基于5-羟色胺信号系统的戊己丸不同配伍方改善结肠运动的机制研究
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摘要
1研究目的
肠易激综合征(Irritable Bowel Syndrome, IBS)是临床上常见的消化道疾病,发病率高,影响患者生活和工作。部分患者是在急性胃肠道感染后出现临床症状,被称为炎症后肠易激综合征(Post inflammation Irritable Bowel Syndrome, PI-IBS).发病机制复杂,尚未阐明,但脑-肠轴的调节功能起着非常重要的作用。5-羟色胺(5-hydroxytryptamine,5-HT)是一种广泛分布在中枢神经系统和胃肠道的脑肠肽,参与脑-肠轴功能的调节,在炎症后肠易激综合征的发病中起着至关重要的作用。戊己丸由黄连、制吴茱萸和土炒白芍组成,具有疏肝理脾,清热和胃的作用,临床上常用来治疗肠易激综合征。本实验以戊己丸的两个配伍方为治疗药物,采用乙酸灌肠致炎和束缚应激法建立炎症后肠易激综合征大鼠模型,从结肠运动功能和中枢边缘系统和结肠中5-HT信号系统功能的角度,进行整体药效和机制的实验研究及大鼠结肠平滑肌细胞的实验,探索戊己丸改善炎症后肠易激综合征结肠运动的药理作用及机制,并观察戊己丸两种配伍比例下在药效及作用机制中的异同,为中医药的配伍理论及临床应用提供依据。
2实验方法与结果
2.1PI-IBS大鼠模型的建立
方法：采用大鼠结肠内灌注40mL·L-1的乙酸1mL,致急性结肠炎症,恢复7天后分别施加束缚应激或寒冷制动应激造成大鼠PI-IBS模型。通过比较两种模型大鼠的结肠运动功能及血清、结肠、脊髓和脑中5-HT的含量,结肠肥大细胞数目和脱颗粒率,选出一种适合进一步实验需求的大鼠模型。
结果：两模型组中大鼠结肠运动明显加快,两组间无统计学差异；血清、结肠和脊髓中5-HT含量显著升高,而脑中则呈下降趋势,且乙酸加束缚应激组下降更为显著；肥大细胞数目虽有所增加,但无统计意义,肥大细胞脱颗粒率均显著升高,且以寒冷制动组更为显著。
结论：乙酸加束缚应激法建立的PI-IBS模型大鼠脑内5-HT水平下降更显著,此模型用于后续实验研究。
2.2戊己丸不同配伍方对PI-IBS结肠运动的药效学研究
方法：PI-IBS大鼠模型采用乙酸灌肠加束缚应激法建立,在应激后,各治疗组分别给予得舒特和相应剂量的戊己丸不同配伍方,模型组和正常对照组给予等体积的生理盐水,给药7天。分别检测建模前、应激后及治疗后的结肠运动功能,以结肠运动指数和运动指数变化率、2h内的排便粒数及玻璃小球排出时间作为观察指标。
结果：
(1)各组大鼠结肠运动指数及运动指数变化率的变化
应激后,各建模组大鼠结肠运动显著加快,差异非常显著(P<0.01)；戊己丸1号方和2号方可使模型大鼠结肠运动指数和结肠运动指数变化率均有所降低,中高剂量组差异显著(P<0.01)；两配伍方高剂量组结肠运动指数及2号方高剂量组运动指数变化率与PC组水平相当；两配伍方的3个剂量组之间亦有明显差异,但同剂量组之间并无差异。
(2)各组大鼠玻璃小球排出时间比较
建模前,各组大鼠玻璃小球排出时间无统计学差异；应激后,各建模组大鼠玻璃小球排出时间明显缩短,与正常组比较差异显著(P<0.01)；戊己丸1号方和2号方可使模型大鼠玻璃小球排出时间有不同程度的延长,1号方和2号方中、高剂量组差异显著(P<0.05,P<0.01)。两配伍方的高剂量组与PC组时间相当。两个配伍方的低、中剂量组与高剂量组有非常显著差异(P<0.01)；同剂量组间无差异。
(3)各组大鼠2h内粪粒数比较
建模前,各组大鼠2h内排出的粪粒数无统计学差异；应激后,各建模组大鼠的粪粒数较正常组显著增多(P<0.01)；戊己丸两配伍方均可不同程度的减少模型大鼠排出的粪粒数,两方的高剂量组差异显著(P<0.05,P<0.01)；中、高剂量组与PC组无差异；2号方低、高剂量组间有极显著差异(P<0.01)。
2.3戊己丸不同配伍方对PI-IBS结肠运动的作用机制研究
方法：治疗7天后,各组大鼠腹腔麻醉,腹主动脉取血,以HPLC-ECD检测血清中5-HT和5-HIAA的含量。同时取结肠及海马、下丘脑和额叶,以HPLC-ECD检测5-HT和5-HIAA的含量；并行5-HT免疫组化染色,并用Image Pro Plus分析软件对免疫反应阳性表达OD值,进行半定量分析。采用免疫荧光染色法,激光共聚焦显微镜检测结肠和海马中5-HT3,4受体的荧光强度；用ELISA试剂盒检测结肠和海马中腺苷酸环化酶、磷酸二酯酶4和钙离子浓度；应用Western Blotting检测结肠中cAMP反应元件结合蛋白磷酸化水平。数据均以均数±标准差(x±s)表示。
结果：
(1)戊己丸不同配伍方对大鼠血清中5-HT和5-HT转化率的影响
模型组大鼠血清中5-HT含量有所增高,但无统计差异；5-HIAA含量、5-HT转化率均显著降低(P<0.05,P<0.01)；1号方高剂量可显著降低血清5-HT含量(#P<0.05)；1号方中、高剂量可明显升高5-HIAA的含量(P<0.05,P<0.01)；除W2-L组外,戊己丸各组可使5-HT转化率有不同程度的提高,差异显著(P<0.05,P<0.01)；2号方高剂量组在升高5-HIAA含量及中、高剂量组提高5-HT转化率的作用优于同剂量1号方。
(2)戊己丸不同配伍方对大鼠结肠中5-HT和5-HT转化率的影响
模型大鼠结肠中5-HT和5-HIAA含量显著增高(P<0.01),而5-HT转化率有下降趋势,但无统计差异：戊己丸不同配伍方可使5-HT含量显著下降,中、高剂量可使5-HIAA的含量明显下降(P<0.05,P<0.01),药效与剂量成正比；5-HT转化率无统计学差异；两配伍方的高剂量降低5-HT含量的作用优于得舒特：两方之间无差异。
(3)戊己丸不同配伍方对大鼠结肠中5-HT表达的影响
M组大鼠结肠粘膜下神经丛和肌间神经丛5-HT阳性神经表达显著增高(P<0.01)；戊己丸能使其明显下降(P<0.01),作用效果与剂量成正比；两方同剂量组间无统计差异。
(4)戊己丸不同配伍方对PI-IBS大鼠结肠5-HT3R,5-HT4R的影响
模型大鼠结肠肌层5-HT3R和5-HT4R的荧光强度显著上调(P<0.01)；两个配伍方均可使其明显下调(P<0.01),且作用效果与剂量成正相关；1号方中、高剂量下调5-HT3R的作用及低剂量降低5-HT4R的效果优于2号方同剂量(P<0.05,P<0.01)。
(5)戊己丸不同配伍方对PI-IBS大鼠结肠Ca2+,AC,PDE4的影响
模型大鼠结肠Ca2+,AC的含量显著升高,PDE4则明显下降(P<0.01)；1号方和2号方均可显著下调Ca2+,AC,并上调PDE4(P<0.05,P<0.01)；2号方中、高剂量组对Ca2+, AC的下调作用与PC相当；但升高PDE4的作用优于得舒特(P<0.01)；2号方下调Ca2+, AC及低剂量上调PDE4的作用优于同剂量1号方。
(6)戊己丸不同配伍方对PI-IBS大鼠结肠CREB磷酸化的影响
各组CREB的表达无差异,但M组大鼠CREB蛋白磷酸化水平显著增高(P<0.01)；除1号方低剂量外,各剂量均可明显使其下调(P<0.05,P<0.01)；两方同剂量组间无差异。
(7)各组大鼠结肠肥大细胞数目和脱颗粒率的变化
M组大鼠结肠肥大细胞数目和脱颗粒率均明显升高(P<0.01)；戊己丸各剂量均可使其显著下降(P<0.01),作用效果与剂量正相关；两方的高剂量组肥大细胞数目及1号方高剂量组脱颗粒率与PC组相当；两方同剂量组间无差异。
(8)各组大鼠结肠黏膜嗜铬细胞数目的变化
M组中大鼠结肠黏膜嗜铬细胞数目较NC组显著增多(P<0.01)；戊己丸可使其明显下降(P<0.01),药效与剂量成正比；两方的高剂量下调作用优于得舒特(P<0.05)：两方同剂量组间无差异。
(9)各组大鼠海马中5-HT及5-HT转化率的变化
与NC组比较,模型组中大鼠海马中5-HT含量明显降低,5-HIAA含量和5-HT转化率显著升高(P<0.05,P<0.01)。除1号方低剂量外,戊己丸各剂量均可显著上调5-HT含量；两配伍方的高剂量可明显下调5-HIAA的含量；除2号方低剂量外均可明显降低5-HT转化率(P<0.05,P<0.01),作用效果与剂量成正比。两配伍方同剂量组间无差异。
(10)各组大鼠下丘脑中5-HT及5-HT转化率的变化
与NC组比较,模型组中大鼠下丘脑中5-HT和5-HIAA含量明显降低(P<0.05,P<0.01),5-HT转化率则显著升高(P<0.01)。除1号方低剂量外,戊己丸各剂量均可显著上调5-HT含量和下调5-HT转化率；两配伍方的中剂量可明显上调5-HIAA的含量(P<0.05,P<0.01)。1号方的高剂量升高5-HT含量的作用优于得舒特,两配伍方同剂量组间无差异。
(11)各组大鼠额叶中5-HT及5-HT转化率的变化
与NC组比较,模型组中大鼠额叶中5-HT含量较正常组明显降低(P<0.01),5-HIAA含量和5-HT转化率则明显升高(P<0.01)。戊己丸的中、高剂量可显著升高5-HT含量及降低5-HT转化率；各剂量均可明显下调5-HIAA的含量(P<0.05,P<0.01)。两配伍方同剂量组间无差异。
(12)下丘脑、额叶及海马中5-HT阳性表达
M组的大鼠海马、下丘脑中5-HT阳性表达量较NC组显著下降,而额叶中则明显升高(P<0.01)；戊己丸可明显上调海马、下丘脑中5-HT表达,除1号方低剂量外均可显著下调额叶中表达(P<0.05,P<0.01),药效与剂量正相关；两配伍方的中、高剂量组在海马中的作用,除1号方低剂量外在下丘脑中的上调作用及1号方的高剂量组和2号方中、高剂量在额叶中的下调作用均优于得舒特(P<0.05,P<0.01)；2号方的高剂量上调下丘脑中5-HT表达量及中、高剂量组降低额叶中5-HT表达量的作用优于同剂量1号方(P<0.05,P<0.01)。
(13)戊己丸不同配伍方对大鼠海马中5-HT3R,5-HT4R的影响
M组大鼠5-HT3R和5-HT4R的表达量较NC组均显著下降(P<0.01)；戊己丸可使其明显上调(P<0.01),作用效果与剂量成正比；1号方的高剂量、2号方中、高剂量上调5-HT3R和5-HT4R的作用优于得舒特(P<0.05,P<0.01)；2号方低、中剂量对5-HT3R及中、高剂量对5-HT4R的上调作用强于同剂量水平的1号方(P<0.05,P<0.01)。
(14)戊己丸不同配伍方对大鼠海马中AC, PDE4,Ca2+浓度的影响
与NC组比较,M组海马中钙离子浓度和PDE4含量明显升高,AC活性显著降低(P<0.01)；戊己丸可显著下调钙离子和PDE4及上调AC活性(P<0.05,P<0.01),作用效果与剂量正相关；1号方低、高剂量降低钙离子的作用优于同剂量2号方,1号方的高剂量对AC, PDE4的作用优于2号方的高剂量(P<0.05)。2.4戊己丸不同配伍方对大鼠结肠平滑肌细胞5-HT3,4受体及下游信号通路的影响
方法：采用Bitar的方法分离培养大鼠结肠平滑肌细胞,并用a-actin免疫组化法进行鉴定,应用第二代平滑肌细胞进行实验。应用免疫荧光法和钙离子探针检测戊己丸对5-HT3R、5-HT4R的激动剂和抑制剂引起的平滑肌细胞膜上3、4受体表达及胞内钙离子浓度的影响；应用ELISA试剂盒检测戊己丸对5-HT4R的激动剂和抑制剂、AC激动剂和抑制剂引起的cAMP、PKA的影响。
结果：
(1)1号方和2号方对正常大鼠结肠平滑肌细胞5-HT3,4受体的表达及Ca2+浓度均具有抑制作用,能显著拮抗苯基双胍和替加色罗引起的3、4受体荧光强度和胞内Ca2+升高,亦能明显促进阿洛司琼和GR113808引起的3、4受体表达和胞内Ca2+下降,2号方的作用效果优于1号方。
(2)戊己丸1号方和2号方可显著下调正常平滑肌细胞的cAMP和PKA水平,也可明显抑制替加色罗和Forskolin引起的cAMP和PKA升高,并可促进GR113808和SQ22536引起的cAMP和PKA降低,且2号方作用优于1号方。
3结论
戊己丸不同配伍方对炎症后肠易激综合征的结肠运动有明确的改善作用,其作用机制可能是通过脑-肠轴的双向调节作用,影响结肠和脑中5-HT、5-HT3R、5-HT4R、cAMP、PKA、Ca2+及结肠中CREB磷酸化水平、MC和EC的数量和功能状态而实现的。
Objective
Irritable Bowel Syndrome (IBS) is a clinical commonly encountered disease of digestive tract with high incidence and influences the life and work of patients. Part of patients got the symptoms of IBS after recovering from acute inflammation of gastrointestinal and it is called post inflammation irritable bowel syndrome (PI-IBS). The pathogenesis of PI-IBS is very complicated and still unclear, but the the regulation effects of brain-gut axis have a very important function im its mechanism.5-hydroxytryptamine (5-HT) is a kind of brain-gut petide which wildly distributed in central nerve system and gastrointestinal tract, participates in regulation of brain-gut axis and has fatal effects in the pathogenesis of PI-IBS. Wuji pill, which was constituted of huanglian, wuzhuyu and baishao, with the effects of Shuganlipi and Qingrehewei, was commonly used in treatment of IBS. In this study, Wuji pill with two different compatibilities were used as therapeutic drugs to deal with the rats of PI-IBS which was established by intracolonic instillation of acetic acid with restraint stress. The colon motility and the function of5-HT signal system in central fringe system and colon were observed to study the in vivo pharmacodynamics actin and mechanism of Wuji pill with two different compatibilities, then apply the ex vivo experiments with rat colon smooth muscle cells to explore the mechanism of Wuji pill with two different compatibilities in improving the colonic motility of PI-IBS, furthermore compared the similarities and differences of Wuji pill with two different compatibilities and provided foundations for compatibility theory of traditional chinese medicine and its clinical application.
Methods and Results
1Induction of PI-IBS rat model
Method:The PI-IBS rat models were established by by intracolonic instillation of1ml4%acetic acid to induce acute inflammation of colon, after7days post-enema, the rats were wrap-restraint or immobilized with cold. Compared the colonic motility, contents of5-HT (in serum, colon, spinal cord and brain), quantity of mast cells and its degranulation rate in colon, in order to choose a suitable model for further study.
Results:The colonic motility of the two models were significantly accelerated, but no differences between each other; the content of5-HT were remarkably increased in serum, colon and spinal cord but with a downtrend in brain, moreover the wrap-restraint group had notable difference with normal group; the quantity of mast cells was increased but had no differences and the degranulation rate in two model groups were much more higher than normal group, especially in the immobilized with cold group.
Conclusion:The PI-IBS rat model established by intracolonic instillation acetic acid with wrap-restraint has advantages in decreasing the content of5-HT in brain, so it was chose to further study.
2Study of pharmacodynamics of Wuji pill on the colonic motility of PI-IBS
Motheds:PI-IBS rat model was established by intracolonic instillation of acetic acid with wrap-restraint stress, rats in each group were given Dicetel and different dose of Wuji pill of two compatibilities respectively after stress, and the rats in model and normal control group were given saline of same volume for7days. The colonic motility index, the number of feces defecated in2hours and the discharge time of glass beads were measured before enema, after stress and after treated for7days to evaluate the function of colon movement.
Results:
There were no differences in motility index (MI) between groups before enema. After stress, the MI and the change rate of MI in model groups were significantly increased(P<0.01). The middle-dose and high-dose of two compatibilities can remarkably decrease the MI and the change rate of MI(P<0.01). The effects of high-dose Wuji pill on motility index and high-dose NO.2Wuji pill were similar with Dicetel. There were notable differences between different dose and no differences between the same doses of two compatibilities.
There were no differences in the discharge time of glass beads between groups before enema. After stress, the discharge time in model groups were obviously shorter than normal control group(P<0.01). The middle-dose and high-dose of two compatibilities can significantly prolong the discharge time(P<0.05, P<0.01). There were no differences between positive control group and high-dose Wuji pill groups. There were notable differences between different dose(P<0.01) and no differences between the same dose of two compatibilities.
There were no statistical differences in the number of feces defecated in2hours between groups before enema. After stress, the number of feces defecated was notably increased compared with normal control group(P<0.01). The high-dose of two compatibilities can remarkably decreased the feces defecated number(P<0.05, P<0.01). The effects of middle-dose and high-dose Wuji pill were similar with Dicetel in reducing the feces number. There was obviously difference between low-dose and high-dose group of NO.2Wuji pill(P<0.01).
3The mechanism study of Wuji pill on colonic motility of PI-IBS
Methods:After treated for7days, all the rats were anaesthetized and then collected the blood, colon, hippocampus, hypothalamus and frontal lobe. Apply high performance liquid chromatography with electron capture detector to test the content of5-HT and immunohistochemistry stain to measure the5-HT positive expression, the result were investigated by qualitatively and quantitatively by means of image pro plus software through analyzing the opacity density(OD). Immunofluorescence stain was used to evaluate the5-HT receptor3and4in colon and hippocampus and the results were analyzed by laser scanning confocal microscope. The adenyl cyclase, phosphodiesterase4and calcium ion concentration in colon and hippocampus were tested by ELISA and the phosphorylation level of c AMP-response element binding protein was investigated by western blotting. All the data was expressed as mean±standard deviation.
Results:
The content of5-HT in serum of model group was increased but no difference with normal control group, the content of5-HIAA and the percent conversion of5-HT were significantly decreased(P<0.05, P<0.01). High-dose of NO.1Wuji pill can remarkably decrease the content of5-HT, middle-dose and high-dose of NO.1Wuji pill can notably increase the content of5-HIAA, the two compatibilities all can obviously improve the percent conversion of5-HT except low-dose of NO.2Wuji pill (P<0.05, P<0.01). The effects of high-dose of NO.2Wuji pill on increasing5-HIAA, middle-dose and high-dose of NO.2Wuji pill on improving the percent conversion of5-HT were better than the same dose of NO.1Wuji pill.
The content of5-HT and5-HIAA in colon of model group were notably increased (P<0.01) and the percent conversion of5-HT was decreasing, but no difference was observed. Two compatibilities can remarkably decrease the content of5-HT, Middle-dose and High-dose can obviously decrease the5-HIAA(P<0.05, P<0.01) and no differences in percent conversion of5-HT. The two compatibilities had preponderance in High-dose compared with Dicetel, but no differences between the same doses of the two compatibilities.
The5-HT positive expression in colon submucous nerve plexus and syenteric nerve plexus of model group was notably increased (P<0.01), two compatibilities can decrease it remarkably(P<0.01) and the effect was directly related to dose, but no statistical differences were observed between the same dose of two compatibilities.
The fluorescence intensity of5-HT receptor3and4were remarkably increased in colon muscularis of model group (P<0.01), two compatibilities can decrease it notably(P<0.01) and the effect was directly related to dose. The effects of Middle-dose and High-dose of NO.1Wuji pill on reducing5-HT receptor3and Low-dose on reducing5-HT receptor4were better than NO.2Wuji pill (P<0.05, P <0.01).
The calcium ion and the activity of adenyl cyclase were increased significantly and the phosphodiesterase4was obviously decreased (P<0.01) in colon of model group, Wuji pill had predominant effects on down regulating Ca2+, AC and up regulating PDE4(P<0.05, P<0.01). NO.2Wuji pill of Middle-dose and High-dose had the similar effects with Dicetel on down regulating Ca2+and AC, better effect on up regulating PDE4than Dicetel (P<0.01). Compared with the same dose of NO.1Wuji pill, NO.2had better effects on down regulating Ca2+, AC and Low-dose of NO.2can up regulating PDE4remarkably.
There were no differences in expression of CREB in each group, but the phosphorylation level of CREB in model group were increased significantly (P<0.01). Wuji pill can obviously down regulate it (P<0.05,P<0.01) except Low-dose of NO.1Wuji pill, and there was no difference in the same dose of two compatibilities.
The quantity of mast cells and enterochromaffin cells, the degranulation rate of mast cell were increased obviously (P<0.01) in colon of model group. The down regulating effects of Wuji pill were remarkable (P<0.01) and directly related to dose. High-dose of Wuji pill had the similar effect on decreasing the quantity of mast cell and enterochromaffin cells while the high-dose of NO.1Wuji pill also had no difference with Dicetel on down regulating the degranulation rate of mast cell and there was no difference in the same dose of two compatibilities.
The content of5-HT was obviously decreased while the content of5-HIAA and the percent conversion of5-HT were increased significantly in hippocampus of model group (P<0.05, P<0.01). Each dose of Wuji pill can up regulate5-HT and the High-dose can down regulate5-HIAA notably while two compatibilities can decrease the percent conversion of5-HT significantly (P<0.05, P<0.01) except the Low-dose of NO.2Wuji pill, and the effects were directly related to dose. There was no difference in the same dose of two compatibilities.
The content of5-HT and5-HIAA was obviously decreased(P<0.05, P<0.01) while the percent conversion of5-HT were increased significantly in hypothalamus of model group (P<0.01). Each dose of Wuji pill can up regulate5-HT and decrease the percent conversion of5-HT significantly except the Low-dose of NO.1Wuji pill while the Middle-dose of two compatibilities can up regulate5-HIAA notably (P<0.05, P<0.01). The effect of High-dose of NO.1Wuji pill on increasing the content of5-HT was better than Dicetel, but there was no difference in the same dose of two compatibilities.
The content of5-HT was obviously decreased while the content of5-HIAA and the percent conversion of5-HT were increased significantly in frontal lobe of model group (P<0.01). Each dose of Wuji pill can down regulate5-HIAA while the Middle-dose and High-dose can up regulate5-HT notably and decrease the percent conversion of5-HT significantly (P<0.05, P<0.01). There was no difference in the same dose of two compatibilities.
The positive expression of5-HT were decreased in hippocampus and hypothalamus while it was increased obviously in frontal lobe (P<0.01)in model group. Wuji pill can significantly increase it in hippocampus and hypothalamus, and also can decrease it in frontal lobe (P<0.05, P<0.01) except Low-dose of NO.1Wuji pill, and the effects were directly related to dose. The up regulation effects of Middle&High-dose and the down regulation effects of High-dose of NO.1Wuji pill and Middle&High-dose of NO.2Wuji pill were better than Dicetel (P<0.05, P<0.01). The up regulation effects of High-dose of NO.2Wuji pill in hypothalamus and the down regulation effects of Middle&High-dose of NO.2Wuji pill were better than the same dose of NO.1Wuji pill (P<0.05, P<0.01).
The fluorescence intensity of5-HT receptor3and4were remarkably decreased in hippocampus of model group (P<0.01), two compatibilities can increase it notably(P<0.01) and the effect was directly related to dose. The effects of Middle&High-dose of NO.2and High-dose of NO.1Wuji pill on increasing5-HT receptor3&4were better than Dicetel (P<0.05, P<0.01). The effects of Low&Middle-dose of NO.2on increasing5-HT receptor3and Middle&High-dose of NO.2Wuji pill on increasing5-HT receptor4were better than the same dose of NO.1(P<0.05, P<0.01)
The calcium ion and the phosphodiesterase4were increased significantly and the activity of adenyl cyclase was obviously decreased (P<0.01) in hippocampus of model group. Wuji pill had predominant effects on down regulating Ca2+PDE4and up regulating AC (P<0.05, P<0.01), and the effects were directly related to dose. Low&High-dose of NO.1Wuji pill had better effects on reducing Ca2+and High-dose of NO.1Wuji pill had better regulations on AC and PDE4than the same dose of NO.2Wuji pill (P<0.05).
4Influence of Wuji pill on5-HT receptor3&4and downstream signaling pathway of rat colon smooth muscle cells
Methods:According to Bitar's methods, the rat colon smooth muscle cells (SMC) were isolated and cultured, and then identified by a-actin immunohistochemistry stain. Furthermore, tested the5-HT receptor3&4of the SMC membrane and Ca2+by immunofluorescence stain and calcium ion probe to evaluate the influence of Wuji pill on the change of5-HT receptor3&4caused by agonist and antagonist of5-HT receptor3&4. The cAMP and PKA level were measured by ELISA to estimate the effect of Wuji pill on the change of cAMP and PKA caused by agonist and antagonist of5-HT receptor4and AC.
Results:
Two compatibilities can decrease the expression of5-HT receptor3&4and the concentration of Ca2+of the SMC significantly, furthermore inhibit the elevation of5-HT receptor3&4and Ca2+caused by phenylbiguanide and Tegaserod dramatically, also obviously promote the reduction of5-HT receptor3&4and Ca2+caused by Alosetron and GR113808. The effect of NO.2Wuji pill was better than NO.1.
Two compatibilities can down regulate the level of cAMP and PKA of the SMC significantly, furthermore obviously inhibit the elevation of cAMP and PKA caused by Tegaserod and Forskolin, and also dramatically promote the reduction of cAMP and PKA caused by GRl13808and SQ22536. The effect of NO.2Wuji pill was better than NO.1.
Conclusion
The two compatibilities of Wuji pill had definite effects on colonic motility of post inflammation irritable bowel syndrome and its mechanism is probably via bi-directional regulation of brain-gut axis, then affecting the level of5-HT, the expression of5-HT receptor3&4, the content of cAMP, PKA, Ca2+in colon and brain as well as the phosphorylational level of CREB, the quantity and condition of MC and EC in colon.
肠易激综合征(Irritable Bowel Syndrome, IBS)是临床上常见的消化道疾病,发病率高,影响患者生活和工作。部分患者是在急性胃肠道感染后出现临床症状,被称为炎症后肠易激综合征(Post inflammation Irritable Bowel Syndrome, PI-IBS).发病机制复杂,尚未阐明,但脑-肠轴的调节功能起着非常重要的作用。5-羟色胺(5-hydroxytryptamine,5-HT)是一种广泛分布在中枢神经系统和胃肠道的脑肠肽,参与脑-肠轴功能的调节,在炎症后肠易激综合征的发病中起着至关重要的作用。戊己丸由黄连、制吴茱萸和土炒白芍组成,具有疏肝理脾,清热和胃的作用,临床上常用来治疗肠易激综合征。本实验以戊己丸的两个配伍方为治疗药物,采用乙酸灌肠致炎和束缚应激法建立炎症后肠易激综合征大鼠模型,从结肠运动功能和中枢边缘系统和结肠中5-HT信号系统功能的角度,进行整体药效和机制的实验研究及大鼠结肠平滑肌细胞的实验,探索戊己丸改善炎症后肠易激综合征结肠运动的药理作用及机制,并观察戊己丸两种配伍比例下在药效及作用机制中的异同,为中医药的配伍理论及临床应用提供依据。
2实验方法与结果
2.1PI-IBS大鼠模型的建立
方法：采用大鼠结肠内灌注40mL·L-1的乙酸1mL,致急性结肠炎症,恢复7天后分别施加束缚应激或寒冷制动应激造成大鼠PI-IBS模型。通过比较两种模型大鼠的结肠运动功能及血清、结肠、脊髓和脑中5-HT的含量,结肠肥大细胞数目和脱颗粒率,选出一种适合进一步实验需求的大鼠模型。
结果：两模型组中大鼠结肠运动明显加快,两组间无统计学差异；血清、结肠和脊髓中5-HT含量显著升高,而脑中则呈下降趋势,且乙酸加束缚应激组下降更为显著；肥大细胞数目虽有所增加,但无统计意义,肥大细胞脱颗粒率均显著升高,且以寒冷制动组更为显著。
结论：乙酸加束缚应激法建立的PI-IBS模型大鼠脑内5-HT水平下降更显著,此模型用于后续实验研究。
2.2戊己丸不同配伍方对PI-IBS结肠运动的药效学研究
方法：PI-IBS大鼠模型采用乙酸灌肠加束缚应激法建立,在应激后,各治疗组分别给予得舒特和相应剂量的戊己丸不同配伍方,模型组和正常对照组给予等体积的生理盐水,给药7天。分别检测建模前、应激后及治疗后的结肠运动功能,以结肠运动指数和运动指数变化率、2h内的排便粒数及玻璃小球排出时间作为观察指标。
结果：
(1)各组大鼠结肠运动指数及运动指数变化率的变化
应激后,各建模组大鼠结肠运动显著加快,差异非常显著(P<0.01)；戊己丸1号方和2号方可使模型大鼠结肠运动指数和结肠运动指数变化率均有所降低,中高剂量组差异显著(P<0.01)；两配伍方高剂量组结肠运动指数及2号方高剂量组运动指数变化率与PC组水平相当；两配伍方的3个剂量组之间亦有明显差异,但同剂量组之间并无差异。
(2)各组大鼠玻璃小球排出时间比较
建模前,各组大鼠玻璃小球排出时间无统计学差异；应激后,各建模组大鼠玻璃小球排出时间明显缩短,与正常组比较差异显著(P<0.01)；戊己丸1号方和2号方可使模型大鼠玻璃小球排出时间有不同程度的延长,1号方和2号方中、高剂量组差异显著(P<0.05,P<0.01)。两配伍方的高剂量组与PC组时间相当。两个配伍方的低、中剂量组与高剂量组有非常显著差异(P<0.01)；同剂量组间无差异。
(3)各组大鼠2h内粪粒数比较
建模前,各组大鼠2h内排出的粪粒数无统计学差异；应激后,各建模组大鼠的粪粒数较正常组显著增多(P<0.01)；戊己丸两配伍方均可不同程度的减少模型大鼠排出的粪粒数,两方的高剂量组差异显著(P<0.05,P<0.01)；中、高剂量组与PC组无差异；2号方低、高剂量组间有极显著差异(P<0.01)。
2.3戊己丸不同配伍方对PI-IBS结肠运动的作用机制研究
方法：治疗7天后,各组大鼠腹腔麻醉,腹主动脉取血,以HPLC-ECD检测血清中5-HT和5-HIAA的含量。同时取结肠及海马、下丘脑和额叶,以HPLC-ECD检测5-HT和5-HIAA的含量；并行5-HT免疫组化染色,并用Image Pro Plus分析软件对免疫反应阳性表达OD值,进行半定量分析。采用免疫荧光染色法,激光共聚焦显微镜检测结肠和海马中5-HT3,4受体的荧光强度；用ELISA试剂盒检测结肠和海马中腺苷酸环化酶、磷酸二酯酶4和钙离子浓度；应用Western Blotting检测结肠中cAMP反应元件结合蛋白磷酸化水平。数据均以均数±标准差(x±s)表示。
结果：
(1)戊己丸不同配伍方对大鼠血清中5-HT和5-HT转化率的影响
模型组大鼠血清中5-HT含量有所增高,但无统计差异；5-HIAA含量、5-HT转化率均显著降低(P<0.05,P<0.01)；1号方高剂量可显著降低血清5-HT含量(#P<0.05)；1号方中、高剂量可明显升高5-HIAA的含量(P<0.05,P<0.01)；除W2-L组外,戊己丸各组可使5-HT转化率有不同程度的提高,差异显著(P<0.05,P<0.01)；2号方高剂量组在升高5-HIAA含量及中、高剂量组提高5-HT转化率的作用优于同剂量1号方。
(2)戊己丸不同配伍方对大鼠结肠中5-HT和5-HT转化率的影响
模型大鼠结肠中5-HT和5-HIAA含量显著增高(P<0.01),而5-HT转化率有下降趋势,但无统计差异：戊己丸不同配伍方可使5-HT含量显著下降,中、高剂量可使5-HIAA的含量明显下降(P<0.05,P<0.01),药效与剂量成正比；5-HT转化率无统计学差异；两配伍方的高剂量降低5-HT含量的作用优于得舒特：两方之间无差异。
(3)戊己丸不同配伍方对大鼠结肠中5-HT表达的影响
M组大鼠结肠粘膜下神经丛和肌间神经丛5-HT阳性神经表达显著增高(P<0.01)；戊己丸能使其明显下降(P<0.01),作用效果与剂量成正比；两方同剂量组间无统计差异。
(4)戊己丸不同配伍方对PI-IBS大鼠结肠5-HT3R,5-HT4R的影响
模型大鼠结肠肌层5-HT3R和5-HT4R的荧光强度显著上调(P<0.01)；两个配伍方均可使其明显下调(P<0.01),且作用效果与剂量成正相关；1号方中、高剂量下调5-HT3R的作用及低剂量降低5-HT4R的效果优于2号方同剂量(P<0.05,P<0.01)。
(5)戊己丸不同配伍方对PI-IBS大鼠结肠Ca2+,AC,PDE4的影响
模型大鼠结肠Ca2+,AC的含量显著升高,PDE4则明显下降(P<0.01)；1号方和2号方均可显著下调Ca2+,AC,并上调PDE4(P<0.05,P<0.01)；2号方中、高剂量组对Ca2+, AC的下调作用与PC相当；但升高PDE4的作用优于得舒特(P<0.01)；2号方下调Ca2+, AC及低剂量上调PDE4的作用优于同剂量1号方。
(6)戊己丸不同配伍方对PI-IBS大鼠结肠CREB磷酸化的影响
各组CREB的表达无差异,但M组大鼠CREB蛋白磷酸化水平显著增高(P<0.01)；除1号方低剂量外,各剂量均可明显使其下调(P<0.05,P<0.01)；两方同剂量组间无差异。
(7)各组大鼠结肠肥大细胞数目和脱颗粒率的变化
M组大鼠结肠肥大细胞数目和脱颗粒率均明显升高(P<0.01)；戊己丸各剂量均可使其显著下降(P<0.01),作用效果与剂量正相关；两方的高剂量组肥大细胞数目及1号方高剂量组脱颗粒率与PC组相当；两方同剂量组间无差异。
(8)各组大鼠结肠黏膜嗜铬细胞数目的变化
M组中大鼠结肠黏膜嗜铬细胞数目较NC组显著增多(P<0.01)；戊己丸可使其明显下降(P<0.01),药效与剂量成正比；两方的高剂量下调作用优于得舒特(P<0.05)：两方同剂量组间无差异。
(9)各组大鼠海马中5-HT及5-HT转化率的变化
与NC组比较,模型组中大鼠海马中5-HT含量明显降低,5-HIAA含量和5-HT转化率显著升高(P<0.05,P<0.01)。除1号方低剂量外,戊己丸各剂量均可显著上调5-HT含量；两配伍方的高剂量可明显下调5-HIAA的含量；除2号方低剂量外均可明显降低5-HT转化率(P<0.05,P<0.01),作用效果与剂量成正比。两配伍方同剂量组间无差异。
(10)各组大鼠下丘脑中5-HT及5-HT转化率的变化
与NC组比较,模型组中大鼠下丘脑中5-HT和5-HIAA含量明显降低(P<0.05,P<0.01),5-HT转化率则显著升高(P<0.01)。除1号方低剂量外,戊己丸各剂量均可显著上调5-HT含量和下调5-HT转化率；两配伍方的中剂量可明显上调5-HIAA的含量(P<0.05,P<0.01)。1号方的高剂量升高5-HT含量的作用优于得舒特,两配伍方同剂量组间无差异。
(11)各组大鼠额叶中5-HT及5-HT转化率的变化
与NC组比较,模型组中大鼠额叶中5-HT含量较正常组明显降低(P<0.01),5-HIAA含量和5-HT转化率则明显升高(P<0.01)。戊己丸的中、高剂量可显著升高5-HT含量及降低5-HT转化率；各剂量均可明显下调5-HIAA的含量(P<0.05,P<0.01)。两配伍方同剂量组间无差异。
(12)下丘脑、额叶及海马中5-HT阳性表达
M组的大鼠海马、下丘脑中5-HT阳性表达量较NC组显著下降,而额叶中则明显升高(P<0.01)；戊己丸可明显上调海马、下丘脑中5-HT表达,除1号方低剂量外均可显著下调额叶中表达(P<0.05,P<0.01),药效与剂量正相关；两配伍方的中、高剂量组在海马中的作用,除1号方低剂量外在下丘脑中的上调作用及1号方的高剂量组和2号方中、高剂量在额叶中的下调作用均优于得舒特(P<0.05,P<0.01)；2号方的高剂量上调下丘脑中5-HT表达量及中、高剂量组降低额叶中5-HT表达量的作用优于同剂量1号方(P<0.05,P<0.01)。
(13)戊己丸不同配伍方对大鼠海马中5-HT3R,5-HT4R的影响
M组大鼠5-HT3R和5-HT4R的表达量较NC组均显著下降(P<0.01)；戊己丸可使其明显上调(P<0.01),作用效果与剂量成正比；1号方的高剂量、2号方中、高剂量上调5-HT3R和5-HT4R的作用优于得舒特(P<0.05,P<0.01)；2号方低、中剂量对5-HT3R及中、高剂量对5-HT4R的上调作用强于同剂量水平的1号方(P<0.05,P<0.01)。
(14)戊己丸不同配伍方对大鼠海马中AC, PDE4,Ca2+浓度的影响
与NC组比较,M组海马中钙离子浓度和PDE4含量明显升高,AC活性显著降低(P<0.01)；戊己丸可显著下调钙离子和PDE4及上调AC活性(P<0.05,P<0.01),作用效果与剂量正相关；1号方低、高剂量降低钙离子的作用优于同剂量2号方,1号方的高剂量对AC, PDE4的作用优于2号方的高剂量(P<0.05)。2.4戊己丸不同配伍方对大鼠结肠平滑肌细胞5-HT3,4受体及下游信号通路的影响
方法：采用Bitar的方法分离培养大鼠结肠平滑肌细胞,并用a-actin免疫组化法进行鉴定,应用第二代平滑肌细胞进行实验。应用免疫荧光法和钙离子探针检测戊己丸对5-HT3R、5-HT4R的激动剂和抑制剂引起的平滑肌细胞膜上3、4受体表达及胞内钙离子浓度的影响；应用ELISA试剂盒检测戊己丸对5-HT4R的激动剂和抑制剂、AC激动剂和抑制剂引起的cAMP、PKA的影响。
结果：
(1)1号方和2号方对正常大鼠结肠平滑肌细胞5-HT3,4受体的表达及Ca2+浓度均具有抑制作用,能显著拮抗苯基双胍和替加色罗引起的3、4受体荧光强度和胞内Ca2+升高,亦能明显促进阿洛司琼和GR113808引起的3、4受体表达和胞内Ca2+下降,2号方的作用效果优于1号方。
(2)戊己丸1号方和2号方可显著下调正常平滑肌细胞的cAMP和PKA水平,也可明显抑制替加色罗和Forskolin引起的cAMP和PKA升高,并可促进GR113808和SQ22536引起的cAMP和PKA降低,且2号方作用优于1号方。
3结论
戊己丸不同配伍方对炎症后肠易激综合征的结肠运动有明确的改善作用,其作用机制可能是通过脑-肠轴的双向调节作用,影响结肠和脑中5-HT、5-HT3R、5-HT4R、cAMP、PKA、Ca2+及结肠中CREB磷酸化水平、MC和EC的数量和功能状态而实现的。
Objective
Irritable Bowel Syndrome (IBS) is a clinical commonly encountered disease of digestive tract with high incidence and influences the life and work of patients. Part of patients got the symptoms of IBS after recovering from acute inflammation of gastrointestinal and it is called post inflammation irritable bowel syndrome (PI-IBS). The pathogenesis of PI-IBS is very complicated and still unclear, but the the regulation effects of brain-gut axis have a very important function im its mechanism.5-hydroxytryptamine (5-HT) is a kind of brain-gut petide which wildly distributed in central nerve system and gastrointestinal tract, participates in regulation of brain-gut axis and has fatal effects in the pathogenesis of PI-IBS. Wuji pill, which was constituted of huanglian, wuzhuyu and baishao, with the effects of Shuganlipi and Qingrehewei, was commonly used in treatment of IBS. In this study, Wuji pill with two different compatibilities were used as therapeutic drugs to deal with the rats of PI-IBS which was established by intracolonic instillation of acetic acid with restraint stress. The colon motility and the function of5-HT signal system in central fringe system and colon were observed to study the in vivo pharmacodynamics actin and mechanism of Wuji pill with two different compatibilities, then apply the ex vivo experiments with rat colon smooth muscle cells to explore the mechanism of Wuji pill with two different compatibilities in improving the colonic motility of PI-IBS, furthermore compared the similarities and differences of Wuji pill with two different compatibilities and provided foundations for compatibility theory of traditional chinese medicine and its clinical application.
Methods and Results
1Induction of PI-IBS rat model
Method:The PI-IBS rat models were established by by intracolonic instillation of1ml4%acetic acid to induce acute inflammation of colon, after7days post-enema, the rats were wrap-restraint or immobilized with cold. Compared the colonic motility, contents of5-HT (in serum, colon, spinal cord and brain), quantity of mast cells and its degranulation rate in colon, in order to choose a suitable model for further study.
Results:The colonic motility of the two models were significantly accelerated, but no differences between each other; the content of5-HT were remarkably increased in serum, colon and spinal cord but with a downtrend in brain, moreover the wrap-restraint group had notable difference with normal group; the quantity of mast cells was increased but had no differences and the degranulation rate in two model groups were much more higher than normal group, especially in the immobilized with cold group.
Conclusion:The PI-IBS rat model established by intracolonic instillation acetic acid with wrap-restraint has advantages in decreasing the content of5-HT in brain, so it was chose to further study.
2Study of pharmacodynamics of Wuji pill on the colonic motility of PI-IBS
Motheds:PI-IBS rat model was established by intracolonic instillation of acetic acid with wrap-restraint stress, rats in each group were given Dicetel and different dose of Wuji pill of two compatibilities respectively after stress, and the rats in model and normal control group were given saline of same volume for7days. The colonic motility index, the number of feces defecated in2hours and the discharge time of glass beads were measured before enema, after stress and after treated for7days to evaluate the function of colon movement.
Results:
There were no differences in motility index (MI) between groups before enema. After stress, the MI and the change rate of MI in model groups were significantly increased(P<0.01). The middle-dose and high-dose of two compatibilities can remarkably decrease the MI and the change rate of MI(P<0.01). The effects of high-dose Wuji pill on motility index and high-dose NO.2Wuji pill were similar with Dicetel. There were notable differences between different dose and no differences between the same doses of two compatibilities.
There were no differences in the discharge time of glass beads between groups before enema. After stress, the discharge time in model groups were obviously shorter than normal control group(P<0.01). The middle-dose and high-dose of two compatibilities can significantly prolong the discharge time(P<0.05, P<0.01). There were no differences between positive control group and high-dose Wuji pill groups. There were notable differences between different dose(P<0.01) and no differences between the same dose of two compatibilities.
There were no statistical differences in the number of feces defecated in2hours between groups before enema. After stress, the number of feces defecated was notably increased compared with normal control group(P<0.01). The high-dose of two compatibilities can remarkably decreased the feces defecated number(P<0.05, P<0.01). The effects of middle-dose and high-dose Wuji pill were similar with Dicetel in reducing the feces number. There was obviously difference between low-dose and high-dose group of NO.2Wuji pill(P<0.01).
3The mechanism study of Wuji pill on colonic motility of PI-IBS
Methods:After treated for7days, all the rats were anaesthetized and then collected the blood, colon, hippocampus, hypothalamus and frontal lobe. Apply high performance liquid chromatography with electron capture detector to test the content of5-HT and immunohistochemistry stain to measure the5-HT positive expression, the result were investigated by qualitatively and quantitatively by means of image pro plus software through analyzing the opacity density(OD). Immunofluorescence stain was used to evaluate the5-HT receptor3and4in colon and hippocampus and the results were analyzed by laser scanning confocal microscope. The adenyl cyclase, phosphodiesterase4and calcium ion concentration in colon and hippocampus were tested by ELISA and the phosphorylation level of c AMP-response element binding protein was investigated by western blotting. All the data was expressed as mean±standard deviation.
Results:
The content of5-HT in serum of model group was increased but no difference with normal control group, the content of5-HIAA and the percent conversion of5-HT were significantly decreased(P<0.05, P<0.01). High-dose of NO.1Wuji pill can remarkably decrease the content of5-HT, middle-dose and high-dose of NO.1Wuji pill can notably increase the content of5-HIAA, the two compatibilities all can obviously improve the percent conversion of5-HT except low-dose of NO.2Wuji pill (P<0.05, P<0.01). The effects of high-dose of NO.2Wuji pill on increasing5-HIAA, middle-dose and high-dose of NO.2Wuji pill on improving the percent conversion of5-HT were better than the same dose of NO.1Wuji pill.
The content of5-HT and5-HIAA in colon of model group were notably increased (P<0.01) and the percent conversion of5-HT was decreasing, but no difference was observed. Two compatibilities can remarkably decrease the content of5-HT, Middle-dose and High-dose can obviously decrease the5-HIAA(P<0.05, P<0.01) and no differences in percent conversion of5-HT. The two compatibilities had preponderance in High-dose compared with Dicetel, but no differences between the same doses of the two compatibilities.
The5-HT positive expression in colon submucous nerve plexus and syenteric nerve plexus of model group was notably increased (P<0.01), two compatibilities can decrease it remarkably(P<0.01) and the effect was directly related to dose, but no statistical differences were observed between the same dose of two compatibilities.
The fluorescence intensity of5-HT receptor3and4were remarkably increased in colon muscularis of model group (P<0.01), two compatibilities can decrease it notably(P<0.01) and the effect was directly related to dose. The effects of Middle-dose and High-dose of NO.1Wuji pill on reducing5-HT receptor3and Low-dose on reducing5-HT receptor4were better than NO.2Wuji pill (P<0.05, P <0.01).
The calcium ion and the activity of adenyl cyclase were increased significantly and the phosphodiesterase4was obviously decreased (P<0.01) in colon of model group, Wuji pill had predominant effects on down regulating Ca2+, AC and up regulating PDE4(P<0.05, P<0.01). NO.2Wuji pill of Middle-dose and High-dose had the similar effects with Dicetel on down regulating Ca2+and AC, better effect on up regulating PDE4than Dicetel (P<0.01). Compared with the same dose of NO.1Wuji pill, NO.2had better effects on down regulating Ca2+, AC and Low-dose of NO.2can up regulating PDE4remarkably.
There were no differences in expression of CREB in each group, but the phosphorylation level of CREB in model group were increased significantly (P<0.01). Wuji pill can obviously down regulate it (P<0.05,P<0.01) except Low-dose of NO.1Wuji pill, and there was no difference in the same dose of two compatibilities.
The quantity of mast cells and enterochromaffin cells, the degranulation rate of mast cell were increased obviously (P<0.01) in colon of model group. The down regulating effects of Wuji pill were remarkable (P<0.01) and directly related to dose. High-dose of Wuji pill had the similar effect on decreasing the quantity of mast cell and enterochromaffin cells while the high-dose of NO.1Wuji pill also had no difference with Dicetel on down regulating the degranulation rate of mast cell and there was no difference in the same dose of two compatibilities.
The content of5-HT was obviously decreased while the content of5-HIAA and the percent conversion of5-HT were increased significantly in hippocampus of model group (P<0.05, P<0.01). Each dose of Wuji pill can up regulate5-HT and the High-dose can down regulate5-HIAA notably while two compatibilities can decrease the percent conversion of5-HT significantly (P<0.05, P<0.01) except the Low-dose of NO.2Wuji pill, and the effects were directly related to dose. There was no difference in the same dose of two compatibilities.
The content of5-HT and5-HIAA was obviously decreased(P<0.05, P<0.01) while the percent conversion of5-HT were increased significantly in hypothalamus of model group (P<0.01). Each dose of Wuji pill can up regulate5-HT and decrease the percent conversion of5-HT significantly except the Low-dose of NO.1Wuji pill while the Middle-dose of two compatibilities can up regulate5-HIAA notably (P<0.05, P<0.01). The effect of High-dose of NO.1Wuji pill on increasing the content of5-HT was better than Dicetel, but there was no difference in the same dose of two compatibilities.
The content of5-HT was obviously decreased while the content of5-HIAA and the percent conversion of5-HT were increased significantly in frontal lobe of model group (P<0.01). Each dose of Wuji pill can down regulate5-HIAA while the Middle-dose and High-dose can up regulate5-HT notably and decrease the percent conversion of5-HT significantly (P<0.05, P<0.01). There was no difference in the same dose of two compatibilities.
The positive expression of5-HT were decreased in hippocampus and hypothalamus while it was increased obviously in frontal lobe (P<0.01)in model group. Wuji pill can significantly increase it in hippocampus and hypothalamus, and also can decrease it in frontal lobe (P<0.05, P<0.01) except Low-dose of NO.1Wuji pill, and the effects were directly related to dose. The up regulation effects of Middle&High-dose and the down regulation effects of High-dose of NO.1Wuji pill and Middle&High-dose of NO.2Wuji pill were better than Dicetel (P<0.05, P<0.01). The up regulation effects of High-dose of NO.2Wuji pill in hypothalamus and the down regulation effects of Middle&High-dose of NO.2Wuji pill were better than the same dose of NO.1Wuji pill (P<0.05, P<0.01).
The fluorescence intensity of5-HT receptor3and4were remarkably decreased in hippocampus of model group (P<0.01), two compatibilities can increase it notably(P<0.01) and the effect was directly related to dose. The effects of Middle&High-dose of NO.2and High-dose of NO.1Wuji pill on increasing5-HT receptor3&4were better than Dicetel (P<0.05, P<0.01). The effects of Low&Middle-dose of NO.2on increasing5-HT receptor3and Middle&High-dose of NO.2Wuji pill on increasing5-HT receptor4were better than the same dose of NO.1(P<0.05, P<0.01)
The calcium ion and the phosphodiesterase4were increased significantly and the activity of adenyl cyclase was obviously decreased (P<0.01) in hippocampus of model group. Wuji pill had predominant effects on down regulating Ca2+PDE4and up regulating AC (P<0.05, P<0.01), and the effects were directly related to dose. Low&High-dose of NO.1Wuji pill had better effects on reducing Ca2+and High-dose of NO.1Wuji pill had better regulations on AC and PDE4than the same dose of NO.2Wuji pill (P<0.05).
4Influence of Wuji pill on5-HT receptor3&4and downstream signaling pathway of rat colon smooth muscle cells
Methods:According to Bitar's methods, the rat colon smooth muscle cells (SMC) were isolated and cultured, and then identified by a-actin immunohistochemistry stain. Furthermore, tested the5-HT receptor3&4of the SMC membrane and Ca2+by immunofluorescence stain and calcium ion probe to evaluate the influence of Wuji pill on the change of5-HT receptor3&4caused by agonist and antagonist of5-HT receptor3&4. The cAMP and PKA level were measured by ELISA to estimate the effect of Wuji pill on the change of cAMP and PKA caused by agonist and antagonist of5-HT receptor4and AC.
Results:
Two compatibilities can decrease the expression of5-HT receptor3&4and the concentration of Ca2+of the SMC significantly, furthermore inhibit the elevation of5-HT receptor3&4and Ca2+caused by phenylbiguanide and Tegaserod dramatically, also obviously promote the reduction of5-HT receptor3&4and Ca2+caused by Alosetron and GR113808. The effect of NO.2Wuji pill was better than NO.1.
Two compatibilities can down regulate the level of cAMP and PKA of the SMC significantly, furthermore obviously inhibit the elevation of cAMP and PKA caused by Tegaserod and Forskolin, and also dramatically promote the reduction of cAMP and PKA caused by GRl13808and SQ22536. The effect of NO.2Wuji pill was better than NO.1.
Conclusion
The two compatibilities of Wuji pill had definite effects on colonic motility of post inflammation irritable bowel syndrome and its mechanism is probably via bi-directional regulation of brain-gut axis, then affecting the level of5-HT, the expression of5-HT receptor3&4, the content of cAMP, PKA, Ca2+in colon and brain as well as the phosphorylational level of CREB, the quantity and condition of MC and EC in colon.
引文
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