乌头汤对膝骨关节炎模型大鼠滑膜组织TLR4/NF-κB信号通路的影响
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摘要
背景:前期研究表明乌头汤可有效抑制膝骨关节炎炎症反应,但具体机制尚不明确。目的:观察乌头汤对膝骨关节炎大鼠滑膜组织TLR4/NF-κB通路相关调节因子的影响,探讨乌头汤治疗膝骨关节炎的作用机制。方法:SPF级雄性SD大鼠36只,购自上海斯莱克实验动物有限责任公司。实验方案经福建中医药大学动物实验伦理委员会批准。大鼠适应性喂养1周后,采用随机数字表法分为空白组、模型组和治疗组。模型组和治疗组均采用改良Hulth法复制双膝膝骨关节炎模型,术后1周空白组和模型组给予生理盐水,按照10 mL/(kg·d)的量进行灌胃;治疗组给予乌头汤,按照4.2 g/(kg·d)的药量进行灌胃。治疗8周后,取大鼠膝关节滑膜组织。ELISA法检测诱导型一氧化氮合酶、肿瘤坏死因子α和白细胞介素6含量变化;Real-time PCR法检测TLR4、MyD88、TRAF6和NF-κBp65mRNA表达;Westernblot法检测TLR4、MyD88、TRAF6和NF-κB p65蛋白表达。结果与结论:①ELISA结果显示,与模型组相比,乌头汤可有效抑制诱导型一氧化氮合酶、肿瘤坏死因子α和白细胞介素6表达(P<0.01);②Real-timePCR结果表明,乌头汤能抑制TLR4、MyD88、TRAF6和NF-κB p65 mRNA表达(P <0.01);③Western blot结果与Real-time PCR结果趋势一致,即乌头汤能抑制TLR4、MyD88、TRAF6和NF-κB p65蛋白表达(P <0.01);④结果提示,乌头汤可通过调控TLR4/NF-κB信号通路,抑制膝骨关节炎滑膜炎症反应,从而起到治疗膝骨关节炎作用。
BACKGROUND: Preliminary study has shown that Wutou Decoction can effectively inhibit inflammatory reaction in knee osteoarthritis. But its underling mechanism remains unclear.OBJECTIVE: To observe the effect of Wutou Decoction on the related regulatory factors in TLR4/NF-κB signaling pathway in synovium of rats with knee osteoarthritis, so as to explore its anti-inflammatory mechanism on synovitis of knee osteoarthritis.METHODS: Thirty-six male Sprague-Dawley rats, SPF grade, were provided by Shanghai Slack Laboratory Animals Co., Ltd., and the study was approved by the Experimental Animal Ethics Committee of Fujian University of Traditional Chinese Medicine. After 1 week of acclimation,the rats were randomly divided into blank, model and treatment groups. The rats in the model and treatment groups were used for establishing osteoarthritis model in both knees by modified Hulth method. One week later, the blank and model groups received 10 mL/(kg·d) saline via gavage. The experiment group received 4.2 g/(kg·d) Wutou Decoction. After 8 weeks of treatment, the rat synovial tissue was obtained. The contents of inducible nitric oxide synthase, tumor necrosis factor-α and interleukin-6 were measured by ELISA. The mRNA expression levels of TLR4, MyD88, TRAF6 and NF-κB p65 were measured by real-time PCR. The protein levels of TLR4, MyD88, TRAF6 and NF-κB p65 were measured by western blot assay.RESULTS AND CONCLUSION:(1) ELISA results showed that Wutou Decoction could effectively inhibit the expression of inducible nitric oxide synthase, tumor necrosis factor-α and interleukin-6 compared with the model group(P < 0.01).(2) Real-time PCR results suggested that Wutou Decoction could inhibit the mRNA expression of TLR4, MyD88, TRAF6 and NF-κB p65 compared with the model group(P < 0.01).(3)The results of western blot assay were in agreement with the results of real-time PCR. That was, Wutou Decoction could inhibit the protein expression of TLR4, MyD88, TRAF6 and NF-κB p65(P < 0.01).(4) These results suggest that Wutou Decoction can inhibit the inflammatory reaction in synovitis of knee osteoarthritis by regulating TLR4/NF-κB signaling pathway, which may, in part, explain its clinical efficacy in the treatment of synovitis of knee osteoarthritis.
BACKGROUND: Preliminary study has shown that Wutou Decoction can effectively inhibit inflammatory reaction in knee osteoarthritis. But its underling mechanism remains unclear.OBJECTIVE: To observe the effect of Wutou Decoction on the related regulatory factors in TLR4/NF-κB signaling pathway in synovium of rats with knee osteoarthritis, so as to explore its anti-inflammatory mechanism on synovitis of knee osteoarthritis.METHODS: Thirty-six male Sprague-Dawley rats, SPF grade, were provided by Shanghai Slack Laboratory Animals Co., Ltd., and the study was approved by the Experimental Animal Ethics Committee of Fujian University of Traditional Chinese Medicine. After 1 week of acclimation,the rats were randomly divided into blank, model and treatment groups. The rats in the model and treatment groups were used for establishing osteoarthritis model in both knees by modified Hulth method. One week later, the blank and model groups received 10 mL/(kg·d) saline via gavage. The experiment group received 4.2 g/(kg·d) Wutou Decoction. After 8 weeks of treatment, the rat synovial tissue was obtained. The contents of inducible nitric oxide synthase, tumor necrosis factor-α and interleukin-6 were measured by ELISA. The mRNA expression levels of TLR4, MyD88, TRAF6 and NF-κB p65 were measured by real-time PCR. The protein levels of TLR4, MyD88, TRAF6 and NF-κB p65 were measured by western blot assay.RESULTS AND CONCLUSION:(1) ELISA results showed that Wutou Decoction could effectively inhibit the expression of inducible nitric oxide synthase, tumor necrosis factor-α and interleukin-6 compared with the model group(P < 0.01).(2) Real-time PCR results suggested that Wutou Decoction could inhibit the mRNA expression of TLR4, MyD88, TRAF6 and NF-κB p65 compared with the model group(P < 0.01).(3)The results of western blot assay were in agreement with the results of real-time PCR. That was, Wutou Decoction could inhibit the protein expression of TLR4, MyD88, TRAF6 and NF-κB p65(P < 0.01).(4) These results suggest that Wutou Decoction can inhibit the inflammatory reaction in synovitis of knee osteoarthritis by regulating TLR4/NF-κB signaling pathway, which may, in part, explain its clinical efficacy in the treatment of synovitis of knee osteoarthritis.
引文
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[6]韦嵩,申茜茸,李晓昊,等.经筋微创松解术治疗膝骨关节炎对TLR4/MyD88/NF-κB信号转导通路的影响[J].中华中医药杂志,2018,33(10):4637-4641.
[7]曹云祥.基于miRNA-21/TLR4/MAPK/NF-κB信号通路探讨新风胶囊改善RA心功能的机制研究[D].南京:南京中医药大学,2018.
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[9]焦永亮,刘慧敏,顾海伦,等.白藜芦醇对肥胖者骨性关节炎软骨细胞的作用及对TLR4、NF-κBmRNA表达的影响[J].东南大学学报,2015,34(3):342-346.
[10]付长龙,梅阳阳,叶锦霞,等.乌头汤治疗寒湿痹阻型膝骨关节炎:与双氯芬酸钠的比较[J].风湿病与关节炎,2017,6(4):12-16.
[11]林洁,陈俊,付长龙,等.乌头汤治疗骨关节炎的系统评价[J].风湿病与关节炎,2017,6(5):35-38,50.
[12]郑春松,林洁,付长龙,等.基于计算机模拟乌头汤治疗疼痛的药效物质基础与分子作用机制[J].福建中医药大学学报,2016,1(26):33-37.
[13]陈俊,叶锦霞,林洁,等.乌头汤对膝骨关节炎大鼠细胞因子IL-1β、IL-6、TNF-α、MMP-3的影响[J].福建中医药,2017,18(5):27-29.
[14]刘献祥,李西海,周江涛.改良Hulth造模法复制膝骨关节炎的实验研究[J].中国中西医结合杂志,2005,25(12):1104-1108.
[15]黄继汗,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2004,9(9):1069-1072.
[16]Wakamatsu K,Nanki T,Miyasaka N,et al.Effect of a small molecule inhibitor of nuclear factor-kappaB nuclear translocation in a murine model of arthritis and cultured human synovial cells.Arthritis Res Ther.2005;7:1348-1359.
[17]Kosinska MK,Liebisch G,Lochnit G,et al.A Lipidomic Study of Phospholipid Classes and Species in Human Synovial Fluid.Arthritis Rheum.2013;65(9):2323-2333.
[18]Ritter SY,Subbaiah R,Bebek G,et al.Proteomic analysis of synovial fluid from the osteoarthritic knee:comparison withrtranscriptome analyses of joint tissues.Arthritis Rheum.2013;65(4):981-992.
[19]张鹏,石关桐,郑昱新.膝骨关节炎滑膜细胞体外培养及生物学特性观察[J].中国骨伤,2006,19(11):656-658.
[20]Deng LL,Yuan D,Zhou ZY,et al.Saponins from Panax japonicus attenuate age-related neuroinflammation via regulation of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways.Neural Regen Res.2017;12(11):1877-1884.
[21]陈宁,叶静,汤特,等.共有序列寡核苷酸竞争结合NF-κB以减少TNF产生的实验研究[J].中国病理生理杂志,2003,19(5):619-621.
[22]Rigoglou S,Papavassiliou AG.The NF-κB signaling pathway in osteoarthritis.Int J Biochem Cell Biol.2013;45(11):2580-2584.
[23]Yang H,Cheng X,Yang YL,et al.Ramulus Cinnamomi extract attenuates neuroinflammatory responses via downregulating TLR4/MyD88 signaling pathway in BV2 cells.Neural Regen Res.2017;12(11):1860-1864.
[24]Kawai T,Akira S.The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors.Nature Immunology.2010;11(5):373-384.
[25]Bobacz K,Sunk IG,Hofstaetter JG,et al.Toll-like receptors and chondrocytes:the lipopolysaccharide-induced decrease in cartilage matrixsynthesis is dependent on the presence of toll-like receptor 4 and antagonized by bone morphogenetic protein 7.Arthritis Rheum.2007;56(6):1880-1893.
[26]Bahtiar A,Nurazizah M,Roselina T,et al.Ethanolic extracts of babandotanleaves(Ageratum conyzoides L.)prevents inflammation and proteoglycan degradation by inhibiting TNF-αand MMP-9 on osteoarthritis rats induced by monosodium iodoacetate.Asian Pac J Trop Med.2017;10(3):270-277.
[27]Campo GM,Avenoso A,Campo S,et al.Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes.Biochimie.2009;92(2):204-215.
[28]钦丹萍,周毅骏,孙佩娜,等.雷公藤多苷对溃疡性结肠炎大鼠TLR4/MyD88非依赖信号通路的作用研究[J].中国中药杂志,2016,41(6):1093-1099.
[29]Bortolotto V,Grilli M.Not only a bad guy:potential proneurogenic role of the RAGE/NF-κB axis in Alzheimer's disease brain.Neural Regen Res.2016;11(12):1924-1925.
[30]Li T,Hu J,Thomas JA,et al.Differential induction of apoptosis by LPS and taxol in monocytic cells.Molecular Immunology.2005;42(9):1049-1055.
[31]Kopp EB,Medzhitov R.The Toll-receptor family and control of innate immunity.Current Opinion in Immunology.1999;11(1):13-18.
[32]Modlin RL,Brightbill HD,Godowski PJ.The toll of innate immunity on microbial pathogens.N Engl J Med.1999;340(23):1834-1835.
[33]Dunne A,Carpenter S,Brikos C,et al.IRAK1 and IRAK4 promote phosphorylation,ubiquitination,and degradation of MyD88 adaptor-like(Mal).J Biol Chem.2010;285(24):18276-18282.
[2]Radstake TR,Roelofs MF,Jenniskens YM,et al.Expression of toll-like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin-12 and interleukin-18 via interferon-gamma.Arthritis Rheum.2004;50(12):3856-3865.
[3]Sohn DH,Sokolove J,Sharpe O,et al.Plasma proteins present in osteoarthritic synovial fluid can stimulate cytokine production via Toll-like receptor 4.Arthritis Res Ther.2012;14(1):R7.
[4]Yamamoto Y,Gaynor RB.Role of the NF-kappa B pathway in the pathogenesis of human disease states.CurrMol Med.2001;1(3):287-296.
[5]王永福,林剑,詹宏刚.CD14/TLR-4-NF-κB信号通路参与骨关节炎发病机制的研究[J].实用骨科杂志,2013,19(6):528-530.
[6]韦嵩,申茜茸,李晓昊,等.经筋微创松解术治疗膝骨关节炎对TLR4/MyD88/NF-κB信号转导通路的影响[J].中华中医药杂志,2018,33(10):4637-4641.
[7]曹云祥.基于miRNA-21/TLR4/MAPK/NF-κB信号通路探讨新风胶囊改善RA心功能的机制研究[D].南京:南京中医药大学,2018.
[8]董小君.风湿宁对风寒湿痹证CIA模型TLR4/NF-κB信号通路的作用机制研宄[D].成都:成都中医药大学,2018.
[9]焦永亮,刘慧敏,顾海伦,等.白藜芦醇对肥胖者骨性关节炎软骨细胞的作用及对TLR4、NF-κBmRNA表达的影响[J].东南大学学报,2015,34(3):342-346.
[10]付长龙,梅阳阳,叶锦霞,等.乌头汤治疗寒湿痹阻型膝骨关节炎:与双氯芬酸钠的比较[J].风湿病与关节炎,2017,6(4):12-16.
[11]林洁,陈俊,付长龙,等.乌头汤治疗骨关节炎的系统评价[J].风湿病与关节炎,2017,6(5):35-38,50.
[12]郑春松,林洁,付长龙,等.基于计算机模拟乌头汤治疗疼痛的药效物质基础与分子作用机制[J].福建中医药大学学报,2016,1(26):33-37.
[13]陈俊,叶锦霞,林洁,等.乌头汤对膝骨关节炎大鼠细胞因子IL-1β、IL-6、TNF-α、MMP-3的影响[J].福建中医药,2017,18(5):27-29.
[14]刘献祥,李西海,周江涛.改良Hulth造模法复制膝骨关节炎的实验研究[J].中国中西医结合杂志,2005,25(12):1104-1108.
[15]黄继汗,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2004,9(9):1069-1072.
[16]Wakamatsu K,Nanki T,Miyasaka N,et al.Effect of a small molecule inhibitor of nuclear factor-kappaB nuclear translocation in a murine model of arthritis and cultured human synovial cells.Arthritis Res Ther.2005;7:1348-1359.
[17]Kosinska MK,Liebisch G,Lochnit G,et al.A Lipidomic Study of Phospholipid Classes and Species in Human Synovial Fluid.Arthritis Rheum.2013;65(9):2323-2333.
[18]Ritter SY,Subbaiah R,Bebek G,et al.Proteomic analysis of synovial fluid from the osteoarthritic knee:comparison withrtranscriptome analyses of joint tissues.Arthritis Rheum.2013;65(4):981-992.
[19]张鹏,石关桐,郑昱新.膝骨关节炎滑膜细胞体外培养及生物学特性观察[J].中国骨伤,2006,19(11):656-658.
[20]Deng LL,Yuan D,Zhou ZY,et al.Saponins from Panax japonicus attenuate age-related neuroinflammation via regulation of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways.Neural Regen Res.2017;12(11):1877-1884.
[21]陈宁,叶静,汤特,等.共有序列寡核苷酸竞争结合NF-κB以减少TNF产生的实验研究[J].中国病理生理杂志,2003,19(5):619-621.
[22]Rigoglou S,Papavassiliou AG.The NF-κB signaling pathway in osteoarthritis.Int J Biochem Cell Biol.2013;45(11):2580-2584.
[23]Yang H,Cheng X,Yang YL,et al.Ramulus Cinnamomi extract attenuates neuroinflammatory responses via downregulating TLR4/MyD88 signaling pathway in BV2 cells.Neural Regen Res.2017;12(11):1860-1864.
[24]Kawai T,Akira S.The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors.Nature Immunology.2010;11(5):373-384.
[25]Bobacz K,Sunk IG,Hofstaetter JG,et al.Toll-like receptors and chondrocytes:the lipopolysaccharide-induced decrease in cartilage matrixsynthesis is dependent on the presence of toll-like receptor 4 and antagonized by bone morphogenetic protein 7.Arthritis Rheum.2007;56(6):1880-1893.
[26]Bahtiar A,Nurazizah M,Roselina T,et al.Ethanolic extracts of babandotanleaves(Ageratum conyzoides L.)prevents inflammation and proteoglycan degradation by inhibiting TNF-αand MMP-9 on osteoarthritis rats induced by monosodium iodoacetate.Asian Pac J Trop Med.2017;10(3):270-277.
[27]Campo GM,Avenoso A,Campo S,et al.Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes.Biochimie.2009;92(2):204-215.
[28]钦丹萍,周毅骏,孙佩娜,等.雷公藤多苷对溃疡性结肠炎大鼠TLR4/MyD88非依赖信号通路的作用研究[J].中国中药杂志,2016,41(6):1093-1099.
[29]Bortolotto V,Grilli M.Not only a bad guy:potential proneurogenic role of the RAGE/NF-κB axis in Alzheimer's disease brain.Neural Regen Res.2016;11(12):1924-1925.
[30]Li T,Hu J,Thomas JA,et al.Differential induction of apoptosis by LPS and taxol in monocytic cells.Molecular Immunology.2005;42(9):1049-1055.
[31]Kopp EB,Medzhitov R.The Toll-receptor family and control of innate immunity.Current Opinion in Immunology.1999;11(1):13-18.
[32]Modlin RL,Brightbill HD,Godowski PJ.The toll of innate immunity on microbial pathogens.N Engl J Med.1999;340(23):1834-1835.
[33]Dunne A,Carpenter S,Brikos C,et al.IRAK1 and IRAK4 promote phosphorylation,ubiquitination,and degradation of MyD88 adaptor-like(Mal).J Biol Chem.2010;285(24):18276-18282.