糖酵解关键酶基因表达对胶原诱导关节炎大鼠及其糖代谢的机制研究

Rheumatoid arthritis is a common rheumatic immune disease, more and more studies have found abnormal glucose metabolism in rheumatoid arthritis, the probability of occurrence of diabetes than normal people increased. At present, the research on RA is mainly focused on immune, genetic and cell biology, and glucose metabolism pathway on the pathogenesis of RA is currently reported very little. In recent years, studies have found that glucose metabolism in the pathogenesis of RA plays an important role. Studies have shown that RA synovial tissue increased glycolytic activity. In our early study, we found that the levels of IL-6 and TNF-α in serum of collagen-induced arthritis rats were higher than those of control group, the level of insulin decreased and the expression of Caspase-3 was increased. However, the mechanism of apoptosis of pancreatic islet cells in CIA rats was unknow. In order to clarify the abnormality of glucose metabolism in CIA rats, our group will detect the gene expression of the key enzyme of glycolysis in synovial cells and islet β-cell in experimental group and control group, and the effect of glucose metabolism pathway on RA. Detect the relationship of key enzyme of glycolysis gene expression with synovial inflammation and islet apoptosis, further explore the role of glucose metabolism in rheumatoid arthritis, find new markers and targets of RA early diagnosis and treatment.

类风湿关节炎是常见的风湿免疫疾病，越来越多的研究发现类风湿关节炎存在糖代谢异常，发生糖尿病的概率也较正常人升高。目前对RA的研究主要集中于免疫、遗传和细胞生物学等方面，而葡萄糖代谢通路对RA发病机制的研究报道极少。近年，有研究发现葡萄糖代谢在RA的发病机制中具有重要作用。研究表明RA滑膜组织中糖酵解活性增高。本课题组既往研究发现胶原诱导关节炎大鼠血清中IL-6、TNF-α水较对照组升高，胰岛素水平下降，细胞凋亡蛋白Caspase-3表达增加，但是对于CIA大鼠胰岛细胞凋亡的发生机制未深入研究。为明确CIA大鼠并发糖代谢异常情况，本课题组将检测实验组与对照组滑膜细胞、胰岛β细胞糖酵解关键酶基因表达情况及糖代谢通路对RA发生的影响，明确糖酵解关键酶基因表达与滑膜炎症、胰岛凋亡的关系，进一步探索糖代谢途径在类风湿关节炎发生作用，为RA早期诊断寻找新的标记物及RA的治疗新靶点。

RA是慢性炎症性自身免疫性疾病，RA的发病机制至今仍未完全清楚，多种原因及致病因素都可能发挥作用。越来越多的研究表明，糖代谢在类风湿关节炎的发病机制中起着重要作用。本研究通过建立胶原诱导关节炎大鼠（CIA）模型，检测CIA大鼠体内炎症状态、CIA大鼠滑膜细胞、胰岛β细胞糖酵解关键酶基因表达差异，明确CIA大鼠体内炎症水平及胰岛凋亡状态及糖酵解关键酶表达情况，明确糖酵解关键酶基因表达与滑膜炎症、增殖、胰岛凋亡的关系，探讨糖代谢异常对RA的发病作用。本研究采用WB、免疫组化等技术对CIA及对照组两组中凋亡相关指标Fas、Cleaved Caspase3以及Bax/Bcl2表达水平进行分析，结果表明：与对照组相比，CIA组中胰腺组织中Fas、Cleaved Caspase3以及Bax/Bcl2均明显上调（P < 0.01），CIA组关节炎模型中胰腺组织出现典型的凋亡模式。ELISA法检测两组大鼠血清中细胞因子表达，CIA大鼠血清中TNFα、IL-1β、IL-6、Leptin、Resistin和MCP-1细胞因子的含量均明显高于对照组。取CIA组及对照组大鼠滑膜组织进行蛋白质组学技术，在定量到的蛋白质中，我们发现在CIA与对照组比较，有465个蛋白表达发生上调，368个蛋白表达发生下调，CIA组大鼠滑膜组织中糖酵解代谢相关酶磷酸果糖-2-激酶1（PFKFB1）及L-乳酸脱氢酶B链均显著减少，糖酵解代谢调节相关酶蛋白激酶C delta型及AMPK β1显著增加，蛋白激酶A C-β显著减少，增殖过程相关蛋白细胞周期蛋白依赖性激酶5激活蛋白2（CDK5R2）显著增加，同时调节细胞凋亡及细胞自噬的蛋白Ubiquilin-1显著增加。上调蛋白中筛选出糖异生限速酶磷酸烯醇式丙酮酸羧激酶2（PCK2），并在滑膜细胞和组织中进行WB及免疫组化验证。PCK2介导了NFκB和AKT\/MAPK信号通路中的蛋白质磷酸化，这是控制炎症级联激活的主要信号通路，AKT\/MAPK可介导NF-κB激活，导致促炎症因子生成增加，介导炎症反应，从而导致RA炎症病理过程。