基于AMPK-TSC信号探索四神丸干预能量代谢调控免疫记忆性T细胞分化治疗溃疡性结肠炎的作用机制研究

It is hotspot of immunotherapy that energy metabolism regulate the differentiation of memory T cell (Tm), which disorder is an important character of ulcerative colitis (UC). All hinted that this is an effective therapeutic strategy of UC treated by regulating energy metabolism to improve the differentiation of Tm. AMPK can regulate the differentiation of Tm via regulating fatty acid oxidation of mitochondria and TSC signaling pathway. Sishen Wan (SSW) has a potential in regulating energy metabolism, and regulating immune to effectively treat UC. However there is hardly reported that SSW treated via regulating energy metabolism and the differentiation of Tm. So we propose a scientific hypothesis that SSW maybe effectively treats UC by regulating energy metabolism and the differentiation of Tm via AMPK-TSC signaling pathway. In the present project, mice with chronic relapsed colitis were administrated SSW for 10 days. We will verify the effect of SSW, test the differentiation of Tm, the level of energy metabolism and activation of ATPase, and separate CD4+Tm、CD8+Tm cells to screen related genes of FAO and glycolytic pathway, while we will analyze expressions of the upstream and downstream proteins of AMPK-TSC signaling pathway. Our objects are to explore the immunological mechanism of SSW treated UC by regulating energy metabolism and the differentiation via AMPK-TSC pathway. These are helpful to expand and perfect the pharmacological action from energy metabolism and the differentiation of Tm.

能量代谢调控记忆性T细胞(Tm)的分化水平是免疫治疗学的研究热点，其发生紊乱也是溃疡性结肠炎(UC)发病的重要特征之一，提示干预能量代谢调控Tm分化是治疗UC的有效策略之一。AMPK可通过调节线粒体脂肪酸氧化(FAO)和TSC信号途径共同干预Tm分化。四神丸有调控能量代谢潜力且可调节免疫有效治疗UC，但能否从调控两者二者相关性出发治疗UC鲜见报道。故我们提出四神丸可能干预AMPK-TSC信号调节能量代谢影响Tm分化治疗UC的科学假设。本课题采用四神丸治疗UC小鼠模型，验证四神丸疗效，检测Tm分化水平，分析能量代谢水平和ATP酶活性，并分离CD4+Tm、CD8+Tm细胞，采用PCR array筛选FAO和糖酵解过程相关基因，检测其AMPK-TSC信号上下游蛋白活性，旨在初次从AMPK-TSC信号探明四神丸干预能量代谢调控Tm分化治疗UC的作用机制，以完善四神丸治疗UC的免疫学机制。

溃疡性结肠炎（ulcerative colitis，UC）是一种以腹痛、腹泻和黏液脓血便为主要临床表现的直肠和结肠慢性非特异性炎症性疾病。免疫记忆细胞功能紊乱、能量代谢紊乱是溃疡性结肠炎发病的重要特征之一。四神丸作为温脾补肾的代表方，广泛应用于临床，具有疗效好、复发率低的特点，研究显示四神丸全方及各组成药物均有调控组织或细胞能量代谢的作用，且可有效调控机体的免疫状态治疗溃疡性结肠炎。但四神丸能否从调控记忆T细胞和细胞能量代谢的相关性出发治疗溃疡性结肠炎，国内外鲜见报道。. 本课题采用经典的葡聚糖硫酸钠（DSS）诱导结肠炎动物模型，经四神丸给药治疗7天后，发现四神丸可以明显改善溃疡性结肠炎小鼠状态，修复小鼠结肠黏膜；可通过调节记忆T细胞及其CD4+、CD8+亚群水平治疗DSS诱导结肠炎，如升高结肠炎小鼠结肠中CD45RA－CD62L+CCR7+Tcm、CD45RA+CD62L+CCR7+Tcm细胞水平，显著降低CD45RA－CD62L－CCR7－Tem细胞水平。再进一步根据细胞表面标记物区分CD4+和CD8+亚群，均显示有不同程度的升高或下降。能明显提升小鼠结肠组织中Ca2+-Mg2+-ATPase、Na+-K+-ATPase及ATP含量活力值，显著降低LDH活力值。升高DSS诱导的结肠炎小鼠结肠粘膜AMPKα、p-AMPKα、TSC1、TSC2、Raptor、Kif2a、4E-BP2、p70S6K蛋白表达水平，降低PI3K、AKT、p-Akt、Rheb、T-bet、HIF-1a、c-Myc蛋白表达水平。调控DSS诱导的结肠炎小鼠结肠粘膜ATP相关的mRNA和miRNA基因表达。. 以上结果提示四神丸治疗溃疡性结肠炎作用可能与AMPK-TSC信号的活化、PI3K-Akt信号的抑制及其下游mTOR信号所调控能量代谢有着密切的的内在联系，其内在机制可能是通过上述机制提升ATP合成酶亚基表达及ATP水平以干预细胞能量代谢途径，进而调控记忆T细胞分化来实现治疗目的。