虎杖醇提物通过NLRP3/ASC/caspase-1轴干预小鼠急性痛风性关节炎的作用研究
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摘要
通过NLRP3/ASC/caspase-1轴探讨虎杖醇提物(ethanolic extract of Polygonum cuspidatum, PCE)对C57BL/6小鼠急性痛风性关节炎的干预作用及机制。采用PCE高、中、低3个剂量,以秋水仙碱为阳性对照,小鼠采用踝关节注射尿酸钠晶体(MSU)造模。动物模型通过PCE干预后,采用HE染色观察滑膜组织形态学变化;采用ELISA法测定踝关节滑膜组织分泌的IL-1β,IL-6和TNF-α浓度;蛋白印迹(Western blot)法测定滑膜组织中NLRP3,ASC,caspase-1蛋白表达情况;RT-PCR法测定滑膜组织中NLRP3,ASC,caspase-1 mRNA表达情况。结果表明,与正常组比较,模型组小鼠踝关节肿胀度明显增加,滑膜组织IL-1β,IL-6,TNF-α表达水平显著升高,NLRP3,ASC,caspase-1蛋白表达和mRNA水平显著升高;与模型组比较,虎杖醇提物组能够显著降低小鼠踝关节肿胀度,关节滑膜IL-1β,IL-6和TNF-α水平以及NLRP3/ASC/caspase-1轴蛋白和mRNA表达均显著降低。该研究初步探明虎杖醇提物能防治小鼠急性痛风性关节炎,其机制可能是调控NLRP3/ASC/caspase-1轴在基因和蛋白水平上的表达。
The aim of this paper was to study the effect and mechanism of alcohol extract from Polygonum cuspidatum(PCE) on acute gouty arthritis in C57 BL/6 mice through NLRP3/ASC/caspase-1 axis. The model mice which injected with ankle joint injection of sodium urate crystals(MSU) were orally administrated with three different concentration of PCE, with colchicine as positive control. HE staining was used for observing the morphological changes of synovial tissue; concentration of IL-1β, IL-6 and TNF-α secreted by synovial tissue of the ankle joint were detected by ELISA; mRNA and protein expression of NLRP3, ASC and caspase-1 in synovial tissue were detected by RT-PCR and Western blot respectively. The results showed that the swelling degree of ankle joint in model mice were significantly elevated; expression of IL-1β, IL-6 and TNF-α were significantly increased; mRNA and protein expression of NLRP3, ASC and caspase-1 also significant increase, compared with normal control group. The swelling degree of ankle joint significantly relief; expression of IL-1β, IL-6 and TNF-α in joint synovium significantly decrease; mRNA and protein expression of NLRP3, ASC and caspase-1 were significantly decrease in PCE treatment group compared with model group. Our research implied that alcohol extract from P. cuspidatum had positive effect on acute gouty arthritis in mice, and the regulation of NLRP3/ASC/caspase-1 axis may be its mechanism.
The aim of this paper was to study the effect and mechanism of alcohol extract from Polygonum cuspidatum(PCE) on acute gouty arthritis in C57 BL/6 mice through NLRP3/ASC/caspase-1 axis. The model mice which injected with ankle joint injection of sodium urate crystals(MSU) were orally administrated with three different concentration of PCE, with colchicine as positive control. HE staining was used for observing the morphological changes of synovial tissue; concentration of IL-1β, IL-6 and TNF-α secreted by synovial tissue of the ankle joint were detected by ELISA; mRNA and protein expression of NLRP3, ASC and caspase-1 in synovial tissue were detected by RT-PCR and Western blot respectively. The results showed that the swelling degree of ankle joint in model mice were significantly elevated; expression of IL-1β, IL-6 and TNF-α were significantly increased; mRNA and protein expression of NLRP3, ASC and caspase-1 also significant increase, compared with normal control group. The swelling degree of ankle joint significantly relief; expression of IL-1β, IL-6 and TNF-α in joint synovium significantly decrease; mRNA and protein expression of NLRP3, ASC and caspase-1 were significantly decrease in PCE treatment group compared with model group. Our research implied that alcohol extract from P. cuspidatum had positive effect on acute gouty arthritis in mice, and the regulation of NLRP3/ASC/caspase-1 axis may be its mechanism.
引文
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[17] 马鸿斌,王苗慧,孙红旭.清热排毒胶囊对急性痛风性关节炎模型大鼠核因子κB和白细胞介素-1β的影响[J].中国中医药信息杂志, 2014, 21(12): 42.
[18] 康乐,苗明三,刘慧娟,等.基于痛风病临床病症特点的动物模型分析[J].中国中药杂志,2018,43(22):4547.
[19] Yokose Kohei, Sato Shuzo, Asano Tomoyuki, et al. TNF-α potentiates uric acid-induced interleukin-1β (IL-1β) secretion in human neutrophils[J]. Mod Rheumatol,2018,28(3):513.
[20] 党万太,周京国,谢文光,等. NLRP3炎性体在痛风性关节炎患者炎症反应中的机制研究[J].中国免疫学杂志,2014,30(3):373.
[21] Punzi I, Calo L, Plobani M. Clinical significance of cytokine determination in synovial fluid[J].Crit Rev Clin Lab Sci,2002,39(1):63.
[22] 党万太,王婧,谢文光,等.sCD14在痛风性关节炎患者炎症反应中的变化及其意义[J].西安交通大学学报:医学版,2015,36(4):514.
[2] Chowalloor P V, Keen H I. A systematic review of ultrasonography in gout and asymp tomatic hyperuricaemia[J]. Ann Rheum Dis,2013,72(5):638.
[3] Singh J A, Saag K G, Bridges S L Jr, et al. 2015 American college of rheumatology guideline for the treatment of rheumatoid arthritis[J]. Arthritis Care Res (Hoboken),2016,68(1):1.
[4] 邓智标.血清C-反应蛋白和胱抑素C联合检测对痛风患者早期肾功能损伤的诊断价值[J].中国卫生产业, 2014, 12(26):179.
[5] Zhou M Z, Li S N, Song L, et al. 4-(2-(4-chlorophenyl)-1-((4-chlorophenyl)amino)ethyl) benzene-1,3-diol is a potential agent for gout therapy as a dual inhibitor of XOD and NLRP3[J]. Phytomedicine, 2018, 42:9.
[6] Yang Gabsik, Yeon Sang Hyeon, Lee Hye Eun, et al. Suppression of NLRP3 inflammasome by oral treatment with sulforaphane alleviates acute gouty inflammation[J]. Rheumatology (Oxford), 2018, 57(4):727.
[7] 金晓敏,张晓熙,郭璐,等.基于NLRP3炎性体轴探讨土茯苓总黄酮对痛风性关节炎的作用和机制[J].中国实验方剂学杂志,2018,24(4):90.
[8] 中国药典.一部[S]. 2015:208.
[9] 樊慧婷,丁世兰,林洪生.中药虎杖的药理研究进展[J].中国中药杂志,2013,38(6):2545.
[10] 陶晓璇,李刚,张增珠,等.秋水仙碱致不良反应的文献分析[J].中国药业,2016,25(2):84.
[11] 朱春霞,顾祖莲,杨博,等. 虎杖苷-桂皮醛对MSU诱导的THP-1细胞痛风性炎症模型的影响及机制[J].中药材,2017,40(7):1710.
[12] 顾袓莲.寒温配伍虎杖-桂枝药对干预急性痛风炎症中TLRs/MyD88分子信号通路机制研究[D].广州:广东药学院,2015.
[13] 任丽. 虎杖提取物治疗痛风性关节炎的初步成药性研究[D].遵义:遵义医学院,2017.
[14] 任丽,欧水平,陈灵,等.虎杖提取物及其有效部位的大鼠抗痛风性关节炎试验[J].中国实验方剂学杂志,2016,22(19):111.
[15] Reber Laurent L, Marichal Thomas, Sokolove Jeremy, et al. Contribution of mast cell-derived interleukin-1β to uric acid crystal-induced acute arthritis in mice[J]. Arthritis Rheumatol,2014,66(10):2881.
[16] Martinon Fabio. Mechanisms of uric acid crystal-mediated autoinflammation[J]. Immunol Rev, 2010, 233(1): 218.
[17] 马鸿斌,王苗慧,孙红旭.清热排毒胶囊对急性痛风性关节炎模型大鼠核因子κB和白细胞介素-1β的影响[J].中国中医药信息杂志, 2014, 21(12): 42.
[18] 康乐,苗明三,刘慧娟,等.基于痛风病临床病症特点的动物模型分析[J].中国中药杂志,2018,43(22):4547.
[19] Yokose Kohei, Sato Shuzo, Asano Tomoyuki, et al. TNF-α potentiates uric acid-induced interleukin-1β (IL-1β) secretion in human neutrophils[J]. Mod Rheumatol,2018,28(3):513.
[20] 党万太,周京国,谢文光,等. NLRP3炎性体在痛风性关节炎患者炎症反应中的机制研究[J].中国免疫学杂志,2014,30(3):373.
[21] Punzi I, Calo L, Plobani M. Clinical significance of cytokine determination in synovial fluid[J].Crit Rev Clin Lab Sci,2002,39(1):63.
[22] 党万太,王婧,谢文光,等.sCD14在痛风性关节炎患者炎症反应中的变化及其意义[J].西安交通大学学报:医学版,2015,36(4):514.