痛风汤对急性痛风性关节炎模型大鼠踝关节滑膜组织中NLRP3炎症小体及NETs的影响  

Effect of Tongfeng Decoction(痛风汤)on NLRP3 Inflammatory Vesicles and Neutrophil Extracellular Traps in Synovial Tissues of Ankle Joints in Acute Gouty Arthritis Model Rats

作　　者：邓雍钲 孟庆良 马俊福 崔家康 李凯 王赓丰 宋晨阳 卞华[3] DENG Yongzheng;MENG Qingliang;MA Junfu;CUI Jiakang;LI Kai;WANG Gengfeng;SONG Chenyang;BIAN Hua(College of Traumatiology and Orthopedics,Henan University of Chinese Medicine,Zhengzhou,450053;Henan Province Hospital of TCM(The Second Affiliated Hospital of Henan University of Chinese Medicine);Nanyang Institute of Technology)

机构地区：[1]河南中医药大学骨伤学院,河南省郑州市450053 [2]河南省中医院(河南中医药大学第二附属医院) [3]南阳理工学院

出　　处：《中医杂志》2024年第17期1816-1824,共9页Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(81874456);河南省中医药传承与创新人才工程(仲景工程)中医药学科拔尖人才项目;河南省中医药科学研究专项(20-21ZYZD05);河南省卫生健康委国家中医临床研究基地科研专项(2021JDZX2079)。

摘　　要：目的探讨痛风汤防治急性痛风性关节炎的可能作用机制。方法将60只Wistar雄性大鼠按照随机数字表法分为正常组、模型组、秋水仙碱组及痛风汤低、中、高剂量组,每组10只。痛风汤低、中、高剂量组分别给予痛风汤11.34、22.68、45.36 g/(kg·d)灌胃,秋水仙碱组给予秋水仙碱3.15×10^(-4)g/(kg·d)灌胃,正常组和模型组给予生理盐水10 ml/(kg·d)灌胃,连续7天。第5天灌胃1 h后,除正常组外其余各组大鼠参照改良Coderre法对右侧后肢踝关节建立急性痛风性关节炎模型;正常组大鼠在相同位置注射0.2 ml生理盐水。分别于造模前及造模6 h、12 h、24 h、48 h后测量大鼠踝关节肿胀度;采用HE染色观察踝关节滑膜组织病理学形态变化;免疫印迹法和RT-qPCR法分别检测踝关节滑膜组织中NOD样受体蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、半胱氨酸天冬氨酸蛋白酶1(Caspase-1)、白细胞介素1β(IL-1β)蛋白及mRNA表达水平;免疫组化法检测踝关节滑膜组织中Caspase-1、IL-1β阳性表达;ELISA法检测血清中肿瘤坏死因子α(TNF-α)、IL-1β、白细胞介素6(IL-6)含量;免疫荧光染色法观察踝关节滑膜组织中中性粒细胞胞外诱捕网(NETs)形成情况。结果与正常组比较,模型组大鼠各时间点关节肿胀度升高,踝关节滑膜组织中炎症细胞浸润及滑膜细胞层数明显增多,NLRP3、Caspase-1、ASC、IL-1β蛋白及mRNA表达均升高,Caspase-1和IL-1β阳性表达升高,NETs形成增多,血清中TNF-α、IL-6、IL-1β升高(P<0.05)。与模型组比较,秋水仙碱组与痛风汤高、中剂量组踝关节滑膜细胞增生及炎症细胞浸润均有改善;秋水仙碱组与痛风汤高、中、低剂量组在造模24 h、48 h后关节肿胀度,踝关节滑膜组织中Caspase-1、IL-1β阳性表达和NETs形成,以及血清中TNF-α、IL-6、IL-1β含量均降低;秋水仙碱与痛风汤高剂量组踝关节滑膜组织中NLRP3、ASC、Caspase-1、IL-1β蛋白及mRNA�Objective To explore the possible mechanism of Tongfeng Decoction(痛风汤)for preventing and treating acute gouty arthritis.Methods Sixty Wistar male rats were divided into normal group,model group,colchicine group and low-,medium-and high-dose Tongfeng Decoction groups according to the random number table,10 rats in each group.Tongfeng Decoction of 11.34,22.68 and 45.36 g/kg were given by gavage to low-,medium-and highdose Tongfeng Decoction groups,colchicine 3.15×10~(-4)g/(kg·d)to the colchicine group,and normal saline 10 ml/(kg·d)to the normal group and model group respectively for 7 consecutive days.After 1 hour of gavage on day 5,rats in all groups except the normal group were modelled as acute gouty arthritis in the ankle joint of the right hind limb with the modified Coderre's method;rats in the normal group were injected with 0.2 ml of normal saline at the same location.The swelling degree of the ankle joint was measured before modelling and after 6 h,12 h,24 h and 48 h of modelling,respectively.HE staining was used to observe the histopathological and morphological changes in the synovial tissue of the ankle joint;immunoblotting and RT-qPCR were used to detect NOD-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein(ASC),cysteine aspartate protease 1(Caspase-1),and interleukin 1β(IL-1β)protein and mRNA expression levels in ankle synovial tissues,respectively;immunohistochemistry was used to detect the positive expression of Caspase-1 and IL-1βin ankle synovial tissues;ELISA was used to detect the tumour necrosis factor alpha(TNF-α),IL-1β,and interleukin in serum;immunofluorescence staining was used to observe the formation of neutrophil extracellular traps(NETs)in the synovial tissues of the ankle joints.Results Compared with the normal group,rats in the model group showed elevated joint swelling at all time points,significantly increased inflammatory cell infiltration and the number of synovial cell layers in the synovial tissues of the ankle joints,elevated NLRP3,Caspase-1,A

关 键 词：痛风性关节炎 踝关节 NOD样受体蛋白3 炎症小体 中性粒细胞胞外诱捕网 痛风汤 

分 类 号：R285.5[医药卫生—中药学] R-332[医药卫生—中医学]