白头翁汤对溃疡性结肠炎大鼠的疗效及免疫机制的影响
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摘要
目的:探讨白头翁汤对溃疡性结肠炎(UC)大鼠的治疗效果及免疫机制。方法:采用2,4-二硝基氯苯(DNCB)加醋酸/乙醇复合法建立UC大鼠模型,将造模成功的大鼠随机分成模型组、美沙拉嗪组和白头翁汤高、中、低剂量组,正常大鼠为对照组。美沙拉嗪组给予美沙拉嗪(0. 5 g·kg-1)灌胃,白头翁汤高、中、低剂量组分别给予白头翁汤(10,5,2. 5 g·kg-1)灌胃,其余各组给予双蒸水。连续给药7 d后,观察各组大鼠一般情况和疾病活动指数;大鼠麻醉后腹主动脉取血,处死大鼠,取结肠测长度,肉眼观察结肠黏膜损伤程度及大体评分;光镜下观察结肠组织病理形态学变化及病理评分;采用分光光度计比色法检测血清和结肠组织匀浆液中性粒细胞髓过氧化物酶(MPO)活性;酶联免疫吸附测定(ELISA)法测定血清和结肠组织匀浆液P-选择素、巨噬细胞移动抑制因子(MIF)和血栓素B2(TXB2)水平;免疫组织化学法及蛋白免疫印迹法(Western blot)技术检测结肠组织Toll样受体4(TLR4)和核转录因子-κB(NF-κB)蛋白表达。结果:与正常组比较,模型组出现稀便、腹泻及血便等症状,DAI,结肠大体评分及病理学评分显著增高(P <0. 01);白头翁汤组明显改善UC症状,DAI,结肠大体形态及病理学评分显著下降(P <0. 01)。模型组血清和结肠组织中MPO,P-选择素,MIF和TXB2的含量较正常组显著升高;白头翁汤高、中剂量组能显著降低MPO,P-选择素,MIF和TXB2的水平。模型组结肠组织中TLR4和NF-κB蛋白表达量均显著高于正常组;高、中、低剂量组均能明显抑制TLR4和NF-κB的表达。结论:白头翁汤可能通过抑制TLR4/NF-κB信号通路,下调P-选择素,MPO,MIF,TXB2的水平,促进肠道黏膜的的修复,减轻结肠炎症反应。
Objective: To investigate the therapeutic effect and immune mechanism of Baitouweng Tang on ulcerative colitis( UC) rats. Method: The mode of UC rats was made of 2,4-dinitrochlorobenzene( DNCB)/ethanol enema. Rats were randomly divided into control group,model group,mesalazine group,and high-dose,middle-dose and low-dose Baitouweng Tang groups. The mesalazine group were administered with mesalazine( 0. 5 g·kg-1). Baitouweng Tang groups were given Baitouweng Tang( 10,5,2. 5 g·kg-1),while the other groups were given double steaming water. After 7 days of continuous administration,the general condition and disease activity index of rats in each group were observed. After anesthesia in rats,blood was taken from the abdominal aorta. Then the rats were put to death,and the length and morphological observation of the colon were measured. Ultraviolet spectrophotometry detection was used to detect the activities of myeloperoxidase( MPO) in blood and colon tissue. The levels of P-selectin,macrophage migration inhibitory factor( MIF) and thromboxane B2( TXB2) in blood and colon tissues were detected by enzyme-linked immunosorbent assay( ELISA).Immunohistochemistry and Western blot methods were undertaken to determine the expressions of Toll-like receptor4( TLR4) and nuclear transcription factor-κB( NF-κB) proteins in colon tissue. Result: Compared with the model group,the rats in model group showed severe symptoms,such as loose stools,diarrhea and bloody stools,while Baitouweng Tang obviously ameliorated them. Moreover,Baitouweng Tang significantly reduced DAI,colon general and pathological scores,which were high in model group( P < 0. 01). The levels of MPO,P-selectin,MIF and TXB2 in the serum and colon tissues of the model group were obviously increased( P < 0. 01),and Baitouweng Tang could reverse them. Similarly,the expressions of TLR4 and NF-κB in colons of model group were markedly higher than those in control group( P < 0. 01). However,Baitouweng Tang group showed lower expressions than the model group( P < 0. 01). Conclusion: Baitouweng Tang could inhibit TLR4/NF-κB signaling pathway in treatment of ulcerative colitis,and reduce the expressions of P-selectin,MPO,MIF and TXB2,and thus promoting intestinal mucosal repair and improving intestinal function.
Objective: To investigate the therapeutic effect and immune mechanism of Baitouweng Tang on ulcerative colitis( UC) rats. Method: The mode of UC rats was made of 2,4-dinitrochlorobenzene( DNCB)/ethanol enema. Rats were randomly divided into control group,model group,mesalazine group,and high-dose,middle-dose and low-dose Baitouweng Tang groups. The mesalazine group were administered with mesalazine( 0. 5 g·kg-1). Baitouweng Tang groups were given Baitouweng Tang( 10,5,2. 5 g·kg-1),while the other groups were given double steaming water. After 7 days of continuous administration,the general condition and disease activity index of rats in each group were observed. After anesthesia in rats,blood was taken from the abdominal aorta. Then the rats were put to death,and the length and morphological observation of the colon were measured. Ultraviolet spectrophotometry detection was used to detect the activities of myeloperoxidase( MPO) in blood and colon tissue. The levels of P-selectin,macrophage migration inhibitory factor( MIF) and thromboxane B2( TXB2) in blood and colon tissues were detected by enzyme-linked immunosorbent assay( ELISA).Immunohistochemistry and Western blot methods were undertaken to determine the expressions of Toll-like receptor4( TLR4) and nuclear transcription factor-κB( NF-κB) proteins in colon tissue. Result: Compared with the model group,the rats in model group showed severe symptoms,such as loose stools,diarrhea and bloody stools,while Baitouweng Tang obviously ameliorated them. Moreover,Baitouweng Tang significantly reduced DAI,colon general and pathological scores,which were high in model group( P < 0. 01). The levels of MPO,P-selectin,MIF and TXB2 in the serum and colon tissues of the model group were obviously increased( P < 0. 01),and Baitouweng Tang could reverse them. Similarly,the expressions of TLR4 and NF-κB in colons of model group were markedly higher than those in control group( P < 0. 01). However,Baitouweng Tang group showed lower expressions than the model group( P < 0. 01). Conclusion: Baitouweng Tang could inhibit TLR4/NF-κB signaling pathway in treatment of ulcerative colitis,and reduce the expressions of P-selectin,MPO,MIF and TXB2,and thus promoting intestinal mucosal repair and improving intestinal function.
引文
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[16]庾国桢,彭皓均,黄秀芳,等.黄连素对Aβ25-35诱导的HT22细胞TLR 4/NF-κB信号通路的影响[J].中国实验方剂学杂志,2018,24(24):164-170.
[17]曹世霞,祝捷,张三印,等.秦皮总香豆素对急性痛风性关节炎大鼠模型IL-1β、IL-8、TNF-α的影响[J].四川中医,2011,29(3):68-70.
[18]胡天穹,邵中一,王曜.清肠化湿方联合美沙拉嗪治疗活动期轻中度溃疡性结肠炎的临床疗效[J].世界华人消化杂志,2016,24(36):4824-4829.
[19]PAN Y M, ZHU J C, WANG H S, et al.Antioxidantactivity of ethanolic extract of Cortex fraxini and use in peanutoil[J]. Food Chemistry,2007,103(3):913-918.
[20]Opavsky M A, Martino T, Rabinovitch M, et al.Enhanced ERK-1/2 activation in mice susceptible to coxsachievirus-induced myocarditis[J]. J Clin Invest,2002,109(12):1561-1569.
[21]王燕颖,孙丽芳,禹晶,等. MPO与TGF-β1在溃疡性结肠炎中的表达[J].中国医师杂志,2009,11(1):31-33.
[22]杨权,陈守国,许宁.溃疡性结肠炎患者血浆P选择素的测定及其意义[J].海南医学,2010,21(2):13-14.
[23]韩红,夏冰,曾宪昌.炎症性肠病的血栓前状态[J].临床血液学杂志,2003,16(2):91-93.
[24]HAN L, LIU D L, ZENG Q K, et al. The neuroprotective effects and probable mechanisms of ligustilide and its degradative products on intracerebral hemorrhage in mice[J]. Int Immunopharmacol,2018,63:43-57.
[25]Roger T,David J,Glauser M P,et al. MIF regulates innate immune responses through modulation of Toll-like recepor 4[J]. Nature,2001,414(6866):920-924.
[2]Bamias G,Arseneau K O,Cominelli F,et al. Mouse models of inflammatory bowel disease for investigating mucosal immunity in the intestine[J]. Curr Opin Gastroenterol,2018,33(6):411-416.
[3]Choy M C,Visvanathan K,De Cruz P,et al. An overview of the innate and adaptive immune system in inflammatory bowel disease[J]. Inflamm Bowel Dis,2017,23(1):2-13.
[4]De Mesquita M B, Civitelli F, Levine A.Epidemiology,genes and inflammatory bowel diseases in childhood[J]. Dig Liver Dis,2008,40(1):3-11.
[5]Ma B,Hottiger M O. Crosstalk between Wnt/betacatenin and Nf-kappab signaling pathway during inflammation[J]. Front Immuno,2016,dio:10. 3389/fimmu. 2016. 00378.
[6]JING H,Lee S. Nf-kappaB in cellular senescence and cancer treatment[J]. Mol Cells,2014,37(3):189-195.
[7]Baker R G, Hayden M S, Ghosh S. Nf-kappab,inflammation,and metabolic disease[J]. Cell Metab,2011,13(1):11-22.
[8]WANG Y,SUN L,SONG Z,et al. Maspin inhibits macrophage phagocytosis and enhances inflammatory cytokine production via activation of NF-κB signaling[J]. Mol Immunol,2017,82:94-103.
[9]赵欣,恽海峰,向桂玲,等.白头翁汤加减联合双歧杆菌制剂对大鼠溃疡性结肠炎的影响[J].中国实验方剂学杂志,2018,24(13):110-116.
[10]江学良,权启镇,王东,等.复合法建立大鼠溃疡性结肠炎模型[J].青海医学院学报,1999,20(4):1-3.
[11]Cooper H S, Murthy S N, Shah R S, et al.Clinicopathologic study of dextran sulfate sodium experimental murine colitis[J]. Lab Invest,1993,69(2):238-249.
[12]Ekstrm G M. Oxazolone-induced colitis in rats:effects of budesonide,cyclosporin A,and 5-aminosalicylic acid[J]. Scand J Gastroenterol,1998,33(2):174-179.
[13]Dieleman L A,Palmen M J,Akol H,et al. Chronic experimental colitis induced by dextran sulphate sodium(DSS)is characterized by Th1 and Th2 cytokines[J].Clin Exp Immunol,1998,114(3):385-391.
[14]Probert C,Hraring S. Infliximab in moderately severe glucocorticoid resistant ulcerative colitis:a ran-domised controlled trial[J]. Gut,2003,52(7):998-1002.
[15]宋立武.自拟解毒凉血汤灌肠联合美沙拉嗪栓治疗直肠型溃疡性结肠炎的临床疗效观察[J].临床合理用药杂志,2016,9(20):51-52.
[16]庾国桢,彭皓均,黄秀芳,等.黄连素对Aβ25-35诱导的HT22细胞TLR 4/NF-κB信号通路的影响[J].中国实验方剂学杂志,2018,24(24):164-170.
[17]曹世霞,祝捷,张三印,等.秦皮总香豆素对急性痛风性关节炎大鼠模型IL-1β、IL-8、TNF-α的影响[J].四川中医,2011,29(3):68-70.
[18]胡天穹,邵中一,王曜.清肠化湿方联合美沙拉嗪治疗活动期轻中度溃疡性结肠炎的临床疗效[J].世界华人消化杂志,2016,24(36):4824-4829.
[19]PAN Y M, ZHU J C, WANG H S, et al.Antioxidantactivity of ethanolic extract of Cortex fraxini and use in peanutoil[J]. Food Chemistry,2007,103(3):913-918.
[20]Opavsky M A, Martino T, Rabinovitch M, et al.Enhanced ERK-1/2 activation in mice susceptible to coxsachievirus-induced myocarditis[J]. J Clin Invest,2002,109(12):1561-1569.
[21]王燕颖,孙丽芳,禹晶,等. MPO与TGF-β1在溃疡性结肠炎中的表达[J].中国医师杂志,2009,11(1):31-33.
[22]杨权,陈守国,许宁.溃疡性结肠炎患者血浆P选择素的测定及其意义[J].海南医学,2010,21(2):13-14.
[23]韩红,夏冰,曾宪昌.炎症性肠病的血栓前状态[J].临床血液学杂志,2003,16(2):91-93.
[24]HAN L, LIU D L, ZENG Q K, et al. The neuroprotective effects and probable mechanisms of ligustilide and its degradative products on intracerebral hemorrhage in mice[J]. Int Immunopharmacol,2018,63:43-57.
[25]Roger T,David J,Glauser M P,et al. MIF regulates innate immune responses through modulation of Toll-like recepor 4[J]. Nature,2001,414(6866):920-924.