加味四君子汤对脾虚型IBS-D的治疗作用及机理的研究
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摘要
背景与目的：
随着脾虚证的中西医结合研究进一步发展,从病证结合的角度进行脾虚证的研究成为新的方向和趋势。自上世纪八十年代以来,脾虚证的现代研究取得了丰硕的成果。大量的临床实践证明,与脾虚证相关的诸多疾病都与机体能量的代谢水平下降密切相关。ATP酶是广泛存在于细胞膜上的一种磷酸酶,能把ATP水解成ADP而释放出磷酸基,提供机体代谢所需的能量,因而ATP酶可以在一定程度上反映机体的能量代谢水平。
肠易激综合征(IBS)是由多种病因引起的以结肠动力学紊乱及内脏敏感性增高为基本病理生理表现的功能性疾病。多项研究显示,IBS患者存在5-HT分泌或分布的异常,5-HT参与了IBS的主要病理生理过程。5-HT通过与不同器官上的膜受体结合而发挥作用,其中胃肠道中主要存在5-HT4两种类型。5-HT通过与这两种受体结合影响和调节IBS患者的胃肠运动、分泌、吸收及感觉等复杂功能,故5-HT及其受体与IBS患者腹痛、腹泻和内脏感觉异常等症状密切相关。
中医药对IBS具有较好的疗效。有研究显示,中医药治疗优势在于有效缓解腹痛、腹泻及便秘等主要临床症状,改善中医临床证候,提高患者生存质量,具有较好的成本效果比。但其诊治IBS病理生理学机制尚不明确。我们推测中医药的这种作用可能通过影响5-HT的浓度、5-HT受体的表达等来实现。因此本课题拟从脾虚型IBS-D患者肠粘膜5-HT及其受体及四君子汤的干预作用入手,来探讨脾虚型IBS-D的发生机理及其四君子汤的干预机制,课题的实施对指导脾虚型IBS-D的临床用药及开发治疗脾虚型IBS-D的有效中药制剂,对进一步充实中医脾胃病辨证治疗体系有重要意义。
方法：
在广州中医药大学第一附属医院收集脾虚型IBS-D病例,按照随机、对照的原则,将符合肠易激综合征罗马Ⅲ诊断标准和脾虚型IBS-D中医诊断标准者的IBS-D受试对象,按照随机数字表法分为阳性对照组(对照组)、四君子汤治疗组(治疗组)。对照组给予马来酸曲美布汀口服,每次0.1g,每日2次,连续治疗一个月。治疗组给予加味四君子汤(由党参20g、茯苓15g、白术15g、甘草6g、黄芪20g)口服,每日一剂,水煎两次分服,连续治疗一个月。另外征集健康自愿者设立健康对照组(正常组),常规饮食,不服用药物。
参照肠易激综合征罗马Ⅲ诊断标准和中国中西医结合学会消化系统疾病专业委员会的《肠易激综合征中西医结合诊治方案(2003版)》制定脾虚型IBS-D患者临床疗效评价标准,所有受试者治疗前后由专业人员指导认真填写临床评估表。经过加味四君子汤或阳性对照组药物治疗一个月后,对受试者进行临床疗效评价。
对脾虚型IBS-D和健康自愿者经电子结肠镜检查,取回盲部及距肛门约25cm处乙状结肠粘膜若干块,约重10-20mg,所有活检标本均立即投入液氮中,l周后同时转入-80℃冰箱中保存。
本次试验最终收集合格病例共39例,其中治疗组20例,对照组19例。治疗组完成疗程后再次结肠镜取标本的病例共11例,对照组完成疗程后再次结肠镜取标本的病例9例,健康对照组收集到结肠粘膜标本共10例。进行指标检测如下：
(1)高效液相色谱法检测正常组、治疗组和对照组治疗前后的受试者肠粘膜5-HT的含量。
(2) RT-PCR法检测正常组、治疗组和对照组治疗前后的受试者肠粘膜5-HL3、5-HT4受体基因表达水平。
(3)无机磷法测定正常组、治疗组和对照组治疗前后的受试者肠粘膜Na+-K+-ATP酶,Ca2+-Mg2+-ATP酶活性。
(4) Fluo-3/Am荧光探针和FCM法检测正常组、治疗组、对照组治疗前后的受试者肠粘膜细胞内钙离子浓度。
采用SPSS统计软件,通过t检验或X2检验对试验结果进行显著性检验。检验结果在P<0.05时认为差异有统计学意义。
结果：
通过对治疗组和对照组在性别、年龄、病程分布等方面进行X2检验等统计学分析,两组在一般情况方面没有统计学差异,具有可比性。
(1)加味四君子汤治疗脾虚型IBS-D总有效率达85%,对照组总有效率为73.68%,治疗组的总有效率高于对照组。两组患者疗效经Wilcoxon秩和检验表明,治疗组与对照组疗效无显著性差异(P=0.5393>0.05)。
(2)两组患者治疗前各症状积分比较,差异无显著性意义(P>0.05)；治疗组治疗后各症状积分均较治疗前有显著下降(P<0.05)；对照组治疗后除腹痛、腹部不适、排便次数增加、大便不成形等症状积分较治疗前有显著下降(P<0.05)外,其他各症状积分治疗前后比较差异无显著性意义(P>0.05)；治疗后两组间比较,差异均有显著性意义(P<0.05),提示治疗组在改善症状积分方面优于对照组。
(3)治疗组及对照组治疗前回盲部结肠粘膜、乙状结肠粘膜5-羟色胺含量均比正常组明显增高(P<0.01),说明5-羟色胺增高与脾虚型IBS-D有一定的联系。经加味四君子汤及对照组药物治疗后,脾虚型IBS-D患者回盲部结肠粘膜、乙状结肠粘膜5-羟色胺含量较治疗前明显降低(P<0.05)。治疗后治疗组与对照组回盲部结肠粘膜、乙状结肠粘膜5-羟色胺含量无显著性差异(P>0.05)。
(4)治疗前治疗组及对照组的5-HT3R和5-HT4R受体mRNA在结肠粘膜的表达显著升高(P<0.01)；治疗后两组的5-HT3R和5-HT4R受体mRNA在结肠粘膜的表达显著降低(P<0.01或P<0.05),并与正常组相比无显著性差异(P>0.05)。
(5)脾虚型IBS-D患者肠粘膜Na+-K+-ATP酶(?)(?)Ca2+-Mg2+-ATP酶与正常组相比均有显著降低(P<0.01)；经加味四君子汤治疗后,患者肠粘膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶均有显著提高(P<0.01)；而经对照组药物治疗后,患者肠粘膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶均与治疗前无显著差异(P>0.05)。
(6)脾虚型IBS-D患者肠粘膜细胞内游离钙离子荧光强度值与正常组相比显著升高(P<0.01)；经加味四君子汤治疗和对照组药物治疗后,患者肠粘膜内游离钙离子荧光强度值均有显著降低(P<0.01)。
结论：
(1)加味四君子汤治疗脾虚型IBS-D的总有效率高于对照组,而且在改善脾虚型IBS-D患者的神疲乏力、面色萎黄、食少纳差、食后腹胀等主要症状上显著优于对照组,且没有明显不良反应。说明四君子汤为主方的中药汤剂能有效改善脾虚型腹泻型IBS患者的临床症状,是对脾虚型IBS-D临床疗效肯定和安全的中药方剂。
(2)脾虚型IBS-D患者存在5-HT分泌及5-HT受体mRNA表达水平的异常。脾虚型IBS-D时5-羟色胺增多,5-HT3R和5-HT4R受体mRNA在结肠粘膜的表达显著升高；脾虚型IBS-D患者的主要症状,如腹痛、腹胀、便溏、大便不尽感等与结肠粘膜5-HT3R、5-HT4R mRNA的表达呈明显正相关,随着加味四君子汤和马来酸曲美布汀的应用,脾虚型IBS-D的主要症状得以改善,也同时伴有结肠粘膜5-HT含量及5-HT3和5-HT4受体mRNA的表达水平明显下降,表明加味四君子汤和马来酸曲美布汀可能通过作用于胃肠道5-羟色胺及其受体而改善患者的主要临床症状。
(3)脾虚型IBS-D患者肠粘膜Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶与正常组相比均有显著降低,经加味四君子汤治疗后,患者肠粘膜Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶均有显著提高。而经对照组药物治疗后,患者均与治疗前无显著差异。表明加味四君子汤可能通过作用于肠粘膜Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶而改善患者的神疲乏力、面色萎黄、食少纳差、食后腹胀等症状。
(4)脾虚型IBS-D患者肠粘膜细胞内游离钙离子荧光强度值与正常组相比显著升高；经加味四君子汤治疗和对照组药物治疗后,患者肠粘膜内游离钙离子荧光强度值均有显著提高。表明加味四君子汤可能与马来酸曲美布汀有相似的功效,即通过作用于细胞膜钙离子通道而维持患者肠粘膜内钙离子浓度处于正常水平。
(5)加味四君子汤对脾虚型IBS-D的治疗作用是多靶点的,除了与马来酸曲美布汀对IB-D的作用机制即通过调节Ca"+通道而使细胞内钙能恢复到较低水平,还可通过改善ATP酶活性,通过降低5-HT的释放和5-HT受体的表达来达到治疗的目的。
(6)脾虚型IBS-D发生的可能机制是：脾虚型IBS-D患者机体的能量生成和转化水平降低,影响到机体ATP酶的活性。当ATP酶活性下降时,可导致大量Ca2+进入肠道细胞内,使钙离子浓度增加,从而激活肠道肠嗜铬细胞,使其进一步释放5-HT等胃肠激素,引起IBS患者出现肠道运动增加、对液体的吸收减少和内脏敏感性增高等异常表现。
Background and objective:
Along with further development of integrated Chinese and Western medicine research on Spleen-deficiency syndrome, studies on Spleen-deficiency syndrome from the perspective of combination of disease and syndrome has become a new direction and trend. Since1980s, modern researches on Spleen-deficiency syndrome has got great achievements. A large number of clinical practices proved that many diseases involved in Spleen-deficiency syndrome are closely associated with declined energy metabolism level. ATP enzymes, a sort of phosphatase, are widely present in cell membrane, which can promote the reaction of ATP being hydrolized into ADP and releasing phosphate, and provide required metabolic energy. So ATP enzymes, to some extent, can represent energy metabolism level.
Irritable bowel syndrome (IBS), caused by various etiological factors, is a functional disease which manifested by colonic motility disorders and increased visceral sensitivity, etc. Many studies have shown that IBS patients had abnormal5-HT secretion or distribution.5-HT participated in the major pathophysiological processes of IBS, and5-HT played different roles by combining with different membrane receptors. In the gastrointestinal tract, there are mainly two kinds of receptors:5-HT3and5-HT4. Combined with the two kinds of receptors,5-HT affected and regulated gastrointestinal motility, secretion, absorption and others in IBS patients, thus5-HT had a close relationship with IBS patients' symptoms such as abdominal pain, diarrhea and visceral sensory abnormalities.
Traditional Chinese medicine (TCM) has a better therapeutic effect on IBS. Studies have shown that TCM had advantages of effectively relieving abdominal pain, diarrhea and constipation and other major clinical symptoms, improving clinical syndromes of TCM, promoting quality of patients'life, and having better cost effectiveness ratio. However, pathophysiology mechanism of TCM's diagnosis and treatment on IBS is still not clear. We hypothesized that TCM's role might be achieved by modulating concentration of5-HT and mRNA expression of5-HT receptors. Therefore, this article planed to study5-HT and its receptors in intestinal mucosal of IBS-D patients with Spleen-deficiency syndrome and Sijunzi Decoction's intervention on them, so as to explore the mechanism of Spleen-deficiency type IBS-D and Sijunzi Decoction's intervention mechanism. This study were beneficial to promote clinical application of the medicine for Spleen-deficiency type IBS-D, to develop effective Chinese herbal medicine of treating Spleen-deficiency type IBS-D, and to further enrich Chinese medicine syndrome differentiation and treatment system on Spleen and Stomach diseases.
Methods:
According to the principles of randomized and controlled method, Spleen-deficiency type IBS-D cases from the first hospital of Guangzhou University of Chinese Medicine were collected. The patients, which met the irritable bowel syndrome diagnostic criteria of Rome Ⅲand TCM diagnosis criteria of spleen-deficiency type IBS-D, were divided into the positive control group (the control group), Sijunzi Decoction treatment group (the treatment group)according to random numbers table method. The control group was given maleic acid Trimebutin in oral,0.1g each time,2times a day, a month of continuous treatment. The treatment group were treated by Modified Sijunzi Decoction, one dose daily,2times a day, continuous treatment for one month. In addition, health volunteers were collected to set up as healthy control group (the normal group), which were given normal diet, without taking drugs.
With reference to IBS diagnostic criteria of Rome III and " Irritable bowel syndrome with integrated traditional Chinese and Western medicine treatment program (2003Edition)" formulated by Digestive diseases professional committee of China Institute of traditional Chinese and Western medicine, we established Spleen-deficiency type IBS-D patients clinical efficacy evaluation criterion. Guided by professionals, all subjects were asked to fill in the clinical evaluation form before and after treatment. After one month period of treatment with modified Sijunzi Decoction or positive control drug, the subjects were given clinical efficacy evaluation.
IBS-D patients with Spleen-deficiency syndrome and healthy volunteers were examined by electronic colonoscopy, and several mucosa, weighing approximately10-20mg, in ileocecal and sigmoid colon, about25cm away from anal, were collected. All biopsy specimens were immediately put into liquid nitrogen, and one week later were moved to a-80℃refrigerator together.
In the research,39cases were collected, including20cases in the treatment group,19cases in the control group. And before and after the treatment course,11cases of intestinal mucosa specimens were collected in the treatment group, and11cases in the control group. As for the healthy control group,10cases of colonic mucosa specimens were collected. The detection indexes are as follows:
(1) High performance liquid chromatography method was used to detect5-HT quantity contained in intestinal mucosa of the normal group, the treatment group, the control group before and after treatment.
(2) RT-PCR method was used to detect mRNA expression of5-HT3,5-HT4receptor in intestinal mucosa of the normal group, the treatment group, the control group before and after treatment.
(3) Inorganic phosphorus method was used to detect Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme activity of cells in intestinal mucosa of the normal group, the treatment group, the control group before and after treatment
(4) Fluo-3/Am fluorescence probe and FCM assay were used to detect intestinal mucosa intracellular calcium ion concentration of the normal group, the treatment group, the control group before and after treatment.
With SPSS statistical17.0software, the examine results were tested by t inspection or X2inspection. The results are regarded as statistically significant when P<0.05.
Result:
X2inspection and other statistical analysis were used to inspect gender, age, course of disease distribution and other aspects in the treatment group and the control group, there is no statistically significant difference in general.
(1) The total effective rate of Sijunzi Decoction on IBS-D was85%, and that of the control group was73.68%. The effective rate in the treatment group was higher that in the control group. The effective rates of the two groups were inspected with Wilcoxon rank test, which showed that there was no significant difference (P=0.5393>0.05).
(2) There were no significant differences in the symptom scores before treatment between the two groups (P>0.05); the symptom scores after treatment in the treatment group were significantly lower than before treatment (P<0.05); as to the control group, except in the symptoms of abdominal pain, abdominal discomfort, increased defecation frequency, unforming stool, there were no significant differences in the other scores between before treatment and after treatment (P<0.05); and after the treatment, the differences between the two groups were significant (P<0.05), which showed that the treatment group was better than the control group in improving symptoms scores.
(3)5-HT content in ileocecal mucosa and sigmoid colon mucosa of the treatment group and the control group before treatment were significantly higher than that of the normal group (P<0.01), which showed that5-HT have certain connection with Spleen-deficiency type IBS-D. After treated by modified Si junzi Decoction or the medicine for the control group,5-HT content in ileocecal mucosa and sigmoid colon mucosa of the two groups after treatment were significantly higher than before treatment (P<0.05). After treatment,5-HT content in ileocecal mucosa and sigmoid colon mucosa of the treatment group and the control group had no significant difference (P>0.05).
(4) Before treatment,5-HT3R and5-HT4R receptor mRNA expression in colonic mucosa of the treatment group and the control group increased significantly(P<0.01); after the treatment,5-HT3R and5-HT4R receptor mRNA expression in colon mucosa of the two groups were significantly reduced (compared with that of before treatment, P<0.05or P<0.01), and there were no significant differences compared with the normal group after treatment (P>0.05).
(5) Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme activity in intestinal mucosa of IBS-D patients with Spleen-deficiency syndrome decreased significantly compared with normal group (P<0.01); after treatment with modified Sijunzi Decoction, Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme activity in intestinal mucosal of patients in the treatment group significantly improved (P<0.01) while those in intestinal mucosal of patients in the control group after treatment had no significant difference compared with those before treatment (P>0.05).
(6) Intracellular free calcium fluorescence intensity values in intestinal mucosa of IBS-D patients with Spleen-deficiency increased significantly compared with that of the normal group (P<0.01); after treatment with the modified Si junzi Decoction or the drug for the control group, intracellular free calcium fluorescence intensity values in intestinal mucosa of the two groups significantly decreased (P<0.01).
Conclusion:
(1)Total efficiency of modified Sijunzi Decoction treating IBS-D patients with Spleen-deficiency syndrome was higher than that of the control group, and modified Sijunzi Decoction could improve the main symptoms of Spleen-deficiency type IBS-D such as lassitude, sallow complexion, anorexia, abdominal distention after eating and there was no obvious adverse reaction, which was significantly better than the control group. Above results showed that modified Sijunzi Decoction could effectively improve the clinical symptoms of IBS-D patients with Spleen-deficiency syndrome, and it is an effective and secure TCM formula.
(2)5-HT secretion and expression level of5-HT receptors'mRNA in IBS-D patients with Spleen-deficiency syndrom were abnormal. When Spleen-deficiency type IBS-D occurred,5-HT increased, expression level of5-HT3R and5-HT4R mRNA in colonic mucosa increased significantly; and they were positively correlated with the main symptoms of IBS-D patients with Spleen-deficiency syndrome such as abdominal pain, distention, loose stool, incompletion sensation of defecation, etc. After treatment with modified Sijunzi Decoction and maleic acid Trimebutin, the main symptoms of Spleen-deficiency type IBS-D were improved, accompanied by significantly decreased content of5-HT in colonic mucosal and significantly decreased mRNA expression level of significantly decreased5-HT3and5-HT4significantly decreased receptor, which indicated that modified Sijunzi Decoction and maleic acid Trimebutin might improve the major clinical symptoms of patients through affecting5-HT and its receptors in gastrointestinal tract.
(3) Activity of Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme in intestinal mucosa of IBS-D patients with Spleen-deficiency syndrome significantly decreased compared with the normal group. After treatment with modified Sijunzi Decoction, activity of Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme in intestinal mucosa significantly increased. However, After treatment with maleic acid Trimebutin, there were no significant difference before and after treatment. Above results showed that modified Si junzi Decoction might improve the clinical symptoms of patients such as lassitude, sallow complexion, anorexia, abdominal distension after eating through affecting activity of Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme in intestinal mucosa of patients.
(4) Intracellular free calcium fluorescence intensity values in intestinal mucosa of IBS-D patients with Spleen-deficiency syndrome increased significantly compared with the normal group. After treatment with modified Sijunzi Decoction and maleic acid Trimebutin, ntracellular free calcium fluorescence intensity values in intestinal mucosat were significantly improved. Above results showed that modified Sijunzi Decoction might have similar efficacy as Trimebutine, that is, affecting the cell membrane calcium channels so as to maintain the calcium ion concentration of intestinal mucosa in normal level.
(5)Modified Sijunzi Decoction's effect on Spleen-deficiency type IBS-D is multi-target. In addition to affect the cell membrane calcium channels so as to maintain the calcium ion concentration in normal level, it can cure the patients through improving activity of ATPase, and reducing content of5-HT and mRNA expression of5-HT receptors.
(6) A possible mechanism of ocurrence of Spleen-deficiency type IBS-D is: when energy production and conversion level of IBS-D patients with Spleen-deficiency syndrome decreased, activity of ATP enzyme also declined, which could lead to large amount of Ca2+getting into the intestinal cells, so that calcium ion concentration increased. Increasing of calcium ion concentration resulted in activation of intestinal enterochromaffin cells, which further released gastrointestinal hormones including5-HT, thus causing IBS patients'symptoms such as abdominal pain, diarrhea and visceral sensory abnormalities.
随着脾虚证的中西医结合研究进一步发展,从病证结合的角度进行脾虚证的研究成为新的方向和趋势。自上世纪八十年代以来,脾虚证的现代研究取得了丰硕的成果。大量的临床实践证明,与脾虚证相关的诸多疾病都与机体能量的代谢水平下降密切相关。ATP酶是广泛存在于细胞膜上的一种磷酸酶,能把ATP水解成ADP而释放出磷酸基,提供机体代谢所需的能量,因而ATP酶可以在一定程度上反映机体的能量代谢水平。
肠易激综合征(IBS)是由多种病因引起的以结肠动力学紊乱及内脏敏感性增高为基本病理生理表现的功能性疾病。多项研究显示,IBS患者存在5-HT分泌或分布的异常,5-HT参与了IBS的主要病理生理过程。5-HT通过与不同器官上的膜受体结合而发挥作用,其中胃肠道中主要存在5-HT4两种类型。5-HT通过与这两种受体结合影响和调节IBS患者的胃肠运动、分泌、吸收及感觉等复杂功能,故5-HT及其受体与IBS患者腹痛、腹泻和内脏感觉异常等症状密切相关。
中医药对IBS具有较好的疗效。有研究显示,中医药治疗优势在于有效缓解腹痛、腹泻及便秘等主要临床症状,改善中医临床证候,提高患者生存质量,具有较好的成本效果比。但其诊治IBS病理生理学机制尚不明确。我们推测中医药的这种作用可能通过影响5-HT的浓度、5-HT受体的表达等来实现。因此本课题拟从脾虚型IBS-D患者肠粘膜5-HT及其受体及四君子汤的干预作用入手,来探讨脾虚型IBS-D的发生机理及其四君子汤的干预机制,课题的实施对指导脾虚型IBS-D的临床用药及开发治疗脾虚型IBS-D的有效中药制剂,对进一步充实中医脾胃病辨证治疗体系有重要意义。
方法：
在广州中医药大学第一附属医院收集脾虚型IBS-D病例,按照随机、对照的原则,将符合肠易激综合征罗马Ⅲ诊断标准和脾虚型IBS-D中医诊断标准者的IBS-D受试对象,按照随机数字表法分为阳性对照组(对照组)、四君子汤治疗组(治疗组)。对照组给予马来酸曲美布汀口服,每次0.1g,每日2次,连续治疗一个月。治疗组给予加味四君子汤(由党参20g、茯苓15g、白术15g、甘草6g、黄芪20g)口服,每日一剂,水煎两次分服,连续治疗一个月。另外征集健康自愿者设立健康对照组(正常组),常规饮食,不服用药物。
参照肠易激综合征罗马Ⅲ诊断标准和中国中西医结合学会消化系统疾病专业委员会的《肠易激综合征中西医结合诊治方案(2003版)》制定脾虚型IBS-D患者临床疗效评价标准,所有受试者治疗前后由专业人员指导认真填写临床评估表。经过加味四君子汤或阳性对照组药物治疗一个月后,对受试者进行临床疗效评价。
对脾虚型IBS-D和健康自愿者经电子结肠镜检查,取回盲部及距肛门约25cm处乙状结肠粘膜若干块,约重10-20mg,所有活检标本均立即投入液氮中,l周后同时转入-80℃冰箱中保存。
本次试验最终收集合格病例共39例,其中治疗组20例,对照组19例。治疗组完成疗程后再次结肠镜取标本的病例共11例,对照组完成疗程后再次结肠镜取标本的病例9例,健康对照组收集到结肠粘膜标本共10例。进行指标检测如下：
(1)高效液相色谱法检测正常组、治疗组和对照组治疗前后的受试者肠粘膜5-HT的含量。
(2) RT-PCR法检测正常组、治疗组和对照组治疗前后的受试者肠粘膜5-HL3、5-HT4受体基因表达水平。
(3)无机磷法测定正常组、治疗组和对照组治疗前后的受试者肠粘膜Na+-K+-ATP酶,Ca2+-Mg2+-ATP酶活性。
(4) Fluo-3/Am荧光探针和FCM法检测正常组、治疗组、对照组治疗前后的受试者肠粘膜细胞内钙离子浓度。
采用SPSS统计软件,通过t检验或X2检验对试验结果进行显著性检验。检验结果在P<0.05时认为差异有统计学意义。
结果：
通过对治疗组和对照组在性别、年龄、病程分布等方面进行X2检验等统计学分析,两组在一般情况方面没有统计学差异,具有可比性。
(1)加味四君子汤治疗脾虚型IBS-D总有效率达85%,对照组总有效率为73.68%,治疗组的总有效率高于对照组。两组患者疗效经Wilcoxon秩和检验表明,治疗组与对照组疗效无显著性差异(P=0.5393>0.05)。
(2)两组患者治疗前各症状积分比较,差异无显著性意义(P>0.05)；治疗组治疗后各症状积分均较治疗前有显著下降(P<0.05)；对照组治疗后除腹痛、腹部不适、排便次数增加、大便不成形等症状积分较治疗前有显著下降(P<0.05)外,其他各症状积分治疗前后比较差异无显著性意义(P>0.05)；治疗后两组间比较,差异均有显著性意义(P<0.05),提示治疗组在改善症状积分方面优于对照组。
(3)治疗组及对照组治疗前回盲部结肠粘膜、乙状结肠粘膜5-羟色胺含量均比正常组明显增高(P<0.01),说明5-羟色胺增高与脾虚型IBS-D有一定的联系。经加味四君子汤及对照组药物治疗后,脾虚型IBS-D患者回盲部结肠粘膜、乙状结肠粘膜5-羟色胺含量较治疗前明显降低(P<0.05)。治疗后治疗组与对照组回盲部结肠粘膜、乙状结肠粘膜5-羟色胺含量无显著性差异(P>0.05)。
(4)治疗前治疗组及对照组的5-HT3R和5-HT4R受体mRNA在结肠粘膜的表达显著升高(P<0.01)；治疗后两组的5-HT3R和5-HT4R受体mRNA在结肠粘膜的表达显著降低(P<0.01或P<0.05),并与正常组相比无显著性差异(P>0.05)。
(5)脾虚型IBS-D患者肠粘膜Na+-K+-ATP酶(?)(?)Ca2+-Mg2+-ATP酶与正常组相比均有显著降低(P<0.01)；经加味四君子汤治疗后,患者肠粘膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶均有显著提高(P<0.01)；而经对照组药物治疗后,患者肠粘膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶均与治疗前无显著差异(P>0.05)。
(6)脾虚型IBS-D患者肠粘膜细胞内游离钙离子荧光强度值与正常组相比显著升高(P<0.01)；经加味四君子汤治疗和对照组药物治疗后,患者肠粘膜内游离钙离子荧光强度值均有显著降低(P<0.01)。
结论：
(1)加味四君子汤治疗脾虚型IBS-D的总有效率高于对照组,而且在改善脾虚型IBS-D患者的神疲乏力、面色萎黄、食少纳差、食后腹胀等主要症状上显著优于对照组,且没有明显不良反应。说明四君子汤为主方的中药汤剂能有效改善脾虚型腹泻型IBS患者的临床症状,是对脾虚型IBS-D临床疗效肯定和安全的中药方剂。
(2)脾虚型IBS-D患者存在5-HT分泌及5-HT受体mRNA表达水平的异常。脾虚型IBS-D时5-羟色胺增多,5-HT3R和5-HT4R受体mRNA在结肠粘膜的表达显著升高；脾虚型IBS-D患者的主要症状,如腹痛、腹胀、便溏、大便不尽感等与结肠粘膜5-HT3R、5-HT4R mRNA的表达呈明显正相关,随着加味四君子汤和马来酸曲美布汀的应用,脾虚型IBS-D的主要症状得以改善,也同时伴有结肠粘膜5-HT含量及5-HT3和5-HT4受体mRNA的表达水平明显下降,表明加味四君子汤和马来酸曲美布汀可能通过作用于胃肠道5-羟色胺及其受体而改善患者的主要临床症状。
(3)脾虚型IBS-D患者肠粘膜Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶与正常组相比均有显著降低,经加味四君子汤治疗后,患者肠粘膜Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶均有显著提高。而经对照组药物治疗后,患者均与治疗前无显著差异。表明加味四君子汤可能通过作用于肠粘膜Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶而改善患者的神疲乏力、面色萎黄、食少纳差、食后腹胀等症状。
(4)脾虚型IBS-D患者肠粘膜细胞内游离钙离子荧光强度值与正常组相比显著升高；经加味四君子汤治疗和对照组药物治疗后,患者肠粘膜内游离钙离子荧光强度值均有显著提高。表明加味四君子汤可能与马来酸曲美布汀有相似的功效,即通过作用于细胞膜钙离子通道而维持患者肠粘膜内钙离子浓度处于正常水平。
(5)加味四君子汤对脾虚型IBS-D的治疗作用是多靶点的,除了与马来酸曲美布汀对IB-D的作用机制即通过调节Ca"+通道而使细胞内钙能恢复到较低水平,还可通过改善ATP酶活性,通过降低5-HT的释放和5-HT受体的表达来达到治疗的目的。
(6)脾虚型IBS-D发生的可能机制是：脾虚型IBS-D患者机体的能量生成和转化水平降低,影响到机体ATP酶的活性。当ATP酶活性下降时,可导致大量Ca2+进入肠道细胞内,使钙离子浓度增加,从而激活肠道肠嗜铬细胞,使其进一步释放5-HT等胃肠激素,引起IBS患者出现肠道运动增加、对液体的吸收减少和内脏敏感性增高等异常表现。
Background and objective:
Along with further development of integrated Chinese and Western medicine research on Spleen-deficiency syndrome, studies on Spleen-deficiency syndrome from the perspective of combination of disease and syndrome has become a new direction and trend. Since1980s, modern researches on Spleen-deficiency syndrome has got great achievements. A large number of clinical practices proved that many diseases involved in Spleen-deficiency syndrome are closely associated with declined energy metabolism level. ATP enzymes, a sort of phosphatase, are widely present in cell membrane, which can promote the reaction of ATP being hydrolized into ADP and releasing phosphate, and provide required metabolic energy. So ATP enzymes, to some extent, can represent energy metabolism level.
Irritable bowel syndrome (IBS), caused by various etiological factors, is a functional disease which manifested by colonic motility disorders and increased visceral sensitivity, etc. Many studies have shown that IBS patients had abnormal5-HT secretion or distribution.5-HT participated in the major pathophysiological processes of IBS, and5-HT played different roles by combining with different membrane receptors. In the gastrointestinal tract, there are mainly two kinds of receptors:5-HT3and5-HT4. Combined with the two kinds of receptors,5-HT affected and regulated gastrointestinal motility, secretion, absorption and others in IBS patients, thus5-HT had a close relationship with IBS patients' symptoms such as abdominal pain, diarrhea and visceral sensory abnormalities.
Traditional Chinese medicine (TCM) has a better therapeutic effect on IBS. Studies have shown that TCM had advantages of effectively relieving abdominal pain, diarrhea and constipation and other major clinical symptoms, improving clinical syndromes of TCM, promoting quality of patients'life, and having better cost effectiveness ratio. However, pathophysiology mechanism of TCM's diagnosis and treatment on IBS is still not clear. We hypothesized that TCM's role might be achieved by modulating concentration of5-HT and mRNA expression of5-HT receptors. Therefore, this article planed to study5-HT and its receptors in intestinal mucosal of IBS-D patients with Spleen-deficiency syndrome and Sijunzi Decoction's intervention on them, so as to explore the mechanism of Spleen-deficiency type IBS-D and Sijunzi Decoction's intervention mechanism. This study were beneficial to promote clinical application of the medicine for Spleen-deficiency type IBS-D, to develop effective Chinese herbal medicine of treating Spleen-deficiency type IBS-D, and to further enrich Chinese medicine syndrome differentiation and treatment system on Spleen and Stomach diseases.
Methods:
According to the principles of randomized and controlled method, Spleen-deficiency type IBS-D cases from the first hospital of Guangzhou University of Chinese Medicine were collected. The patients, which met the irritable bowel syndrome diagnostic criteria of Rome Ⅲand TCM diagnosis criteria of spleen-deficiency type IBS-D, were divided into the positive control group (the control group), Sijunzi Decoction treatment group (the treatment group)according to random numbers table method. The control group was given maleic acid Trimebutin in oral,0.1g each time,2times a day, a month of continuous treatment. The treatment group were treated by Modified Sijunzi Decoction, one dose daily,2times a day, continuous treatment for one month. In addition, health volunteers were collected to set up as healthy control group (the normal group), which were given normal diet, without taking drugs.
With reference to IBS diagnostic criteria of Rome III and " Irritable bowel syndrome with integrated traditional Chinese and Western medicine treatment program (2003Edition)" formulated by Digestive diseases professional committee of China Institute of traditional Chinese and Western medicine, we established Spleen-deficiency type IBS-D patients clinical efficacy evaluation criterion. Guided by professionals, all subjects were asked to fill in the clinical evaluation form before and after treatment. After one month period of treatment with modified Sijunzi Decoction or positive control drug, the subjects were given clinical efficacy evaluation.
IBS-D patients with Spleen-deficiency syndrome and healthy volunteers were examined by electronic colonoscopy, and several mucosa, weighing approximately10-20mg, in ileocecal and sigmoid colon, about25cm away from anal, were collected. All biopsy specimens were immediately put into liquid nitrogen, and one week later were moved to a-80℃refrigerator together.
In the research,39cases were collected, including20cases in the treatment group,19cases in the control group. And before and after the treatment course,11cases of intestinal mucosa specimens were collected in the treatment group, and11cases in the control group. As for the healthy control group,10cases of colonic mucosa specimens were collected. The detection indexes are as follows:
(1) High performance liquid chromatography method was used to detect5-HT quantity contained in intestinal mucosa of the normal group, the treatment group, the control group before and after treatment.
(2) RT-PCR method was used to detect mRNA expression of5-HT3,5-HT4receptor in intestinal mucosa of the normal group, the treatment group, the control group before and after treatment.
(3) Inorganic phosphorus method was used to detect Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme activity of cells in intestinal mucosa of the normal group, the treatment group, the control group before and after treatment
(4) Fluo-3/Am fluorescence probe and FCM assay were used to detect intestinal mucosa intracellular calcium ion concentration of the normal group, the treatment group, the control group before and after treatment.
With SPSS statistical17.0software, the examine results were tested by t inspection or X2inspection. The results are regarded as statistically significant when P<0.05.
Result:
X2inspection and other statistical analysis were used to inspect gender, age, course of disease distribution and other aspects in the treatment group and the control group, there is no statistically significant difference in general.
(1) The total effective rate of Sijunzi Decoction on IBS-D was85%, and that of the control group was73.68%. The effective rate in the treatment group was higher that in the control group. The effective rates of the two groups were inspected with Wilcoxon rank test, which showed that there was no significant difference (P=0.5393>0.05).
(2) There were no significant differences in the symptom scores before treatment between the two groups (P>0.05); the symptom scores after treatment in the treatment group were significantly lower than before treatment (P<0.05); as to the control group, except in the symptoms of abdominal pain, abdominal discomfort, increased defecation frequency, unforming stool, there were no significant differences in the other scores between before treatment and after treatment (P<0.05); and after the treatment, the differences between the two groups were significant (P<0.05), which showed that the treatment group was better than the control group in improving symptoms scores.
(3)5-HT content in ileocecal mucosa and sigmoid colon mucosa of the treatment group and the control group before treatment were significantly higher than that of the normal group (P<0.01), which showed that5-HT have certain connection with Spleen-deficiency type IBS-D. After treated by modified Si junzi Decoction or the medicine for the control group,5-HT content in ileocecal mucosa and sigmoid colon mucosa of the two groups after treatment were significantly higher than before treatment (P<0.05). After treatment,5-HT content in ileocecal mucosa and sigmoid colon mucosa of the treatment group and the control group had no significant difference (P>0.05).
(4) Before treatment,5-HT3R and5-HT4R receptor mRNA expression in colonic mucosa of the treatment group and the control group increased significantly(P<0.01); after the treatment,5-HT3R and5-HT4R receptor mRNA expression in colon mucosa of the two groups were significantly reduced (compared with that of before treatment, P<0.05or P<0.01), and there were no significant differences compared with the normal group after treatment (P>0.05).
(5) Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme activity in intestinal mucosa of IBS-D patients with Spleen-deficiency syndrome decreased significantly compared with normal group (P<0.01); after treatment with modified Sijunzi Decoction, Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme activity in intestinal mucosal of patients in the treatment group significantly improved (P<0.01) while those in intestinal mucosal of patients in the control group after treatment had no significant difference compared with those before treatment (P>0.05).
(6) Intracellular free calcium fluorescence intensity values in intestinal mucosa of IBS-D patients with Spleen-deficiency increased significantly compared with that of the normal group (P<0.01); after treatment with the modified Si junzi Decoction or the drug for the control group, intracellular free calcium fluorescence intensity values in intestinal mucosa of the two groups significantly decreased (P<0.01).
Conclusion:
(1)Total efficiency of modified Sijunzi Decoction treating IBS-D patients with Spleen-deficiency syndrome was higher than that of the control group, and modified Sijunzi Decoction could improve the main symptoms of Spleen-deficiency type IBS-D such as lassitude, sallow complexion, anorexia, abdominal distention after eating and there was no obvious adverse reaction, which was significantly better than the control group. Above results showed that modified Sijunzi Decoction could effectively improve the clinical symptoms of IBS-D patients with Spleen-deficiency syndrome, and it is an effective and secure TCM formula.
(2)5-HT secretion and expression level of5-HT receptors'mRNA in IBS-D patients with Spleen-deficiency syndrom were abnormal. When Spleen-deficiency type IBS-D occurred,5-HT increased, expression level of5-HT3R and5-HT4R mRNA in colonic mucosa increased significantly; and they were positively correlated with the main symptoms of IBS-D patients with Spleen-deficiency syndrome such as abdominal pain, distention, loose stool, incompletion sensation of defecation, etc. After treatment with modified Sijunzi Decoction and maleic acid Trimebutin, the main symptoms of Spleen-deficiency type IBS-D were improved, accompanied by significantly decreased content of5-HT in colonic mucosal and significantly decreased mRNA expression level of significantly decreased5-HT3and5-HT4significantly decreased receptor, which indicated that modified Sijunzi Decoction and maleic acid Trimebutin might improve the major clinical symptoms of patients through affecting5-HT and its receptors in gastrointestinal tract.
(3) Activity of Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme in intestinal mucosa of IBS-D patients with Spleen-deficiency syndrome significantly decreased compared with the normal group. After treatment with modified Sijunzi Decoction, activity of Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme in intestinal mucosa significantly increased. However, After treatment with maleic acid Trimebutin, there were no significant difference before and after treatment. Above results showed that modified Si junzi Decoction might improve the clinical symptoms of patients such as lassitude, sallow complexion, anorexia, abdominal distension after eating through affecting activity of Na+-K+-ATP enzyme and Ca2+-Mg2+-ATP enzyme in intestinal mucosa of patients.
(4) Intracellular free calcium fluorescence intensity values in intestinal mucosa of IBS-D patients with Spleen-deficiency syndrome increased significantly compared with the normal group. After treatment with modified Sijunzi Decoction and maleic acid Trimebutin, ntracellular free calcium fluorescence intensity values in intestinal mucosat were significantly improved. Above results showed that modified Sijunzi Decoction might have similar efficacy as Trimebutine, that is, affecting the cell membrane calcium channels so as to maintain the calcium ion concentration of intestinal mucosa in normal level.
(5)Modified Sijunzi Decoction's effect on Spleen-deficiency type IBS-D is multi-target. In addition to affect the cell membrane calcium channels so as to maintain the calcium ion concentration in normal level, it can cure the patients through improving activity of ATPase, and reducing content of5-HT and mRNA expression of5-HT receptors.
(6) A possible mechanism of ocurrence of Spleen-deficiency type IBS-D is: when energy production and conversion level of IBS-D patients with Spleen-deficiency syndrome decreased, activity of ATP enzyme also declined, which could lead to large amount of Ca2+getting into the intestinal cells, so that calcium ion concentration increased. Increasing of calcium ion concentration resulted in activation of intestinal enterochromaffin cells, which further released gastrointestinal hormones including5-HT, thus causing IBS patients'symptoms such as abdominal pain, diarrhea and visceral sensory abnormalities.
引文
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