不同时辰艾灸治疗RA的抗炎节律及核心钟基因的调控机制研究

Previous studies have verified that the circadian rhythm of RA is attributed to the diurnal oscillator of related key cytokines and hormones. The disturbed bioclock plays an important role in the RA pathomechanism, while core clock genes regulate bioclock system at the molecular level and interact with the cytokines and hormones related to RA. In addition, moxibustion is able to modulate the expression and rhythm of cytokines, hormones and clock genes. Based on these above study results, we put forward a hypothesis of that moxibustion at different times is capable to benign adjust the expression level and rhythm of clock genes, and then regulate the cytokines and hormones more efficacious, then restrain the RA inflammation reaction better and modulate the RA pathology rhythm. Furthermore we choose two different times via acupuncture chronomedicine theory to intervene moxibustion treatment, and focus on the expression level and circadian pattern of core clock genes. This study will follow the main research line of cytokines and hormones——the expression level and circadian rhythm of core clock genes——effect and rhythm of anti-inflammation, and utilize the advanced technology methods of ELISA、RT-PCR、WESTERN BLOT and the like to measure outcomes. It is anticipated that this research will tentatively reveal the difference of curative effect of moxibustion at different time, and consequently explore the underlining mechanisms of moxibustion on regulating the serum concentration and circadian rhythm of cytokines and hormones as well as the expression quantity and diurnal pattern of core clock genes in depth. This study will exert a significant role in deepening the mechanism research of moxibustion on RA, enriching the scientific connotation further and guiding clinical treatment.

本课题基于“RA临床症状的昼夜节律性归因于细胞因子和激素水平的昼夜振荡，紊乱的生物钟参与了RA的病理机制，核心钟基因在分子水平调控着生物钟系统并与RA相关细胞因子和激素交互影响，艾灸可调节细胞因子、激素、钟基因的表达和节律”等研究结果，提出“不同时辰艾灸可良性调控生物钟基因的表达水平和节律，进而更有效地调节RA细胞因子和激素水平，更好地调整RA病理节律、抑制RA炎症反应”的假说，以针灸时间医学理论为指导选择不同时辰介入艾灸治疗，聚焦核心生物钟基因的表达水平和节律，以“细胞因子、激素——生物钟基因——抗炎效应、节律”为研究主线，运用ELISA、基因定量、蛋白表达等方法技术，观察不同时辰介入艾灸的疗效差异，深入探索艾灸调节RA细胞因子和激素水平节律、调控核心生物钟基因表达水平和节律的潜在机制，对于深化艾灸治疗RA的机理研究、进一步丰富艾灸的科学内涵、指导临床具有重要作用。

类风湿性关节炎（RA）临床表现具有昼夜节律性，艾灸治疗RA疗效确切，但其是否与生物钟基因表达水平和节律相关尚不清楚。本研究以血清炎性细胞因子浓度研究不同时辰艾灸治疗RA的抗炎效应，以不同组织核心生物钟基因表达水平分析其昼夜节律模式，研究不同时辰艾灸对RA大鼠核心钟基因表达水平和节律的影响。结果显示：与空白组相比，模型组大鼠血清IL-1β、IL-18、MMP3、IFN-γ、GSDMD浓度显著升高，滑膜NLRP3、Caspase-1、 GSDMD mRNA表达量明显升高。与模型组相比，艾灸组大鼠血清IL-1β、IL-18、MMP3、IFN-γ浓度水平均明显下降，酉时组大鼠滑膜NLRP3、Caspase-1、GSDMD mRNA表达量明显下降。与辰时组相比，酉时组NLRP3 、GSDMD 、Caspase-1 mRNA表达显著下调。下丘脑中，与空白组相比，模型组大鼠CLOCK、Bmal1 mRNA表达水平下调，模型组PER2、CRY1的表达水平显著下调。滑膜中，与空白组相比，模型组CLOCK、Bmal1、PER2、CRY1 mRNA表达水平显著降低；与模型组相比艾灸组PER2和CRY1 mRNA的表达水平显著上调。在滑膜中，模型组大鼠CLOCK、BMAL1蛋白表达水平与空白组比较显著下调；与模型组相比，艾灸组BMAL1蛋白表达水平均明显升高，辰时组CLOCK蛋白表达水平显著增加。钟基因昼夜节律而言，各组大鼠下丘脑中CLOCK、Bmal1、PER2、CRY1 mRNA的表达皆具有昼夜节律性。滑膜中，模型组CLOCK、Bmal1、PER2、CRY1 mRNA和CLOCK、BMAL1蛋白表达的昼夜节律均丧失。与模型组相比，艾灸组Bmal1振幅均有所升高，峰值时相推迟，艾灸组CLOCK峰值推迟。结果表明：艾灸具有抗炎作用，可减轻足肿胀、下调血清IL-1β、IL-18、MMP3、IFN-γ、GSDMD水平和滑膜NLRP3、Caspase-1、GSDMD mRNA表达水平；可通过上调RA大鼠不同组织中CLOCK、Bmal1、PER2、CRY1 mRNA和CLOCK、Bmal1蛋白的表达水平；可良性调节RA滑膜和下丘脑CLOCK、Bmal1、PER2、CRY1 的mRNA、蛋白的表达节律，从而更好地调整RA病理节律。