肠激安方对IBS-D大鼠肠道通透性的影响
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摘要
目的:观察肠激安方对腹泻型肠易激综合征(IBS-D)大鼠肠道通透性的影响,探讨肠激安方治疗IBS-D的作用机制。方法:SD雄性乳鼠,随机分为5组,分别为正常组,模型组,匹维溴铵组(0.018 g·kg~(-1)),肠激安高、低剂量组(33.48,16.74 g·kg~(-1)),除正常组外,均采用"母婴分离+醋酸刺激+束缚应激"三因素结合的方法建立IBS-D模型。给药后,采用透射电镜观察大鼠肠黏膜超微结构;酶联免疫吸附测定(ELISA)法检测大鼠血浆D-乳酸水平;蛋白免疫印迹法(Western blot)检测大鼠结肠紧密连接蛋白闭锁蛋白(Occludin),闭合蛋白-1(Claudin-1)的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测Occludin,Claudin-1,紧密连接蛋白-1(ZO-1)mRNA表达。结果:与正常组比较,IBS-D模型组大鼠肠黏膜上皮细胞损伤,微绒毛排列较稀疏,紧密连接间隙增宽,有的不甚明显;血浆D-乳酸水平明显升高(P<0.05),结肠中Occludin,Claudin-1 mRNA及蛋白表达降低(P<0.05),ZO-1 mRNA表达降低(P<0.05)。药物治疗后,与模型组比较,各给药组大鼠肠黏膜细胞损伤修复;匹维溴铵组、肠激安方高剂量组血浆D-乳酸水平明显降低(P<0.05),肠激安方低剂量组血浆D-乳酸水平有降低趋势,但不具有统计学差异。各给药组大鼠结肠中Occludin,Claudin-1 mRNA及蛋白表达水平,ZO-1 mRNA表达水平升高(P<0.05)。结论:肠激安方对IBS-D的治疗作用可能是改善肠道通透性实现的。
Objective:To observe the effect of Changji'an prescription on intestinal permeability in diarrhea-predominant irritable bowel syndrome(IBS-D)rats and explore its mechanism for treatment of IBS-D.Method:Male SD neonatal rats were randomly divided into five groups:normal group,model group,pinaverium bromide group(0.018 g·kg~(-1)),high-dose(33.48 g·kg~(-1))and low-dose(16.74 g·kg~(-1))Changji'an prescription groups.Except for the normal group,the IBS-D model was established by the combination of maternal and infant separation+acetic acid stimulation+restraint stress.After drug treatment,the ultrastructure of rat intestinal mucosa was observed by using transmission electron microscopy and the plasma D-lactate level was detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of tight junction proteins Occludin and Claudin-1 were detected by Western blot.The mRNA expression levels of Occludin,Claudin-1 and zonula occluden(ZO)-1 were detected by real time polymerase chain reaction(Real-time PCR).Result:As compared with the normal group,the intestinal mucosal epithelial cells were damaged in IBS-D model group,and the microvilli arrangement was sparse and tight junction was widened,and some were not obvious,and the plasma D-lactate level in IBS-D rats was increased significantly(P<0.05),and the mRNA and protein expression levels of Occludin and Claudin-1 in the colon were decreased and the mRNA expression of ZO-1 was also decreased(P<0.05).After drug treatment,as compared with the model group,all drug-administered groups can repair intestinal mucosal cell damage.Plasma D-lactate level in pinaverium bromide group and high-dose Changji'an prescription group was significantly decreased(P<0.05)while D-lactate level in the low-dose group Changji'an prescription group had a tendency to decrease with no statistical difference.The mRNA and protein expression levels of Occludin and Claudin-1 and the mRNA expression of ZO-1 in the colon of rats in each administration group were higher than those in the model group(P<0.05).Conclusion:The therapeutic effect of Changji'an prescription on IBS-D may be achieved by improving the intestinal permeability.
Objective:To observe the effect of Changji'an prescription on intestinal permeability in diarrhea-predominant irritable bowel syndrome(IBS-D)rats and explore its mechanism for treatment of IBS-D.Method:Male SD neonatal rats were randomly divided into five groups:normal group,model group,pinaverium bromide group(0.018 g·kg~(-1)),high-dose(33.48 g·kg~(-1))and low-dose(16.74 g·kg~(-1))Changji'an prescription groups.Except for the normal group,the IBS-D model was established by the combination of maternal and infant separation+acetic acid stimulation+restraint stress.After drug treatment,the ultrastructure of rat intestinal mucosa was observed by using transmission electron microscopy and the plasma D-lactate level was detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of tight junction proteins Occludin and Claudin-1 were detected by Western blot.The mRNA expression levels of Occludin,Claudin-1 and zonula occluden(ZO)-1 were detected by real time polymerase chain reaction(Real-time PCR).Result:As compared with the normal group,the intestinal mucosal epithelial cells were damaged in IBS-D model group,and the microvilli arrangement was sparse and tight junction was widened,and some were not obvious,and the plasma D-lactate level in IBS-D rats was increased significantly(P<0.05),and the mRNA and protein expression levels of Occludin and Claudin-1 in the colon were decreased and the mRNA expression of ZO-1 was also decreased(P<0.05).After drug treatment,as compared with the model group,all drug-administered groups can repair intestinal mucosal cell damage.Plasma D-lactate level in pinaverium bromide group and high-dose Changji'an prescription group was significantly decreased(P<0.05)while D-lactate level in the low-dose group Changji'an prescription group had a tendency to decrease with no statistical difference.The mRNA and protein expression levels of Occludin and Claudin-1 and the mRNA expression of ZO-1 in the colon of rats in each administration group were higher than those in the model group(P<0.05).Conclusion:The therapeutic effect of Changji'an prescription on IBS-D may be achieved by improving the intestinal permeability.
引文
[1]沈淑华,黄宣,吕宾,等.腹泻型肠易激综合征证型及证候要素的文献研究[J].中华中医药杂志,2013,28(5):1538-1540.
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[3]刘瑶,焦豪妍,刘伟.广藿香油对感染后肠易激综合征大鼠肠黏膜屏障的影响[J].中国实验方剂学杂志,2016,22(21):142-146.
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[9]张庆业,廖小红,范丽霞,等.肝郁脾虚泄泻大鼠模型的建立及评价[J].中国实验动物学报,2014,22(3):21-23.
[10]苏敏.肠安Ⅱ号方对三硝基苯磺酸(TNBS)诱导炎症后肠易激综合征(PI-IBS)大鼠肠黏膜免疫屏障的作用机制研究[D].北京:中国中医科学院,2014.
[11]Camilleri M,Gorman H. Intestinal permeability and irritable bowel syndrome[J]. Neurogastroenterol Motil,2007,19(7):545-552.
[12]HOU Q,HUANG Y,ZHU S,et al. MiR-144 increases intestinal permeability in IBS-D rats by targeting OCLN and ZO1[J]. Cell Physiol Biochem,2017,44(6):2256-2268.
[13]邓明,李国亮,童端,等.丙氨酰谷氨酰胺对重型颅脑损伤患者肠黏膜通透性和血浆二胺氧化酶的研究[J].检验医学与临床,2018,15(4):485-487,491.
[14]王玉玉,张军,郑鹏远,等.双歧杆菌、酪酸梭菌及谷氨酰胺对慢性应激模型小鼠肠黏膜屏障的影响[J].郑州大学学报:医学版,2014,49(2):153-157.
[15]XIAO L,RAO J N,CAO S,et al. Long noncoding RNA SPRY4-IT1 regulates intestinal epithelial barrier function by modulating the expression levels of tight junction proteins[J]. Mol Biol Cell,2016,27(4):617-626.
[16]F?rster C. Tight junctions and the modulation of barrier function in disease[J]. Histochem Cell Biol,2008,130(1):55-70.
[17]Turner J R. Molecular basis of epithelial barrier regulation:from basic mechanisms to clinical application[J]. Am J Pathol,2006,169(6):1901-1909.
[18]Pope J L,Bhat A A,Sharma A,et al. Claudin-1 regulates intestinal epithelial homeostasis through the modulation of Notch-signalling[J]. Gut,2014,63(4):622-634.
[19]Gonzalez J E,Di Geronimo R J,Arthur D E,et al.Remodeling of the tight junction during recover y from exposure to hydrogen peroxide in kidney epithelial cells[J]. Free Radic Biol Med,2009,47(11):1561-1569.
[20]孔武明,龚均,董蕾,等.肠易激综合征患者肠道屏障——紧密连接改变的示踪电镜观察[J].南方医科大学学报,2007,27(8):1167-1169,1172.
[21]YU Y B,ZHAO D Y,QI Q Q,et al. BDNF modulates intestinal barrier integrity through regulating the expression of tight junction proteins[J].Neurogastroenterol Motil,2016,29(3):1-11.
[22]孔武明,李光,龚均.紧密连接蛋白claudin-1在肠易激综合征患者肠黏膜中的表达及意义[J].山东医药,2008,27(8):27-29.
[2]彭珊,赵钢,李春艳.肠易激综合征的发病机制研究进展[J].中国医药导报,2016,13(6):54-57.
[3]刘瑶,焦豪妍,刘伟.广藿香油对感染后肠易激综合征大鼠肠黏膜屏障的影响[J].中国实验方剂学杂志,2016,22(21):142-146.
[4]Bertiaux-Vanda?le N,Youmba S B,Belmonte L,et al.The expression and the cellular distribution of the tight junction proteins are altered in irritable bowel syndrome patients with differences according to the disease subtype[J]. Am J Gastroenterol,2011,106(12):2165-2173.
[5]Piche T,Barbara G,Aubert P,et al. Impaired intestinal barrier integrity in the colon of patients with irritable bowel syndrome:involvement of soluble mediators[J].Gut,2009,58(2):196-201.
[6]ZHOU Q,ZHANG B,Verne G N. Intestinal membrane permeability and hypersensitivity in the irritable bowel syndrome[J]. Pain,2009,146(1):41-46.
[7]唐洪梅,黄樱华,李得堂,等.肠激安方对腹泻型肠易激综合征模型大鼠T淋巴细胞和5-羟色胺的影响[J].广州中医药大学学报,2009,26(2):164-168.
[8]柴玉娜,黄育生,唐洪梅,等.疏肝健脾法肠激安方对IBS-D大鼠免疫功能的影响[J].中国实验方剂学杂志,2016,22(21):93-98.
[9]张庆业,廖小红,范丽霞,等.肝郁脾虚泄泻大鼠模型的建立及评价[J].中国实验动物学报,2014,22(3):21-23.
[10]苏敏.肠安Ⅱ号方对三硝基苯磺酸(TNBS)诱导炎症后肠易激综合征(PI-IBS)大鼠肠黏膜免疫屏障的作用机制研究[D].北京:中国中医科学院,2014.
[11]Camilleri M,Gorman H. Intestinal permeability and irritable bowel syndrome[J]. Neurogastroenterol Motil,2007,19(7):545-552.
[12]HOU Q,HUANG Y,ZHU S,et al. MiR-144 increases intestinal permeability in IBS-D rats by targeting OCLN and ZO1[J]. Cell Physiol Biochem,2017,44(6):2256-2268.
[13]邓明,李国亮,童端,等.丙氨酰谷氨酰胺对重型颅脑损伤患者肠黏膜通透性和血浆二胺氧化酶的研究[J].检验医学与临床,2018,15(4):485-487,491.
[14]王玉玉,张军,郑鹏远,等.双歧杆菌、酪酸梭菌及谷氨酰胺对慢性应激模型小鼠肠黏膜屏障的影响[J].郑州大学学报:医学版,2014,49(2):153-157.
[15]XIAO L,RAO J N,CAO S,et al. Long noncoding RNA SPRY4-IT1 regulates intestinal epithelial barrier function by modulating the expression levels of tight junction proteins[J]. Mol Biol Cell,2016,27(4):617-626.
[16]F?rster C. Tight junctions and the modulation of barrier function in disease[J]. Histochem Cell Biol,2008,130(1):55-70.
[17]Turner J R. Molecular basis of epithelial barrier regulation:from basic mechanisms to clinical application[J]. Am J Pathol,2006,169(6):1901-1909.
[18]Pope J L,Bhat A A,Sharma A,et al. Claudin-1 regulates intestinal epithelial homeostasis through the modulation of Notch-signalling[J]. Gut,2014,63(4):622-634.
[19]Gonzalez J E,Di Geronimo R J,Arthur D E,et al.Remodeling of the tight junction during recover y from exposure to hydrogen peroxide in kidney epithelial cells[J]. Free Radic Biol Med,2009,47(11):1561-1569.
[20]孔武明,龚均,董蕾,等.肠易激综合征患者肠道屏障——紧密连接改变的示踪电镜观察[J].南方医科大学学报,2007,27(8):1167-1169,1172.
[21]YU Y B,ZHAO D Y,QI Q Q,et al. BDNF modulates intestinal barrier integrity through regulating the expression of tight junction proteins[J].Neurogastroenterol Motil,2016,29(3):1-11.
[22]孔武明,李光,龚均.紧密连接蛋白claudin-1在肠易激综合征患者肠黏膜中的表达及意义[J].山东医药,2008,27(8):27-29.