成果主要是确定肝郁脾虚证模型的成功建立，并采用OFT、EPM和NSF实验对动物模型进行相关评价，结果显示模型动物表现为易于激怒，防御姿态明显，大便稀溏等症状，OFT实验距离，中央区进入次数减少，EPM实验总距离、开放臂进入次数停留时间减少，NSF时间延长；痛泻要方治疗能够改善UC模型炎症平衡情况，促使IL-17和IFN-γ呈现降低趋势，而升高IL-4和IL-10的含量，说明其能够通过调节T细胞介导的炎性平衡发挥相关治疗作用；痛泻要方治疗机制与其对粘膜miRNAs的调节相关，研究发现48个miRNAs存在显著差异，其中上调的miRNAs有21个，下调的有27个，结合文献数据，最终确定miR-31、miR-194、miR-326、miR-196、miR-205、miR-215在痛泻要方治疗及UC发病机制中具有重要的作用；针对上述miRNAs对T细胞的分化进行了体内外转染实验，结果显示，miR-31和miR-205主要发挥炎性促进作用，表现为促使T细胞朝以Th1和Th17为主的方向进行分化，miR-194、miR-326促使T细胞朝以Th2和Treg的方向分化，miR-196和miR-215主要促使T细胞分化Treg和Th17，总体表现为炎性抑制作用；差异miRNAs的Go分析和KEGG预测结果显示，信号通路方面主要与axon guidance 信号、Wnt、PI3Akt信号通路相关。本项目的研究意义表明，痛泻要方发挥治疗作用与其对miRNAs的调节相关，miRNAs主要参与对T细胞介导的炎性平衡的调节作用，后续研究值得对差异miRNA调节的信号通路和靶点进行进一步分析。