通络解毒泄浊方通过调控中性粒细胞外诱捕网的形成与释放对痛风性关节炎的治疗作用  

Therapeutic Effect of Tongluo Jiedu Xiezhuo Prescription on Gouty Arthritis by Regulating Formation and Release of NETs

作　　者：刘婷婷 刘树民[1] 于纯淼 王宇[1] 于栋华[1] LIU Tingting;LIU Shumin;YU Chunmiao;WANG Yu;YU Donghua(Institute of Traditional Chinese Medicine,School of Pharmacy,Heilongjiang University of Chinese Medicine,Harbin 150040,China;Hainan Provincial Center for Drug Safety Evaluation and Research,Hainan Medical University,Haikou 570100,China)

机构地区：[1]黑龙江中医药大学中医药研究院,药学院,哈尔滨150040 [2]海南医学院海南省药物安全性评价研究中心,海口570100

出　　处：《中国实验方剂学杂志》2024年第19期73-80,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82074149);黑龙江省自然科学基金项目(LH2022H069);海南省教育厅资助项目(Hnky2023-33)。

摘　　要：目的:探讨通络解毒泄浊方(TLJDXZ)通过调控中性粒细胞外诱捕网(NETs)的形成与释放过程对缓解痛风性关节炎(GA)大鼠炎症反应的作用研究。方法:通过采用氧嗪酸钾溶液联合尿酸钠盐溶液以注射方式建立GA大鼠模型,将72只SD大鼠随机分为空白组、模型组、TLJDXZ低、中、高剂量组(1.58、3.15、6.30 g∙kg^(-1))、秋水仙碱组(0.30 mg∙kg^(-1))。采用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)表征TLJDXZ的化学成分;苏木素-伊红(HE)染色观察大鼠踝关节组织形态学变化;酶联免疫吸附测定法(ELISA)检测血清及组织中炎性因子白细胞介素(IL)-1β、IL-6、IL-8、血尿酸(BUA)、肿瘤坏死因子(TNF)-α、活性氧(ROS)、髓过氧化物酶(MPO)水平;绿色荧光染料(PicoGreen)测定血浆中双链脱氧核糖核酸(ds-DNA)浓度;蛋白免疫印迹法(Western blot)检测瓜氨酸组蛋白-3(Cit-H3)及MPO的蛋白表达;免疫荧光(IF)检测中性粒细胞弹性蛋白酶(NE)及MPO表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测肽酰基精氨酸脱亚胺酶4(PAD4)的mRNA表达。结果:与空白组比较,模型组关节肿胀严重,关节腔可见脱落的细胞碎片、滑膜及结缔组织增生并伴有炎性细胞点状浸润,血循环及关节组织内氧化损伤和炎症明显加重(P<0.05),NETs相关蛋白Cit-H3、MPO、NE、PAD4表达明显上调(P<0.05);与模型组比较,TLJDXZ各剂量组大鼠关节损伤明显减轻,肿胀情况得到缓解,关节腔内炎性浸润情况、软骨及滑膜形态及关节结构明显缓解,TLJDXZ组外周血循环及关节组织内氧化损伤和炎症明显减轻(P<0.05),NETs相关蛋白Cit-H3、MPO、NE及PAD4表达明显下调(P<0.05)。结论:TLJDXZ能有效缓解GA大鼠的关节炎症反应,其作用机制可能与调控NETs形成与释放有关。Objective:To explore the effect of Tongluo Jiedu Xiezhuo prescription(TLJDXZ)on alleviating inflammatory response in rats with gouty arthritis(GA)by regulating the formation and release of neutrophil extracellular traps(NETs).Method:Oxonic acid potassium salt solution combined with uric acid sodium solution was used to establish the GA rat model by injection.Seventy-two SD rats were randomly divided into control model,model group,and low-,medium-,and high-dose(1.58,3.15,and 6.30 g∙kg^(-1))TLJDXZ groups,and colchicine group(0.30 mg∙kg^(-1)).Ultra-high performance liquid chromatography-quadrupole-timeof-flight mass spectrometry(UPLC-Q-TOF-MS)was used to characterize the chemical components of TLJDXZ.HE staining was used to observe the ankle joint tissue structure of rats.Enzyme-linked immunosorbent assay(ELISA)was used to determine the levels of inflammatory factors interleukin(IL)-1β,IL-6,IL-8,blood uric acid(BUA)tumor necrosis factor(TNF)-α,reactive oxygen species(ROS),and myeloperoxidase(MPO)in serum and tissue.PicoGreen staining method was used to measure ds-DNA concentration in plasma.Western blot was used to detect the expression of citrullinated histone H3(Cit-H3)and MPO proteins.Immunofluorescence was used to detect the expression of neutrophil elastase(NE)and MPO.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect peptidylarginine deiminase 4(PAD4)mRNA expression.Result:Compared with control group,the model group had severe joint swelling,with shed cell debris,synovial and connective tissue proliferation,and inflammatory cell punctate infiltration visible in the joint cavity.Blood circulation,oxidative damage,and inflammation in joint tissues significantly worsened(P<0.05).The expression of NETs-related proteins Cit-H3,MPO,NE,and PAD4 was significantly upregulated(P<0.05).Compared with the results in model group,joint damage in the TLJDXZ groups was significantly reduced,and the swelling was alleviated.The inflammatory infiltration in the joint cavity,cartil

关 键 词：通络解毒泄浊方 痛风性关节炎 中性粒细胞外诱捕网 炎症反应 炎性浸润 

分 类 号：R2-0[医药卫生—中医学] R33R289R971.1R684.3