小柴胡汤对CFA大鼠滑膜组织NF-κB信号通路作用的探讨
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摘要
目的:观察小柴胡汤治疗后弗氏完全佐剂(CFA)大鼠滑膜组织核转录因子(NF)-κB信号通路中肿瘤坏死因子受体相关死亡结构域(TARDD),NF-κB抑制蛋白α(IκBα),IκB激酶-α(IKKα),NF-κB p65蛋白的表达。方法:将SPF级健康成年雌性Wistar大鼠应用弗氏完全佐剂和牛Ⅱ型胶原制备CFA动物模型。根据随机数字表,将大鼠随机分为正常组、模型组、小柴胡汤低、中、高剂量组、雷公藤多苷组。各组于造模后7 d开始灌胃给药,正常组及模型组均予注射用水,小柴胡汤低、中、高剂量组(5. 94,11. 88,23. 76 g·kg~(-1)),雷公藤多苷片(0. 006 3 g·kg~(-1)),分别2 mL/次,每天3次灌胃,连续给药28 d后观察实验大鼠踝关节组织病理学及蛋白免疫印迹法(Western blot)检测NF-κB信号通路中TARDD,IKKα,IκBα,NF-κB p65蛋白表达。结果:与正常组比较,模型组大鼠右后踝关节组织病理评分显著增加(P <0. 01),NF-κB信号通路中TARDD,IKKα,IκBα,NF-κB p65蛋白表达显著增高(P <0. 01)。与模型组比较,随着小柴胡汤剂量的增加,实验大鼠右后踝关节组织病理评分显著减少(P <0. 01),在大鼠踝关节组织标本NF-κB信号通路中TARDD,IKKα,IκBα,NF-κB p65关键蛋白的表达上,小柴胡汤低剂量组蛋白表达明显减低(P <0. 05),而小柴胡汤中剂量组、小柴胡汤高剂量组、雷公藤多苷组蛋白表达显著减低(P <0. 01)。结论:研究显示小柴胡汤随着剂量的增加,可以有效地改善滑膜炎症,抑制NF-κB炎症信号通路中关键蛋白的表达,从而阻止炎症而抑制骨侵蚀。
Objective: To observe the expression of tumor necrosis factor receptor-associated death domain(TARDD),nuclear transcription factor-κB inhibiting protein α(IκBα) IκB kinase-α(IKKα) and nuclear transcription factor(NF)-κB p65 protein in the NF-κB signaling pathway of synovial tissues of complete Freund's adjuvant(CFA) rats after treatment with Xiao Chaihutang(XCHT). Method: In animal experiments,SPF health adult female Wistar rats were used to prepare the CFA animal model of rats with rheumatoid arthritis with Freund's complete adjuvant and cattle Ⅱ collagen type. According to the random number table,the rats were randomly divided into the normal group,the model group,the low-dose XCHT group,the medium-dose XCHT group,the high-dose XCHT group,and the Tripterygium glucosides group. The drugs were given at 7 d after the model was built. Both normal group and model group were given water for injection,and low-dose XCHT group(5. 94 g·kg~(-1)),medium-dose XCHT group(11. 88 g·kg~(-1)),high-dose XCHT group(23. 76 g ·kg~(-1)),Tripterygium glucosides group(0. 006 3 g·kg~(-1)) were given corresponding drugs by gavage for three times a day,2 m L/time. The histopathology of rat ankle joint was observed,and the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in the NF-κB signaling pathway in synovial tissue of CFA rats were detected by Western blot.Result: With the increase of the dosage of XCHT,the histopathological score of the right posterior ankle joint of the experimental rats was increased. And in the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in NF-κB signaling pathway in Synovial Tissue of CFA rats,compared with the model group,the statistical results of the lowdose XCHT group showed decreased protein expressions(P < 0. 05) and significant differences. However,the statistical results in the medium-dose XCHT group,the high-dose XCHT group and the tripterygium glucosides group showed decreased protein expression(P < 0. 01) and significant differences. Compared with the normal group,the histopathological score of the right posterior ankle of the model group was significantly increased(P <0. 01),and the protein expressions of TARDD,IKKα,IκB α,NF-κB p65 in the NF-κB signaling pathway were significantly increased(P < 0. 01). Compared with model group,with the increase of dose of Xiao Chaihutang,histopathologic score of posterior ankle of experimental rats significantly reduced(P < 0. 01). In rats ankle tissue samples,TARDD,IKKα,IκBα,NF-κB p65 key protein expressions in the NF-κB signaling pathway and protein expressions in low-dose XCHT group were obviously lower(P < 0. 05),and protein expressions in the medium-dose XCHT group,the high-dose XCHT group and the Tripterygium glucosides group were significantly lower(P <0. 01). Conclusion: This study shows that as the dose of Xiao Chaihutang increases,it could effectively improve synovitis,and suppress the expressions of key proteins in the inflammatory signaling pathway of NF-κB,thereby preventing inflammation and suppressing bone erosion.
Objective: To observe the expression of tumor necrosis factor receptor-associated death domain(TARDD),nuclear transcription factor-κB inhibiting protein α(IκBα) IκB kinase-α(IKKα) and nuclear transcription factor(NF)-κB p65 protein in the NF-κB signaling pathway of synovial tissues of complete Freund's adjuvant(CFA) rats after treatment with Xiao Chaihutang(XCHT). Method: In animal experiments,SPF health adult female Wistar rats were used to prepare the CFA animal model of rats with rheumatoid arthritis with Freund's complete adjuvant and cattle Ⅱ collagen type. According to the random number table,the rats were randomly divided into the normal group,the model group,the low-dose XCHT group,the medium-dose XCHT group,the high-dose XCHT group,and the Tripterygium glucosides group. The drugs were given at 7 d after the model was built. Both normal group and model group were given water for injection,and low-dose XCHT group(5. 94 g·kg~(-1)),medium-dose XCHT group(11. 88 g·kg~(-1)),high-dose XCHT group(23. 76 g ·kg~(-1)),Tripterygium glucosides group(0. 006 3 g·kg~(-1)) were given corresponding drugs by gavage for three times a day,2 m L/time. The histopathology of rat ankle joint was observed,and the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in the NF-κB signaling pathway in synovial tissue of CFA rats were detected by Western blot.Result: With the increase of the dosage of XCHT,the histopathological score of the right posterior ankle joint of the experimental rats was increased. And in the protein expressions of TARDD,IKKα,IκBα,NF-κB p65 in NF-κB signaling pathway in Synovial Tissue of CFA rats,compared with the model group,the statistical results of the lowdose XCHT group showed decreased protein expressions(P < 0. 05) and significant differences. However,the statistical results in the medium-dose XCHT group,the high-dose XCHT group and the tripterygium glucosides group showed decreased protein expression(P < 0. 01) and significant differences. Compared with the normal group,the histopathological score of the right posterior ankle of the model group was significantly increased(P <0. 01),and the protein expressions of TARDD,IKKα,IκB α,NF-κB p65 in the NF-κB signaling pathway were significantly increased(P < 0. 01). Compared with model group,with the increase of dose of Xiao Chaihutang,histopathologic score of posterior ankle of experimental rats significantly reduced(P < 0. 01). In rats ankle tissue samples,TARDD,IKKα,IκBα,NF-κB p65 key protein expressions in the NF-κB signaling pathway and protein expressions in low-dose XCHT group were obviously lower(P < 0. 05),and protein expressions in the medium-dose XCHT group,the high-dose XCHT group and the Tripterygium glucosides group were significantly lower(P <0. 01). Conclusion: This study shows that as the dose of Xiao Chaihutang increases,it could effectively improve synovitis,and suppress the expressions of key proteins in the inflammatory signaling pathway of NF-κB,thereby preventing inflammation and suppressing bone erosion.
引文
[1]Smolen J S,Aletaha D,Barton A,et al.Rheumatoid arthritis[J].Nat Rev Dis Primers,2018,4:18001.
[2]高士宗.素问直解[M].成建军,刘娟,李玉清,校注.北京:中国医药科技出版社,2014:157-158.
[3]王丽敏,张丽艳,谷松.从“少阳主骨”解析痹证发病机制[J].辽宁中医药大学学报,2017,19(12):130-132.
[4]熊江华,李艳.中药复方对类风湿性关节炎干预机制的研究进展[J].中国实验方剂学杂志,2017,23(9):230-233.
[5]张莹,周小莉,戴敏,等.小柴胡汤对胶原诱导性关节炎大鼠血清中IL-17、IL-23及IL-27的影响[J].现代中西医结合杂志,2016,25(13):1391-1394.
[6]张莹,周小莉,邵勤,等.小柴胡汤对胶原诱导性关节炎大鼠的抗炎作用及机制探讨[J].免疫学杂志,2015,31(9):781-785.
[7]陈奇.中药药理实验方法[M].北京:人民卫生出版社,1994:205-208.
[8]朱玉芳,裴银辉,周芳媛,等.Wistar大鼠福氏佐剂性类风湿关节炎动物模型研究[J].华北理工大学学报:医学版,2016,18(5):337-340.
[9]Scott D L.Prognostic factors in early rheumatoid arthritis[J].Rheumatology(Oxford),2000,39(1):24-29.
[10]Makarov S S.NF-kappaB in rheumatoid arthritis:a pivotal regulator of inflammation,hyperplasia,and tissue destruction[J].Arthritis Res,2001,3(4):200-206.
[11]Vincenti M P,Coon C I,Brinckerhoff C E.Nuclear factor kappa B/p50 activates an element in the distal matrix metalloproteinase 1 promoter in interleukin-1betastimulated synovial fibroblasts[J].Arthritis Rheum,1998,41(11):1987-1994.
[12]Bondeson J,Brennan F,Foxwell B,et al.Effective adenoviral transfer of Ikappa B alpha into human fibroblasts and chondrosarcoma cells reveals that the induction of matrix metalloproteinases and proinflammatory cytokines is nuclear factor-kappaBdependent[J].J Rheumatol,2000,27(9):2078-2089.
[13]Bond M,Fabunmi R P,Baker A H,et al.Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines:an absolute requirement for transcription factor NF-kappa B[J].FEBS Lett,1998,435(1):29-34.
[14]Yoshida S,Ono M,Shono T,et al.Involvement of interleukin-8,vascular endothelial growth factor,and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis[J].Mol Cell Biol,1997,17(7):4015-4023.
[15]Elinav E,Nowarski R,Thaiss C A,et al.Inflammation-induced cancer:crosstalk between tumours,immune cells and microorganisms[J].Nat Rev Cancer,2013,13(11):759-771.
[16]Adli M,Merkhofer E,Cogswell P,et al.IKKαand IKKβeach function to regulate NF-κB activation in the TNF-induced/canonical pathway[J].PLo S One,2010,5(2):e9428.
[17]Hsu H,XIONG J,Goeddel D V.The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation[J].Cell,1995,81(4):495-504.
[18]Smolen J S,LandewéR,Bijlsma J,et al.EULARrecommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs:2016 update[J].Ann Rheum Dis,2017,76(6):960-977.
[19]Bellucci E,Terenzi R,La Paglia G M,et al.One year in review 2016:pathogenesis of rheumatoid arthritis[J].Clin Exp Rheumatol,2016,34(5):793-801.
[20]王丽敏,张丽艳,谷松.解析小柴胡汤“和法”调节自身免疫性疾病机制[J].中华中医药学刊,2018,36(1):218-221.
[21]唐小云,刘开蕾,杨美荣.小柴胡汤对免疫抑制小鼠淋巴细胞共刺激分子的免疫调节作用[J].热带医学杂志,2010,10(9):1049-1050.
[22]张军能,张轶.小柴胡颗粒抗肺癌的机制研究[J].湖南中医药大学学报,2010,30(6):46-48.
[23]柒小梅,李英,秦雪梅.小柴胡汤临床应用与药理作用研究进展[J].山西中医学院学报,2007,8(3):59-60.
[24]李延萍,谢微杳.小柴胡加味汤治疗类风湿关节炎活动100例疗效观察[J].中国中医急症,2014,23(5):920-921.
[25]苏青,吴婷婷,黄雅兰,等.黄芩提取物制备过程中化学成分及药效的变化规律分析[J].中国实验方剂学杂志,2018,24(14):1-6.
[26]LV Q W,ZHANG W,SHI Q,et al.Comparison of Tripterygium wilfordii Hook F with methotrexate in the treatment of active rheumatoid arthritis(TRIFRA):a randomised,controlled clinical trial[J].Ann Rheum Dis,2015,74(6):1078-1086.
[2]高士宗.素问直解[M].成建军,刘娟,李玉清,校注.北京:中国医药科技出版社,2014:157-158.
[3]王丽敏,张丽艳,谷松.从“少阳主骨”解析痹证发病机制[J].辽宁中医药大学学报,2017,19(12):130-132.
[4]熊江华,李艳.中药复方对类风湿性关节炎干预机制的研究进展[J].中国实验方剂学杂志,2017,23(9):230-233.
[5]张莹,周小莉,戴敏,等.小柴胡汤对胶原诱导性关节炎大鼠血清中IL-17、IL-23及IL-27的影响[J].现代中西医结合杂志,2016,25(13):1391-1394.
[6]张莹,周小莉,邵勤,等.小柴胡汤对胶原诱导性关节炎大鼠的抗炎作用及机制探讨[J].免疫学杂志,2015,31(9):781-785.
[7]陈奇.中药药理实验方法[M].北京:人民卫生出版社,1994:205-208.
[8]朱玉芳,裴银辉,周芳媛,等.Wistar大鼠福氏佐剂性类风湿关节炎动物模型研究[J].华北理工大学学报:医学版,2016,18(5):337-340.
[9]Scott D L.Prognostic factors in early rheumatoid arthritis[J].Rheumatology(Oxford),2000,39(1):24-29.
[10]Makarov S S.NF-kappaB in rheumatoid arthritis:a pivotal regulator of inflammation,hyperplasia,and tissue destruction[J].Arthritis Res,2001,3(4):200-206.
[11]Vincenti M P,Coon C I,Brinckerhoff C E.Nuclear factor kappa B/p50 activates an element in the distal matrix metalloproteinase 1 promoter in interleukin-1betastimulated synovial fibroblasts[J].Arthritis Rheum,1998,41(11):1987-1994.
[12]Bondeson J,Brennan F,Foxwell B,et al.Effective adenoviral transfer of Ikappa B alpha into human fibroblasts and chondrosarcoma cells reveals that the induction of matrix metalloproteinases and proinflammatory cytokines is nuclear factor-kappaBdependent[J].J Rheumatol,2000,27(9):2078-2089.
[13]Bond M,Fabunmi R P,Baker A H,et al.Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines:an absolute requirement for transcription factor NF-kappa B[J].FEBS Lett,1998,435(1):29-34.
[14]Yoshida S,Ono M,Shono T,et al.Involvement of interleukin-8,vascular endothelial growth factor,and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis[J].Mol Cell Biol,1997,17(7):4015-4023.
[15]Elinav E,Nowarski R,Thaiss C A,et al.Inflammation-induced cancer:crosstalk between tumours,immune cells and microorganisms[J].Nat Rev Cancer,2013,13(11):759-771.
[16]Adli M,Merkhofer E,Cogswell P,et al.IKKαand IKKβeach function to regulate NF-κB activation in the TNF-induced/canonical pathway[J].PLo S One,2010,5(2):e9428.
[17]Hsu H,XIONG J,Goeddel D V.The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation[J].Cell,1995,81(4):495-504.
[18]Smolen J S,LandewéR,Bijlsma J,et al.EULARrecommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs:2016 update[J].Ann Rheum Dis,2017,76(6):960-977.
[19]Bellucci E,Terenzi R,La Paglia G M,et al.One year in review 2016:pathogenesis of rheumatoid arthritis[J].Clin Exp Rheumatol,2016,34(5):793-801.
[20]王丽敏,张丽艳,谷松.解析小柴胡汤“和法”调节自身免疫性疾病机制[J].中华中医药学刊,2018,36(1):218-221.
[21]唐小云,刘开蕾,杨美荣.小柴胡汤对免疫抑制小鼠淋巴细胞共刺激分子的免疫调节作用[J].热带医学杂志,2010,10(9):1049-1050.
[22]张军能,张轶.小柴胡颗粒抗肺癌的机制研究[J].湖南中医药大学学报,2010,30(6):46-48.
[23]柒小梅,李英,秦雪梅.小柴胡汤临床应用与药理作用研究进展[J].山西中医学院学报,2007,8(3):59-60.
[24]李延萍,谢微杳.小柴胡加味汤治疗类风湿关节炎活动100例疗效观察[J].中国中医急症,2014,23(5):920-921.
[25]苏青,吴婷婷,黄雅兰,等.黄芩提取物制备过程中化学成分及药效的变化规律分析[J].中国实验方剂学杂志,2018,24(14):1-6.
[26]LV Q W,ZHANG W,SHI Q,et al.Comparison of Tripterygium wilfordii Hook F with methotrexate in the treatment of active rheumatoid arthritis(TRIFRA):a randomised,controlled clinical trial[J].Ann Rheum Dis,2015,74(6):1078-1086.