独活寄生汤治疗膝骨关节炎的作用机制
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摘要
背景:前期研究表明独活寄生汤可有效抑制膝骨关节炎炎症反应,但其具体机制尚不明确。目的:观察独活寄生汤对膝骨关节炎大鼠软骨、滑膜miR-146a/-155-NF-κB/p38MAPK信号通路相关因子的影响。方法:SD雄性大鼠36只,购于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,批准号为[2018]福中医伦理审字第(014)号。大鼠适应性喂养1周后,随机分为空白组(12只)和造模组(24只)。造模组采用改良Hulth法于大鼠右膝关节复制膝骨关节炎模型,造模后1周将造模大鼠随机分为模型组(12只)和实验组(12只)。空白组和模型组给予生理盐水10 mL/(kg·d)灌胃;实验组给予独活寄生汤9.3 g/(kg·d)灌胃。治疗3个月后,麻醉后切取大鼠右膝关节滑膜组织,ELISA法检测诱导型一氧化氮合酶、环氧合酶2、基质金属蛋白酶3和基质金属蛋白酶13含量变化;取大鼠软骨组织,免疫组织化学法检测NF-κB p65和p38 MAPK蛋白表达;Real-time PCR法检测miR-146a、miR-155、NF-κB p65和p38 MAPK基因表达。结果与结论:①Real-time PCR结果表明独活寄生汤能抑制miR-155、NF-κB p65和p38 MAPK基因表达,促进miR-146a基因表达(P <0.01);②免疫组织化学结果与Real-time PCR结果趋势一致,即独活寄生汤能抑制NF-κB p65和p38 MAPK蛋白表达(P <0.01);③ELISA结果显示独活寄生汤可有效抑制诱导型一氧化氮合酶、环氧合酶2、基质金属蛋白酶3和基质金属蛋白酶13表达;④结果提示,独活寄生汤可通过调控miR-146a/-155-NF-κB/p38 MAPK信号通路,抑制膝骨关节炎炎症反应,从而起到治疗膝骨关节炎作用。
BACKGROUND: Preliminary study has shown that Duhuo Jisheng Decoction can effectively inhibit inflammatory reaction in knee osteoarthritis. But its underling mechanism remains unclear. OBJECTIVE: To observe the effect of Duhuo Jisheng Decoction on the related regulatory factors in miR-146a/-155-NF-κB/p38 MAPK signaling pathway in cartilage and synovium of rats with knee osteoarthritis. METHODS: Thirty-six male rats were provided by Shanghai Slack Laboratory Animals Co., Ltd. The study was approved by the Experimental Animal Ethics Committee of Fujian University of Traditional Chinese Medicine, approval number: [2018](014). After 1 week of acclimation, the rats were randomly divided into blank group(n=12) and model group(n=24). Osteoarthritis model was induced in rat right knees by modified Hulth method. One week later, the rat models were randomly divided into model control group(n=12) and experiment group(n=12). The blank and model control groups received an equivalent amount of normal saline(10 mL/kg·d) intragastrically. The experiment group received a clinical oral dose of Duhuo Jisheng Decoction(9.3 g/kg·d) intragastrically. After three months of treatment, the animals were sacrificed and synovial tissue and cartilage tissue were obtained. The contents of inducible nitric oxide synthase, cyclooxygenase-2, matrix metalloproteinase 3 and matrix metalloproteinase 13 were measured by ELISA. The protein levels of nuclear factor-κB p65 and p38 mitogen-activated protein kinase were detected by immunohistochemistry. The mRNA expression levels of miR-146a, miR-155, nuclear factor-κB p65 and p38 mitogen-activated protein kinase were tested by real-time PCR. RESULTS AND CONCLUSION:(1) Real-time PCR results suggest that Duhuo Jisheng Decoction could inhibit the mRNA expression of miR-155, nuclear factor-κB p65 and p38 mitogen-activated protein kinase, and promote miR-146a mRNA expression(P < 0.01).(2) The results of immunohistochemistry were in agreement with the results of real-time PCR, indicating that Duhuo Jisheng Decoction could inhibit the protein expression of nuclear factor-κB p65 and p38 mitogen-activated protein kinase(P < 0.01).(3) ELISA results manifest that Duhuo Jisheng Decoction could effectively inhibit the expression of inducible nitric oxide synthase, cyclooxygenase-2, matrix metalloproteinase 3 and matrix metalloproteinase 13.(4) These results suggest that Duhuo Jisheng Decoction can inhibit the inflammatory reaction in knee osteoarthritis by regulating the miR-146a/-155-NF-κB/p38 MAPK signaling pathway, which may, in part, explain its clinical efficacy in the treatment of knee osteoarthritis.
BACKGROUND: Preliminary study has shown that Duhuo Jisheng Decoction can effectively inhibit inflammatory reaction in knee osteoarthritis. But its underling mechanism remains unclear. OBJECTIVE: To observe the effect of Duhuo Jisheng Decoction on the related regulatory factors in miR-146a/-155-NF-κB/p38 MAPK signaling pathway in cartilage and synovium of rats with knee osteoarthritis. METHODS: Thirty-six male rats were provided by Shanghai Slack Laboratory Animals Co., Ltd. The study was approved by the Experimental Animal Ethics Committee of Fujian University of Traditional Chinese Medicine, approval number: [2018](014). After 1 week of acclimation, the rats were randomly divided into blank group(n=12) and model group(n=24). Osteoarthritis model was induced in rat right knees by modified Hulth method. One week later, the rat models were randomly divided into model control group(n=12) and experiment group(n=12). The blank and model control groups received an equivalent amount of normal saline(10 mL/kg·d) intragastrically. The experiment group received a clinical oral dose of Duhuo Jisheng Decoction(9.3 g/kg·d) intragastrically. After three months of treatment, the animals were sacrificed and synovial tissue and cartilage tissue were obtained. The contents of inducible nitric oxide synthase, cyclooxygenase-2, matrix metalloproteinase 3 and matrix metalloproteinase 13 were measured by ELISA. The protein levels of nuclear factor-κB p65 and p38 mitogen-activated protein kinase were detected by immunohistochemistry. The mRNA expression levels of miR-146a, miR-155, nuclear factor-κB p65 and p38 mitogen-activated protein kinase were tested by real-time PCR. RESULTS AND CONCLUSION:(1) Real-time PCR results suggest that Duhuo Jisheng Decoction could inhibit the mRNA expression of miR-155, nuclear factor-κB p65 and p38 mitogen-activated protein kinase, and promote miR-146a mRNA expression(P < 0.01).(2) The results of immunohistochemistry were in agreement with the results of real-time PCR, indicating that Duhuo Jisheng Decoction could inhibit the protein expression of nuclear factor-κB p65 and p38 mitogen-activated protein kinase(P < 0.01).(3) ELISA results manifest that Duhuo Jisheng Decoction could effectively inhibit the expression of inducible nitric oxide synthase, cyclooxygenase-2, matrix metalloproteinase 3 and matrix metalloproteinase 13.(4) These results suggest that Duhuo Jisheng Decoction can inhibit the inflammatory reaction in knee osteoarthritis by regulating the miR-146a/-155-NF-κB/p38 MAPK signaling pathway, which may, in part, explain its clinical efficacy in the treatment of knee osteoarthritis.
引文
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[12]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.
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[14] Smeriqlio P,Dhulipala L,Lai JH,et al.Collagen VI Enhances Cartilage Tissue Generation by Stimulating Chondrocyte Proliferation. Tissue Enq Part A. 2015;21(3-4):840-849.
[15]高世超,殷海波,刘宏潇,等.MAPK信号通路在骨关节炎发病机制中的研究进展[J].中国骨伤,2014,27(5):441-444.
[16] Chen F, Castranova V, Shi X. New insights into the role of nuclear factor-kappaB in cell growth regulation. Am J Pathol.2001;159(2):387-397.
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[19] Karin M,Ben-Neriah Y.Phosphorlation meets ubiquition:The control of NF-κB activity. Annu Rev Immunol.2000;18(1):621-623.
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[21] Musikacharoen T, Matsuguchi T, Kikuchi T, et al.NF—kappa B and STAT5 play important roles in the regulation of mouse Toll-like receptor 2 gene expression. J Immunol.2001;166(7):4516.
[22]陈宁,叶静,汤特,等.共有序列寡核苷酸竞争结合NF-κF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.
[23] Rigoglou S,Papavassiliou AG.The NF-κA signaling pathway in osteoarthritis. Int J Biochem Cell Biol.2013;45(11):2580-2584.
[24] Brinckerhoff CE, Matrisian LM. Matrix meatlloproetinases:a tail of frog that became a prince. Nat Rev Mol Cell Biol. 2002;3(3):207-214.
[25] Iliopoulos D,Malizos KN,Oikonomou P,et al.Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks.PLoS One. 2008;3(11):e3740.
[26]冯知涛.RA患者外周血miR-146a、miR-16表达与疾病活动、中医证型相关性及青藤碱制剂对其干预的研究[D].广州:南方医科大学,2013.
[27] Boldin MP, Taganov KD, Rao D S, et al.miR-146αis a significant brake on autoimmunity, myeloproliferation, and cancer in mice. J Exp Med.2011;208(6):1189-1201.
[28] Bluml S, Bonelli M, Niederreiter B, et al.Essential role of micro RNA-155 in the pathogenesis of autoimmune arthritis in mice. Arthritis Rheum.2011;63(5):1281-1288.
[29]张烈,马歆,谢渭根.全身炎症反应患者miR-146a的表达与病情发展相关性研究[J].中华医院感染学杂志, 2013,23(22):5404-5406.
[30] Kurowska-Stolarska M, Alivernini S, Ballantine LE, et al.MicroRNA-155 as aproinflammatory regulator in clinical and experimental arthritis.Proc Natl Acad Sci U S A. 2011;108(27):11193-11198.
[31] O'Connell RM, Kahn D, Gibson WS, et al.MicroRNA-155promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity. 2010;33(4):607-619.
[2]Ceppi M,Pereira PM,Dunand-Sauthier I,et al.MicroRNA-155modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells.Proc Natl Acad Sci USA. 2009;106(8):2735-2740.
[3]Gu SX,Li X,Hamilton JL,et al.MicroRNA-146a reduces IL-1dependent inflammatory responses in the intervertebral disc.Gene.2015;555(2):80-87.
[4]Taganov KD,Boldin MP,Chang KJ,et al.NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses.Proc Natl Acad Sci U S A. 2006;103(33):12481-12486.48
[5]王武炼,叶锦霞,刘献祥,等.独活寄生汤加减内服外洗治疗膝骨性关节炎66例临床观察[J].福建中医药大学学报, 2011,21(2):44-46.
[6]胡慧丽.独活寄生汤加减方治疗膝骨性关节炎临床效果分析[J].临床与实践,2018,16(23):28-30.
[7]郑春松,叶蕻芝,李西海,等.分子对接法预测独活寄生汤对TNF-法和IL-1法作用的活性成分[J].福建中医药大学学报, 2012,22(4):28-30.
[8]Zheng CS, Xu XJ, Ye HZ, et al. Computational approaches for exploring the potential synergy and polypharmacology of Duhuo Jisheng Decoction in the therapy of osteoarthritis.Mol Med Rep. 2013;7(6):1812-1818.
[9]陈巧玉,周鑫,扶世杰.独活寄生汤干预食蟹猴自发性膝骨关节炎模型炎症的作用[J].中国组织工程研究, 2018,22(12):1866-1871.
[10]吴广文,王武炼,潘彩彬,等.独活寄生汤含药血清对大鼠退变软骨细胞线粒体凋亡通路的影响[J].风湿病与关节炎, 2014,3(10):5-9.
[11]刘献祥,李西海,周江涛.改良Hulth造模法复制膝骨性关节炎的实验研究[J].中国中西医结合杂志,2005,25(12):1104-1108.
[12]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.
[13] Soslow RA, Dannenberg AJ, Rush D, et al. Cox-2 is expressed in human pulmonary, colonic, and mammary tumors. Cancer.2000;89:2637-2645.
[14] Smeriqlio P,Dhulipala L,Lai JH,et al.Collagen VI Enhances Cartilage Tissue Generation by Stimulating Chondrocyte Proliferation. Tissue Enq Part A. 2015;21(3-4):840-849.
[15]高世超,殷海波,刘宏潇,等.MAPK信号通路在骨关节炎发病机制中的研究进展[J].中国骨伤,2014,27(5):441-444.
[16] Chen F, Castranova V, Shi X. New insights into the role of nuclear factor-kappaB in cell growth regulation. Am J Pathol.2001;159(2):387-397.
[17] Ghosh S, May M J, Kopp E B. NF-κS and Rel ptoteins:Evolutionarily conserved mediators of immune responses.Annu Rev Immunol. 1998;16(1):225-260.
[18] Baldwin A.The NF-κa and Ian ptoteins:New discoveries and insights. Annu Rev Immunol. 1996;(14):649-681.
[19] Karin M,Ben-Neriah Y.Phosphorlation meets ubiquition:The control of NF-κB activity. Annu Rev Immunol.2000;18(1):621-623.
[20] May MJ, Ghosh S. Signal transduction through NF-κB.Immunol Today. 1998;19(2):80-88.
[21] Musikacharoen T, Matsuguchi T, Kikuchi T, et al.NF—kappa B and STAT5 play important roles in the regulation of mouse Toll-like receptor 2 gene expression. J Immunol.2001;166(7):4516.
[22]陈宁,叶静,汤特,等.共有序列寡核苷酸竞争结合NF-κF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.
[23] Rigoglou S,Papavassiliou AG.The NF-κA signaling pathway in osteoarthritis. Int J Biochem Cell Biol.2013;45(11):2580-2584.
[24] Brinckerhoff CE, Matrisian LM. Matrix meatlloproetinases:a tail of frog that became a prince. Nat Rev Mol Cell Biol. 2002;3(3):207-214.
[25] Iliopoulos D,Malizos KN,Oikonomou P,et al.Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks.PLoS One. 2008;3(11):e3740.
[26]冯知涛.RA患者外周血miR-146a、miR-16表达与疾病活动、中医证型相关性及青藤碱制剂对其干预的研究[D].广州:南方医科大学,2013.
[27] Boldin MP, Taganov KD, Rao D S, et al.miR-146αis a significant brake on autoimmunity, myeloproliferation, and cancer in mice. J Exp Med.2011;208(6):1189-1201.
[28] Bluml S, Bonelli M, Niederreiter B, et al.Essential role of micro RNA-155 in the pathogenesis of autoimmune arthritis in mice. Arthritis Rheum.2011;63(5):1281-1288.
[29]张烈,马歆,谢渭根.全身炎症反应患者miR-146a的表达与病情发展相关性研究[J].中华医院感染学杂志, 2013,23(22):5404-5406.
[30] Kurowska-Stolarska M, Alivernini S, Ballantine LE, et al.MicroRNA-155 as aproinflammatory regulator in clinical and experimental arthritis.Proc Natl Acad Sci U S A. 2011;108(27):11193-11198.
[31] O'Connell RM, Kahn D, Gibson WS, et al.MicroRNA-155promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity. 2010;33(4):607-619.