通便汤对STC大鼠模型结肠组织中PKA/MPKA信号通路的影响
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摘要
目的:探讨通便汤对慢传输型便秘(slow transit constipation,STC)大鼠模型结肠组织中蛋白激酶A(protein kinase A,PKA)/丝裂原活化激酶(mitogen-activated protein kinase,MAPK)信号通路的影响及相关机制。方法:80只SD大鼠随机分为正常组和造模组,正常组20只,造模组60只,雌雄各半;正常组给予普通饲料喂养,模型组给予混有复方苯乙哌啶的饲料,造模时间120 d后,随机选取雌雄对半大鼠正常组10只,造模组20只,测定大鼠24 h排便量、含水量及小肠炭末推进率,观察结肠留存粪便粒数,评价STC大鼠造模是否成功;停药1周后,将造模组40只大鼠随机分为模型组,通便汤组(33 g·kg-1),通便汤+H89组(PKA信号通路阻滞剂,5 mg·kg-1),通便汤+U0126组(MAPK信号通路阻滞剂,0. 1 mg·kg-1)各10只,雌雄各半,药物通便汤干预4周后,测定大鼠24 h排便量、含水量及小肠炭末推进率,观察结肠留存粪便粒数;采用免疫组化(IHC),蛋白免疫印迹法(Western blot),实时荧光定量聚合酶链式反应(Real-time PCR)测定结肠内水通道蛋白3(AQP3),AQP4,PKA及MAPKs信号通路的蛋白及mRNA表达情况。结果:与正常组比较,造模组大鼠24 h排便量、粪便含水量、小肠炭末推进率及结肠存留粪便粒数均显著降低(P <0. 01);与模型组比较,通便汤组排便量、含水量及小肠炭末推进率均增加,结肠留存粪便粒数减少(P <0. 01),AQP3,AQP4显著降低(P <0. 01);与通便汤组比较,通便汤+H89组和通便汤+U0126组AQP3,AQP4,PKA蛋白与mRNA表达降低(P <0. 01);与通便汤+H89组比较,通便汤+U0126组排便量、含水量、小肠炭末推进率及结肠留存粪便粒数,AQP3,AQP4,PKA,MAPK蛋白表达量与mRNA含量无明显差异。结论:采用复方苯乙哌啶成功复制出慢性传输型便秘模型,通便汤可以抑制PKA和MAPK信号通路,从而下调AQP3,AQP4表达,增加肠道蠕动和肠道水分,有效治疗STC。
Objective: To explore the effect of Tongbiantang on protein kinase A(PKA) and mitogenactivated protein kinase(MAPK) signal pathway in colon tissue of slow transit constipation(STC) rats and its related mechanism. Method: Eighty SD rats were randomly divided into blank group and model group,20 rats in blank group,60 rats in model group,half male and half female; blank group was fed with common diet,model group was fed with compound phenylethylpiperidine,after 120 days of modeling,10 rats in blank group and 20 rats in model group were randomly selected,and 2 rats were determined. Four-hour stool volume,water content and small intestinal charcoal powder propelling rate were observed to observe the number of stool particles retained in colon and evaluate the success of STC rat modeling. After 1 week of drug withdrawal,40 rats in model group were randomly divided into model group(33 g · kg-1),Tongbiantang group,Tongbiantang + H89 group(PKA signaling pathway blocker,5 mg · kg-1),Tongbiantang + U0126 group(MPKA signaling pathway blocker,0. 1 mg·kg-1) each. After 4 weeks of intervention with Tongbiantang,the amount of stool excretion,water content and small intestinal charcoal powder propelling rate were measured in 10 rats,and the number of stool grains in colon was observed. The protein content and mRNA expression in aquaporins 3(AQP3),AQP4,PKA and MAPKs signaling pathways in colon was determined by immunohistochemical staining(IHC),Western blot and Real-time fluorescence quantitative PCR(Real-time PCR). Result: Compared with the blank group,the 24-hour stool volume,fecal water content,small intestinal charcoal propelling rate and the number of fecal particles in colon of rats in the model group were significantly decreased(P < 0. 01); compared with the model group,the fecal volume,water content and small intestinal charcoal propelling rate of Tongbiantang group were increased,the number of fecal particles in colon decreased(P < 0. 01),AQP3 and AQP4 were significantly decreased(P <0. 01). Compared with the Tongbiantang group,the expressions of AQP3,AQP4,PKA and mRNA in the Tongbiantang + H89 group and the Tongbiantang + U0126 group were significantly lower(P < 0. 01); compared with the Tongbiantang + H89 group,the stool volume,water content,small intestine charcoal propelling rate and the number of stool particles in colon,AQP3,AQP4,PKA,MPKAs protein table were significantly lower(P <0. 01). There was no significant difference between the amount of mRNA and the content of protein. Conclusion:Tongbiantang can inhibit the PKA and MPKA signal pathways,thus down-regulate the expression of AQP3 and AQP4,increase intestinal peristalsis and intestinal water,and effectively treat STC.
Objective: To explore the effect of Tongbiantang on protein kinase A(PKA) and mitogenactivated protein kinase(MAPK) signal pathway in colon tissue of slow transit constipation(STC) rats and its related mechanism. Method: Eighty SD rats were randomly divided into blank group and model group,20 rats in blank group,60 rats in model group,half male and half female; blank group was fed with common diet,model group was fed with compound phenylethylpiperidine,after 120 days of modeling,10 rats in blank group and 20 rats in model group were randomly selected,and 2 rats were determined. Four-hour stool volume,water content and small intestinal charcoal powder propelling rate were observed to observe the number of stool particles retained in colon and evaluate the success of STC rat modeling. After 1 week of drug withdrawal,40 rats in model group were randomly divided into model group(33 g · kg-1),Tongbiantang group,Tongbiantang + H89 group(PKA signaling pathway blocker,5 mg · kg-1),Tongbiantang + U0126 group(MPKA signaling pathway blocker,0. 1 mg·kg-1) each. After 4 weeks of intervention with Tongbiantang,the amount of stool excretion,water content and small intestinal charcoal powder propelling rate were measured in 10 rats,and the number of stool grains in colon was observed. The protein content and mRNA expression in aquaporins 3(AQP3),AQP4,PKA and MAPKs signaling pathways in colon was determined by immunohistochemical staining(IHC),Western blot and Real-time fluorescence quantitative PCR(Real-time PCR). Result: Compared with the blank group,the 24-hour stool volume,fecal water content,small intestinal charcoal propelling rate and the number of fecal particles in colon of rats in the model group were significantly decreased(P < 0. 01); compared with the model group,the fecal volume,water content and small intestinal charcoal propelling rate of Tongbiantang group were increased,the number of fecal particles in colon decreased(P < 0. 01),AQP3 and AQP4 were significantly decreased(P <0. 01). Compared with the Tongbiantang group,the expressions of AQP3,AQP4,PKA and mRNA in the Tongbiantang + H89 group and the Tongbiantang + U0126 group were significantly lower(P < 0. 01); compared with the Tongbiantang + H89 group,the stool volume,water content,small intestine charcoal propelling rate and the number of stool particles in colon,AQP3,AQP4,PKA,MPKAs protein table were significantly lower(P <0. 01). There was no significant difference between the amount of mRNA and the content of protein. Conclusion:Tongbiantang can inhibit the PKA and MPKA signal pathways,thus down-regulate the expression of AQP3 and AQP4,increase intestinal peristalsis and intestinal water,and effectively treat STC.
引文
[1]刘振华,蔡晓辉,张磊,等.慢性传输型便秘的病因及发病机制[J].医学综述,2007,13(22):1738-1740.
[2] Suares N C,Ford A C. Prevalence of and risk factors for chronic idiopathic constipation in the community:systematic review and Meta-analysis[J]. Am J Gastroenterol,2011,106(9):1582-1591.
[3] WANG S B,Amidi F,YIN S L,et al. Cyclic adenosine monophosphate regulation of aquaporin gene expression in human amnion epithelia[J]. Reprod Sci,2007,14(3):234-240.
[4] Carmosino M,Procino G,Tamma G,et al. Trafficking and phosphorylation dynamics of AQP4 in histaminetreated human gastric cells[J]. Biol Cell,2007,99(1):25-36.
[5] Bell C E,Lariviere N M,Watson P H,et al. Mitogenactivated protein kinase(MAPK)pathways mediate embryonic responses to culture medium osmolality by regulating Aquaporin 3 and 9 expression and localization,as well as embryonic apoptosis[J]. Hum Reprod,2009,24(6):1373-1386.
[6]周兵,王月容.健脾润肠通秘汤治疗中老年功能性便秘[J].中国实验方剂学杂志,2012,18(11):268-270.
[7]徐天舒,钱海华.“通便汤”治疗慢性传输型便秘30例临床观察[J].江苏中医药,2012,44(5):36-37.
[8]颜帅,曾莉,钱海华.养阴润肠汤治疗慢性传输型便秘54例[J].南京中医药大学学报,2013,29(4):335-337.
[9]张荣枝,杨光,钱海华.通便汤治疗便秘的临床对照观察[J].湖北中医药大学学报,2014,16(4):80-82.
[10]钱海华,徐天舒,曾莉,等.通便颗粒调节慢传输型便秘大鼠结肠水通道蛋白3,水通道蛋白8表达的研究[J].中国实验方剂学杂志,2014,20(24):180-184.
[11]施建新.医用实验动物学[M].西安:陕西科学技术出版社,1989:481-421.
[12]何俊堂,刘海峰,房殿春,等.慢传输型便秘大鼠结肠肌电生理变化及其意义[J].解放军医学杂志,2004,10(20):857-858.
[13]宋涛,刘跃亮,赵艳芳,等. c AMP-PKA-CREB信号通路在骨形态发生蛋白9诱导小鼠间充质干细胞成骨分化中的作用[J].中国生物制品学杂志,2014,27(2):189-196.
[14]张树恒,衣服新,倪伟民,等. U0126对实验性脑出血大鼠脑内水通道蛋白4表达的影响[J].辽宁医学院学报,2009,30(1):214-215.
[15] Silberstein C. Functional characterization and localization of AQP3 in the human colon[J]. Braz J Med Biol Res,1999,32(10):1303-1313.
[16] WANG K S, MA T, Filiz F, et al. Colon water transport in transgenic mice lacking aquaporin-4 water channels[J]. Am J Physiol Gastrointest Liver Physiol,2000,279(2):463-470.
[17]许惠娟,许慧慧,滕超,等.痛泻药方对腹泻型肠易激综合征模型大鼠结肠水通道蛋白8表达影响的机制研究[J].中国实验方剂学杂志,2012,18(6):141-144.
[18] Umenishi F, Schrier R W. Hypertonicity-induced aquaporin-1(AQP1)expression is mediated by the activation of MAPK pathways and hypertonicityresponsive element in the AQP1 gene[J]. J Biol Chem,2003,278(18):15765-15770.
[19]陈春玲,李涛平,朱丽华. MAPK信号阻断剂U0126对油酸致急性肺损伤大鼠肺泡Ⅱ型上皮细胞中AQP4表达的影响[J].南方医科大学学报,2009,29(8):1525-1528.
[2] Suares N C,Ford A C. Prevalence of and risk factors for chronic idiopathic constipation in the community:systematic review and Meta-analysis[J]. Am J Gastroenterol,2011,106(9):1582-1591.
[3] WANG S B,Amidi F,YIN S L,et al. Cyclic adenosine monophosphate regulation of aquaporin gene expression in human amnion epithelia[J]. Reprod Sci,2007,14(3):234-240.
[4] Carmosino M,Procino G,Tamma G,et al. Trafficking and phosphorylation dynamics of AQP4 in histaminetreated human gastric cells[J]. Biol Cell,2007,99(1):25-36.
[5] Bell C E,Lariviere N M,Watson P H,et al. Mitogenactivated protein kinase(MAPK)pathways mediate embryonic responses to culture medium osmolality by regulating Aquaporin 3 and 9 expression and localization,as well as embryonic apoptosis[J]. Hum Reprod,2009,24(6):1373-1386.
[6]周兵,王月容.健脾润肠通秘汤治疗中老年功能性便秘[J].中国实验方剂学杂志,2012,18(11):268-270.
[7]徐天舒,钱海华.“通便汤”治疗慢性传输型便秘30例临床观察[J].江苏中医药,2012,44(5):36-37.
[8]颜帅,曾莉,钱海华.养阴润肠汤治疗慢性传输型便秘54例[J].南京中医药大学学报,2013,29(4):335-337.
[9]张荣枝,杨光,钱海华.通便汤治疗便秘的临床对照观察[J].湖北中医药大学学报,2014,16(4):80-82.
[10]钱海华,徐天舒,曾莉,等.通便颗粒调节慢传输型便秘大鼠结肠水通道蛋白3,水通道蛋白8表达的研究[J].中国实验方剂学杂志,2014,20(24):180-184.
[11]施建新.医用实验动物学[M].西安:陕西科学技术出版社,1989:481-421.
[12]何俊堂,刘海峰,房殿春,等.慢传输型便秘大鼠结肠肌电生理变化及其意义[J].解放军医学杂志,2004,10(20):857-858.
[13]宋涛,刘跃亮,赵艳芳,等. c AMP-PKA-CREB信号通路在骨形态发生蛋白9诱导小鼠间充质干细胞成骨分化中的作用[J].中国生物制品学杂志,2014,27(2):189-196.
[14]张树恒,衣服新,倪伟民,等. U0126对实验性脑出血大鼠脑内水通道蛋白4表达的影响[J].辽宁医学院学报,2009,30(1):214-215.
[15] Silberstein C. Functional characterization and localization of AQP3 in the human colon[J]. Braz J Med Biol Res,1999,32(10):1303-1313.
[16] WANG K S, MA T, Filiz F, et al. Colon water transport in transgenic mice lacking aquaporin-4 water channels[J]. Am J Physiol Gastrointest Liver Physiol,2000,279(2):463-470.
[17]许惠娟,许慧慧,滕超,等.痛泻药方对腹泻型肠易激综合征模型大鼠结肠水通道蛋白8表达影响的机制研究[J].中国实验方剂学杂志,2012,18(6):141-144.
[18] Umenishi F, Schrier R W. Hypertonicity-induced aquaporin-1(AQP1)expression is mediated by the activation of MAPK pathways and hypertonicityresponsive element in the AQP1 gene[J]. J Biol Chem,2003,278(18):15765-15770.
[19]陈春玲,李涛平,朱丽华. MAPK信号阻断剂U0126对油酸致急性肺损伤大鼠肺泡Ⅱ型上皮细胞中AQP4表达的影响[J].南方医科大学学报,2009,29(8):1525-1528.