芍药汤对湿热内蕴型溃疡性结肠炎大鼠模型结肠组织中AP-1,TNF-α表达的影响
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摘要
目的:观察芍药汤对湿热内蕴型溃疡性结肠炎(UC)大鼠模型结肠组织中激活蛋白-1(AP-1)与肿瘤坏死因子-α(TNF-α)的基因及蛋白表达的影响,探讨芍药汤治疗UC的作用机制。方法:将60只Wistar大鼠随机分为正常组、模型组、柳氮磺砒啶组及芍药汤低、中、高剂量组,采用病症结合的方法复制湿热内蕴型UC大鼠模型,即高脂高糖辛辣食物及免疫复合法,联合2,4,6-三硝基苯磺酸(TNBS)结合乙醇复合法。造模成功后,分别给予芍药汤低、中、高(6,12,24 g·kg~(-1))灌胃,柳氮磺胺嘧啶1 g·kg~(-1)剂量灌胃,正常组给予等体积生理盐水,连续21 d。实时荧光定量聚合酶链式反应(Real-time PCR)检测结肠组织AP-1,TNF-α mRNA表达,蛋白免疫印迹法(Western blot)检测结肠组织AP-1,TNF-α蛋白表达。结果:与正常组比较,模型组AP-1,TNF-α mRNA及蛋白相对表达量明显升高(P <0. 05);与模型组比较,各干预组AP-1,TNF-α mRNA及蛋白表达量明显下降(P <0. 05),以芍药高剂量组较为明显。结论:芍药汤可抑制湿热型UC大鼠AP-1蛋白刺激TNF-α的表达,可能是其治疗湿热型UC的机制之一。
Objective: To observe the effect of Shaoyaotang on mRNA and protein expressions of colon tissue activated protein-1( AP-1) and tumor necrosis factor-α( TNF-α) of hot and humid-type intrinsic ulcerative colitis( UC) model in rats,in order to explore the mechanism of action of herbaceous peony decoction in the treatment of UC. Method: Totally 60 Wistar rats were randomly divided into blank group,model group,SASP group,and low,medium and high-dose Shaoyaotang groups. The damp-heat intrinsic UC rat model was replicated based on integrated disease and syndrome,namely,high-fat and high-sugar spicy food and immune complex method combined with 2,4,6-trinitrobenzene sulfolnic acid( TNBS) and ethanol complex method. After the successful modeling,low,medium and high-dose Shaoyaotang( 6,12,24 g·kg~(-1)) was given by gavage,and 1 g·kg~(-1) dose of salazol sulfadiazine was given to by gavage. The blank group was given constant volume normal saline for 21 d.Real-time fluorescence quantitative polymerase chain reaction( Real-time PCR) was used to detect mRNA expressions of AP-1 and TNF-α in colon tissues,and Western blot was used to detect protein expressions of AP-1 and TNF-α in colon tissues. Result: Compared with the blank group,relative mRNA and protein expressions of AP-1,TNF-α in the model group were significantly increased( P < 0. 05). Compared with the model group,mRNA and protein expressions of AP-1,TNF-α in the treatment groups were significantly decreased( P < 0. 05).Conclusion: Shaoyaotang can inhibit the expression of TNF-α and stimulate AP-1 protein expression in rats with damp-heat UC.
Objective: To observe the effect of Shaoyaotang on mRNA and protein expressions of colon tissue activated protein-1( AP-1) and tumor necrosis factor-α( TNF-α) of hot and humid-type intrinsic ulcerative colitis( UC) model in rats,in order to explore the mechanism of action of herbaceous peony decoction in the treatment of UC. Method: Totally 60 Wistar rats were randomly divided into blank group,model group,SASP group,and low,medium and high-dose Shaoyaotang groups. The damp-heat intrinsic UC rat model was replicated based on integrated disease and syndrome,namely,high-fat and high-sugar spicy food and immune complex method combined with 2,4,6-trinitrobenzene sulfolnic acid( TNBS) and ethanol complex method. After the successful modeling,low,medium and high-dose Shaoyaotang( 6,12,24 g·kg~(-1)) was given by gavage,and 1 g·kg~(-1) dose of salazol sulfadiazine was given to by gavage. The blank group was given constant volume normal saline for 21 d.Real-time fluorescence quantitative polymerase chain reaction( Real-time PCR) was used to detect mRNA expressions of AP-1 and TNF-α in colon tissues,and Western blot was used to detect protein expressions of AP-1 and TNF-α in colon tissues. Result: Compared with the blank group,relative mRNA and protein expressions of AP-1,TNF-α in the model group were significantly increased( P < 0. 05). Compared with the model group,mRNA and protein expressions of AP-1,TNF-α in the treatment groups were significantly decreased( P < 0. 05).Conclusion: Shaoyaotang can inhibit the expression of TNF-α and stimulate AP-1 protein expression in rats with damp-heat UC.
引文
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[18]易文,陈置丰,石孟琼,等.左金丸阻断溃疡性结肠炎小鼠NF-κB激活MIF,TNF-α及IL-1β,IL-6表达[J].中药药理与临床,2014,30(6):9-13.
[2] Wagner E F. Bone development and inflammatory disease is regulated by AP-1(Fos/Jun)[J]. Ann Rheum Dis,2010,69:86-88.
[3] Thomsen M K,Bakiri L,Hasenfuss S C,et al. JUNB/AP-1 controls IFN-γduring inflammatory liver disease[J]. Eur J Clin Invest,2013,123(12):5258-5268.
[4]刘启鸿,黄文彬,骆云丰,等.溃疡性结肠炎当重湿热[J].陕西中医药大学学报,2018,41(6):13-16.
[5]王移飞,王凤仪,徐兰萍,等.芍药汤经HMGB1调节湿热型溃疡性结肠炎大鼠MyD88和NF-κB的分子机制[J].中国实验方剂学杂志,2018,24(12):86-91.
[6]翁一洁,郑学宝.湿热型溃疡性结肠炎大鼠模型的建立与研究[J].时珍国医国药,2011,22(10):2522-2525.
[7]万明民,杜悦,陈纯苇,等.芍药汤的临床应用及实验研究进展[J].中国中医药现代远程教育,2018,16(23):154-157.
[8]刘琦,罗霞,罗爽,等.芍药苷通过抑制NLRP3炎症小体治疗溃疡性结肠炎小鼠的研究[J].中药新药与临床药理,2018,29(4):409-414.
[9]罗爽,罗霞,刘琦,等.大黄酸对DSS诱导溃疡性结肠炎小鼠的治疗作用及机制探讨[J].中国实验方剂学杂志,2017,23(11):109-113.
[10] Nielsen O H,Bjerrum J T,Herfarth H H,et al. Recent advances using immunomodulators for inflammatory bowel disease[J]. J Clin Pharmacol,2013,53(6):575-588.
[11] Danese S. New therapies for inflammatory bowel disease:from the bench to the bedside[J]. Gut,2012,61:918-932.
[12] CHEN Y,Currie R W. Small interfering RNA knocks down heat shock factor-1(HSF-1)and exacerbatesproinflammatory activation of NF-kappa B and AP-1 in vascular smooth muscle cells[J]. Cardiovasc Res,2006,69:66-75.
[13] CHANG L,Karin M. Mammalian MAP kinase signalling cascades[J]. Nature,2001,410(6824):37-40.
[14] Johnson G L,Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38protein kinases[J]. Science,2002, 298(5600):1911-1912.
[15]陈维雄,陆允敏,陈金联,等. AP-1在小鼠实验性肠炎中的表达[C]//中华医学会.第七次全国消化病学术会议论文汇编(下册):2007年卷.济南:出版社不详,2007:188.
[16] Park S,Regmi S C,Park S Y,et al. Protective effect of7-O-succinyl macrolactin A against intestinal inflammation is mediated through PI3-kinase/Akt/mTOR and NF-κB signaling pathways[J]. Eur J Pharmacol,2014,735:184-192.
[17]王彤,王骁,范焕芳,等.白头翁汤对溃疡性结肠炎模型小鼠结肠黏膜及血清TNF-α,IL-6影响[J].辽宁中医药大学学报,2017,19(2):32-35.
[18]易文,陈置丰,石孟琼,等.左金丸阻断溃疡性结肠炎小鼠NF-κB激活MIF,TNF-α及IL-1β,IL-6表达[J].中药药理与临床,2014,30(6):9-13.