Research On Theraputic Effect And Mechanism Of Modified Sijunzi Decoction On IBS-D With Spleen-deficiency Syndrome

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.