刺血疗法对急性痛风性关节炎大鼠局部IL-1β、IL-10启动子甲基化的影响

The Effects of Pricking Blood Therapy on Promotor Methylation of IL-1β,IL-10 in Rats Local Ankle Joint with Acute Gouty Arthritis Model

目的观察刺血疗法对急性痛风性关节炎大鼠造模关节局部白介素-1β(IL-1β)、白介素-10(IL-10)基因的相对表达及启动子区甲基化水平的影响,探讨刺血疗法治疗急性痛风性关节炎的相关表观遗传调控机制。方法 36只SD大鼠随机分为正常组、模型组、药物组、刺血组。右踝关节腔注射尿酸钠建立急性痛风性关节炎大鼠模型。刺血组在右侧昆仑穴点刺放血,药物组以布洛芬灌胃治疗。评价造模关节肿胀程度,光镜下观察关节腔组织形态学改变,qPCR和焦磷酸测序检测大鼠造模关节局部软骨及其周围软组织IL-1β、IL-10的mRNA表达水平及基因启动子区甲基化水平。结果与模型组、药物组比较,刺血组关节肿胀指数明显降低(P<0.01)。刺血疗法能减少关节腔内尿酸盐结晶沉积,抑制炎性细胞的侵润,改善关节滑膜的组织形态结构。与模型组比较,刺血组IL-1βmRNA的相对表达量明显降低(P<0.01)。但IL-1β基因启动子区甲基化水平与其mRNA表达不呈负相关。与正常组、模型组、药物组比较,刺血组IL-10 mRNA的相对表达量升高(P<0.01),IL-10平均甲基化率降低(P<0.05~0.01)。IL-10基因启动子甲基化水平与其mRNA表达呈显著负相关(r=-0.899,P<0.01)。结论刺血疗法通过DNA甲基化的表观遗传修饰调控IL-10基因表达可能是刺血疗法发挥抗炎效应的重要机制之一。

OBJECTIVE To observe the cross section diameter and the effects of pricking blood therapy on acute gouty ar- thrits rats modet＇s interleukin-1β （IL-1t3）, interleukin -10 （IL-10） mRNA expression and promoter methylation in local ankle, and explore the epigenetics mechanisms of pricking blood therapy＇s treatment. METHODS 36 SD rats were randomly divided into normal control group, urate arthritis group, ibuprofen group and bloodletting group. The acute gouty arthrits model set up by injecting uric acid into the right ankle joint cavity . The ibuprofen group was treated by gastric perfusion of ihuprofen, while the bloodletting group was pricked at the right Kunlun points. The swelling index of the modeling joint was evaluated, and the morphological changes were observed under light microscope. The mRNA expression levels and promoter methylation status of IL-I~ and IL-10 in rats local ankle were detected by qPCR and pyrosequencing detection. RESULTS Compared with the urate arthritis group and ibuprofen group, the joint swelling index in the bloodletting group was decreased （P^0.01） after the treatment. Pricking blood therapy reduced intracellular deposition of uric acid in the joint cavity, ＂and inhibited inflammato- ry cells to improve the synovial membrane tissue. Compared with urate arthritis group, the IL-1β mRNA expression levels of blood group was decreased significantly （P〈0.01）. IL-1β promoter methylation and mRNA expression didn＇t showed a nega- tive correlation. Compared with normal control group, urate arthritis group and ibuprofen group, the expression levels of IL-10 mRNA in blood group increased significantly （P 〈0.01）, and the average methylation rate of IL-10 among blood group de- creased significantly （P〈0.05-0.01）. IL-10 promoter methylation and mRNA expression showed a strong negative correla- tion （r =- 0. 899, P d0.01）. CONCLUSION In epigenetics, the methylation of IL-10 can regulate the gene expression, which can be one of important mechanisms which shows anti-inflammatory effects of pricking blood therapy .