基于链接“糖代谢异常-炎症恶化”关键信号琥珀酸/GPR91的白虎加桂枝汤“祛热痹”机制研究

Recent studies showed that the interaction between glucose metabolism and inflammation is an important segment in the deteriorate of rheumatoid arthritis, and the succinate/GPR91 signaling pathway is the pivotal conditioner of the process. During previous study, We found that the characteristic methylation genes of pyretic arthralgia were mainly concentrated on three directions: abnormal cell structure, inflammation and energy metabolism, and 85% aberrant methylation genes were associated with succinate metabolism and the function of succinate receptor; The classical formula Baihu Guizhi Decoction could recuperate those abnormal methylation and observably inhabited the extremely overexpression of GPR91. These results provide a directly support to our hypothesis that “Baihu Guizhi Decoction alleviates or reduces pyretic arthralgia symptoms by regulating succinate metabolism, reducing or removing succinate accumulation, and preventing IL-1β amplification cascade via the intervening succinate/GPR91 signal”. Based on the hypothesis, we intends to take macrophages, pyretic arthralgia model rats and succinate receptor knockout mice as the research objects, starting with succinate metabolism and the key points in the deteriorate of inflammation, investigating the multileve and multi targets intervention effect of Baihu Guizhi Decoction on succinate/GPR91 signal cascade. Then, To reveal the characteristics and disciplinarian of Baihu Guizhi Decoction and to interpret objectively its "dispelling pyretic arthralgia" molecular mechanisms that succinate/GPR91 pathway connected abnormal glucose metabolism induce the deteriorate of inflammation.

新近发现糖代谢与炎症的相互级联是类风湿性关节炎恶化的一个重要环节，而琥珀酸/GPR91信号途径是该环节的关键调节点。项目前期发现热痹异常甲基化基因主要富集于细胞结构异常、炎症和能量代谢三个方向，且85%的异常甲基化基因与琥珀酸代谢或受体功能有关，热痹经方白虎加桂枝汤能够回调上述异常甲基化并显著抑制关节滑膜GPR91异常高表达，该结果为我们的“白虎加桂枝汤通过调节琥珀酸一个或多个代谢途径，降低或清除琥珀酸的堆积，干预琥珀酸/GPR91信号阻止IL-1β等的炎症放大级联，缓解或减轻热痹症状”的工作假说提供了直接支撑。故本项目拟以滑膜巨噬细胞、热痹大鼠和琥珀酸受体敲出小鼠为载体，从琥珀酸代谢及炎症恶化作用各关键点为切入，考察白虎加桂枝汤对琥珀酸/GPR91信号级联的多层次多靶点的干预效应，揭示白虎加桂枝汤的作用特点和规律，客观阐释其“祛热痹”的糖代谢异常恶化炎症的琥珀酸/GPR91途径分子机制。

本研究结合近年来琥珀酸在RA发生发展中的作用和传统医学对热痹的病机认识，以关节局部琥珀酸堆积为中心，能量代谢异常-炎症恶化为关键点，围绕琥珀酸生成和水解平衡、琥珀酸激活GPR91和HIF-1α介导炎症恶化开展了白虎加桂枝汤“祛热痹”的作用及其机制研究。.动物实验表明热痹模型大鼠关节炎症反应明显，表现为足肿胀增加，滑膜炎性细胞浸润和增生明显，炎性因子IL-1β释放增加；滑膜细胞线粒体膜电位降低，琥珀酸含量增加，琥珀酸下游GPR91和HIF-1α蛋白表达增加。以上结果说明热痹的炎症反应与琥珀酸激活GPR91和HIF-1α有关。而白虎加桂枝汤可明显改善热痹大鼠足肿胀、足围，滑膜组织炎性浸润和增生等症状指标，减少滑膜组织中琥珀酸含量，抑制GPR91和HIF-1α的蛋白表达。因此，白虎加桂枝汤对热痹大鼠的治疗效应可能与减少琥珀酸并抑制其介导的GPR91和HIF-1α信号有关。细胞实验表明，炎性巨噬细胞琥珀酸释放增加，其下游GPR91和HIF-1α蛋白表达增加，而白虎加桂枝汤可显著减少琥珀酸，抑制GPR91和HIF-1α蛋白表达，与体外实验结果一致。进一步说明白虎加桂枝汤抗炎与抑制琥珀酸及其介导的GPR91和HIF-1α蛋白活化有关。.琥珀酸堆积的直接原因是生成途径和水解消除途径之间失衡。本研究发现，热痹模型和炎性巨噬细胞模型内与琥珀酸生成相关的丙酮酸、谷氨酰胺和GABA含量均增加，α-KGDH活性明显增加；而与琥珀酸水解相关的SDH活性降低，富马酸含量减少。白虎桂枝汤可显著降低异常增加的丙酮酸、谷氨酰胺和GABA含量，抑制α-KGDH活性，促进SDH活性和增加富马酸含量。说明白虎加桂枝汤减少琥珀酸异常堆积可能与抑制琥珀酸生成途径并促进水解途径有关。