抗类风关异喹啉类生物碱通过脂肪酸氧化促进Treg细胞生成的机制研究

The infusion of in vitro-expanded autologous regulatory T cells (Tregs) or in vivo induction of Tregs is an efficient strategy of treating autoimmune diseases. Our previous studies have demonstrated that three isoquinoline alkaloids, including sinomenine, tetrandrine and norisoboldine, have distinct anti-rheumatoid mechanisms. They function mainly through activating aryl hydrocarbon receptor (AhR), inducing intestinal generation of Tregs, and therefore reserving systemic immune imbalance. This project aims to elucidate the underlying mechanisms by which these alkaloids induce Tregs via activating AhR based on the crucial role of fatty acid oxidation (FAO) in the fifferentiation and survival of Tregs. Under the condition of Tregs differentiation, the effects of these alkaloids on FAO and Tregs generation are observed simutaneously, and the relationship is assessed. The participation and mechanism of FAO in the alkaloids-induced generation of Tregs are investigated in view of Foxp3 histone acetylation. Moreover, the key signaling pathway for alkaloids enhancing FAO of Tregs is explored on the basis of the central role of LKB1. Finally, the intermediate effect of AhR in the enhancement of FAO, induction of Treg generation and consequent amelioration of arthritis by the alkaloids is confirmed by using gene knockout thchnique or specific blockers. The findings will increase our understandings of the anti-arthritis mechanisms of the three alkaloids, and contribute to the expansion of their clinical uses in other immune diseases as natural inducers of Tregs.

体外扩增后回输或体内诱生Treg细胞是治疗自身免疫性疾病的新策略。前期研究发现，三种异喹啉类生物碱（青藤碱、粉防己碱和去甲异波尔定）具有独特的抗类风关机制，主要通过激活AhR，诱导肠道Treg细胞生成，逆转全身性免疫失衡状态。本课题基于脂肪酸氧化在Treg细胞分化和存活中的重要作用，探究上述生物碱活化AhR后诱生Treg细胞的机制。在Treg细胞分化过程中，动态考察生物碱对脂肪酸氧化的促进作用及其与诱生Treg细胞的相关性；从促进Foxp3组蛋白乙酰化角度，阐明脂肪酸氧化在生物碱促进Treg细胞生成中的参与和机制；依据LKB1的核心调控作用，揭示生物碱促进Treg细胞脂肪酸氧化的关键信号途径；采用基因敲除/特异性拮抗剂等，证实AhR在生物碱促进脂肪酸氧化、诱生Treg细胞、改善关节炎中的介导作用。加深对生物碱抗类风关机制的认识，促进其作为天然的Treg细胞诱生剂用于其他免疫性疾病的治疗。

课题组前期研究发现，具有抗类风湿性关节炎活性的三种异喹啉类生物碱（青藤碱、去甲异波尔定和粉防己碱）在体内外均能诱导调节性T细胞（Treg细胞）形成，但其作用机制尚不清楚。本项目从调节能量代谢的角度，探索异喹啉类生物碱的作用机制。. 体外比较研究发现，去甲异波尔定和粉防己碱诱导Treg细胞分化的作用较强，而青藤碱的作用较弱。UHPLC-QTOF/MS代谢组学分析显示，在Treg细胞分化中去甲异波尔定和粉防己碱优先干预脂肪酸氧化（FAO）途径，升高相关代谢物的含量。氧消耗率（OCR）和FAO相关酶mRNA表达检测发现，去甲异波尔定和粉防己碱在Treg细胞分化的早期促进FAO，而抑制或基因敲减FAO限速酶CPT1明显减弱化合物对Treg细胞分化的促进作用。去甲异波尔定和粉防己碱升高Treg细胞中乙酰CoA水平，增强FOXP3启动子和CNS2区域的H3K27乙酰化，而敲减CPT1表达明显减弱两个化合物对Treg细胞生成和组蛋白乙酰化的促进作用及其抗胶原关节炎活性。此外，体内外研究发现，去甲异波尔定和粉防己碱通过激活芳烃受体（AhR），诱导肝激酶B1（LKB1）磷酸化，促进FAO及后续的Treg细胞形成。. 上述工作揭示了异喹啉类生物碱诱导Treg细胞形成从而改善类风湿性关节炎的机制，提示AhR和LKB1等有望成为疾病治疗的潜在靶标。