清肠化湿方对TNBS大鼠结肠组织PPAR-γ/p38 MAPK的影响
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摘要
目的 :观察清肠化湿方(QHD)对三硝基苯磺酸(TNBS)诱导的大鼠结肠炎结肠组织中过氧化物酶体增殖物激活受体(PPAR-γ)和p38丝裂原活化蛋白激酶(p38 MAPK)表达的影响,探讨其治疗溃疡性结肠炎(UC)的可能作用机制。方法:采用80只Wistar大鼠建立UC大鼠模型,随机分为8组,分别为空白对照组、模型对照组、QHD低剂量组、QHD中剂量组、QHD高剂量组、柳氮磺胺吡啶(SASP)组、柳氮磺胺吡啶+双酚A-二甘氨酸醚(SASP+BADGE)组及中药中剂量+BADGE组,每组10只。除空白对照组以生理盐水灌肠外,其余组采用TNBS/乙醇灌肠造UC大鼠模型。以柳氮磺胺吡啶(SASP)为阳性对照,同时联合使用PPAR-γ抑制剂双酚A-二甘氨酸醚(BADGE),给药7 d后病理检测各组大鼠结肠组织的损伤情况,并通过蛋白质印迹法(Western blot)和实时荧光定量多聚核苷酸链式反应(Real-time PCR)实验分别检测大鼠结肠组织中PPAR-γ、p38 MAPK的蛋白和基因表达,同时检测各组大鼠结肠组织中黏蛋白2(MUC2)和三叶因子3(TFF3)的表达情况。结果:清肠化湿方可上调TNBS大鼠结肠组织PPAR-γ的表达(P<0.01),同时抑制p38 MAPK的活性(P<0.01),并促进结肠组织中MUC2和TFF3表达(P<0.01),当联用PPAR-γ抑制剂BADGE后其对p38 MAPK的抑制作用减弱(P<0.05)。结论:清肠化湿方对TNBS大鼠结肠炎有明显的保护作用,其作用机制可能与激活PPAR-γ信号通路、抑制p38 MAPK的激活、减轻炎症反应、升高结肠组织中MUC2与TFF3的表达水平、参与肠黏膜的修复有关。
Objective:To investigate the efficacy of Qingchang Huashi decoction(QHD) on ulcerative colitis induced by 2,4,6- trinitrobenzen sulfonic acid(TNBS) in Wistar rats and determine the expression of PPAR- γ and p38 MAPK in colonic mucosa of rats in further to study the function and mechanism of QHD in rats with ulcerative colitis. Methods:Eighty male Wistar rats were used to establish UC models and divided randomly into eight groups:the normal group,model group,QHD- low group,QHD- medium group,QHD- high group,SASP group,SASP+ BADGE group and the QHD- medium+ BADGE group. Each group had ten rats. TNBS was used in seven groups except for the normal group to establish ulcerative colitis model in rats. SASP and BADGE which were the inhibitors of PPAR- γ were used. After 7 days,the colonic pathological morphology was observed. Western blot and Real- time PCR(RT- PCR) were performed to detect the protein levels and m RNA levels of PPAR- γ and p38 MAPK. The expressions of MUC2 and TFF3 in colonic mucosa were also measured by RT- PCR. Results:Compared with the model group,QHD could effectively improve the life quality,reduce colon tissue inflammatory cells infiltration,enhance colonic mucosal tissue repair and promote the recovery of UC rats.QHD could increase PPAR- γ expression and inhibit p38 MAPK activity. In addition,QHD was able to increase significantly the expressions of MUC2 and TFF3 in colonic mucosa. Conclusion:QHD had obvious protective effect on UC. One of the mechanisms might be that QHD could active PPAR- γsignaling and inhibit p38 MAPK activation,increase the expression of MUC2 and TFF3 in colonic tissue to participate in restoration of intestinal mucosa,and inhibit the release of inflammatory cells.
Objective:To investigate the efficacy of Qingchang Huashi decoction(QHD) on ulcerative colitis induced by 2,4,6- trinitrobenzen sulfonic acid(TNBS) in Wistar rats and determine the expression of PPAR- γ and p38 MAPK in colonic mucosa of rats in further to study the function and mechanism of QHD in rats with ulcerative colitis. Methods:Eighty male Wistar rats were used to establish UC models and divided randomly into eight groups:the normal group,model group,QHD- low group,QHD- medium group,QHD- high group,SASP group,SASP+ BADGE group and the QHD- medium+ BADGE group. Each group had ten rats. TNBS was used in seven groups except for the normal group to establish ulcerative colitis model in rats. SASP and BADGE which were the inhibitors of PPAR- γ were used. After 7 days,the colonic pathological morphology was observed. Western blot and Real- time PCR(RT- PCR) were performed to detect the protein levels and m RNA levels of PPAR- γ and p38 MAPK. The expressions of MUC2 and TFF3 in colonic mucosa were also measured by RT- PCR. Results:Compared with the model group,QHD could effectively improve the life quality,reduce colon tissue inflammatory cells infiltration,enhance colonic mucosal tissue repair and promote the recovery of UC rats.QHD could increase PPAR- γ expression and inhibit p38 MAPK activity. In addition,QHD was able to increase significantly the expressions of MUC2 and TFF3 in colonic mucosa. Conclusion:QHD had obvious protective effect on UC. One of the mechanisms might be that QHD could active PPAR- γsignaling and inhibit p38 MAPK activation,increase the expression of MUC2 and TFF3 in colonic tissue to participate in restoration of intestinal mucosa,and inhibit the release of inflammatory cells.
引文
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[18]沈洪,刘智群,朱荃,等.清肠化湿方对溃疡性结肠炎NF-κB/Tolls通路的影响及其机制[J].中国中西医结合杂志,2013,33(9):1216-1220.
[19]朱磊,沈洪,刘丽,等.清肠化湿方干预治疗对溃疡性结肠炎急性活动期小鼠结肠组织黏膜修复及COX-2表达影响的实验研究[J].辽宁中医杂志,2013,40(11):2371-2373.
[20]吴昊,沈洪,朱磊,等.清肠化湿方对小鼠溃疡性结肠炎模型结肠黏膜上皮细胞Caspase-3、ZO-1的影响[J].世界中西医结合杂志,2014,9(5):473-491.
[21]顾培青,沈洪,刘丽,等.清肠化湿方对溃疡性结肠炎模型大鼠结肠TLR4、NF-κB p65蛋白表达的影响[J].中国中医急症,2010,19(1):99-101.
[2]Roth S,Spalinger M R,Gottier C,et al.Bilberry-Derived anthocyanins modulate cytokine expression in the intestine of patients with ulcerative colitis[J].PLo S One,2016,11(5):e0154817.
[3]沈晨,张声生,崔超,等.清肠化湿法治疗活动期溃疡性结肠炎近期疗效观察[J].北京中医药,2013,32(6):415-419.
[4]沈洪,张声生,王垂杰,等.中药分期序贯治疗轻中度溃疡性结肠炎临床观察[J].中华中医药杂志,2012,27(7):1788-1791.
[5]沈洪,张声生,王垂杰,等.中药分期序贯方案对轻中度溃疡性结肠炎血沉和C反应蛋白的影响[J].中国中西医结合消化杂志,2011,31(2):101-103.
[6]Loddo I,Romano C.Inflammatory bowel disease:genetics,epigenetics,and pathogenesis[J].Front Immunol,2015,6(6):551.
[7]Zhang,Y Z,Li Y Y.Inflammatory bowel disease:pathogenesis[J].World J Gastroenterol,2014,20(1):91-99.
[8]Speca S,Dubuquoy L,Desreumaux P.Peroxisome proliferator-activated receptor gamma in the colon:inflammation and innate antimicrobial immunity[J].J Clin Gastroenterol,2014,48(Suppl1):S23-S27.
[9]Dubuquoy L,Jansson E A,Deeb S,et al.Impaired expression of peroxisome proliferator-activated receptor gamma in ulcerative colitis[J].Gastroenterology,2003,124(5):1265-1276.
[10]Dubuquoy L,Rousseaux C,Thuru X,et al.PPAR-gamma as a new therapeutic target in inflammatory bowel diseases[J].Gut,2006,55(9):1341-1349.
[11]Robinson M J,Cobb M H.Mitegen-activated protein kinase pathways[J].Curr Opin Cell Biol,1997,9(2):180-186.
[12]Hommes D W,Peppelenbosch M P,Van Deventer S J.Mitogenactivated protein(MAP)kinase signal transduction pathways and novel anti-inflammatory Targets[J].Gut,2003,52(1):144-151.
[13]Pang X,Liu J,Li Y,et al.Emodin inhibits homocysteine-induced C-reactive protein generation in vascular smooth muscle Cells by Regulating PPARγExpression and ROS-ERK1/2/p38Signal Pathway[J].PLo S One,2015,10(7):e0131295.
[14]Miner-Williams W M,Moughan P J.Intestinal barrier dysfunction:implications for chronic inflammatory conditions of the Bowel[J].Nutr Res Rev,2016,18:1-20.
[15]Shigeshiro Mizuki,Tanabe Soichi,Suzuki Takuya.Repeated exposure to water immersion stress reduces the Muc2 gene level in the rat colon via two distinct mechanisms[J].Brain,Behavior and Immunity,2012,26(7):1061-1065.
[16]Belovari T,Bijelic N,Tolusic Levak M,et al.Trefoil factor family peptides TFF1 and TFF3 in the nervous tissues of developing mouse embryo[J].Bosnian Journal of Basic Medical Sciences,2015,15(1):33-37.
[17]翟金海,沈洪,陈兰.清肠化湿方对UC模型大鼠结肠黏膜ICAM 1表达的影响[J].时珍国医国药,2014,25(4):799-801.
[18]沈洪,刘智群,朱荃,等.清肠化湿方对溃疡性结肠炎NF-κB/Tolls通路的影响及其机制[J].中国中西医结合杂志,2013,33(9):1216-1220.
[19]朱磊,沈洪,刘丽,等.清肠化湿方干预治疗对溃疡性结肠炎急性活动期小鼠结肠组织黏膜修复及COX-2表达影响的实验研究[J].辽宁中医杂志,2013,40(11):2371-2373.
[20]吴昊,沈洪,朱磊,等.清肠化湿方对小鼠溃疡性结肠炎模型结肠黏膜上皮细胞Caspase-3、ZO-1的影响[J].世界中西医结合杂志,2014,9(5):473-491.
[21]顾培青,沈洪,刘丽,等.清肠化湿方对溃疡性结肠炎模型大鼠结肠TLR4、NF-κB p65蛋白表达的影响[J].中国中医急症,2010,19(1):99-101.