The Experimental And Clinical Study On The Effect Of Jianpijieduhuazhuofang On Irritable Bowel Syndrome

Objective: The irritable bowel syndrome (IBS) were one kind of intestinal irritable disease because of intestinal hypersensitivity, is refers to a group of the functionality intestines disorder syndrome including the abdominal pain, the distension of the abdomen, the diarrhea, which of causes and the mechanism is not yet expounded until now.Speeds up unceasingly along with the rhythm of life with the social competition intensely, the incidence of IBS of world is rising day by day, which has interfered with people's normal work, the study and the life seriously.The IBS cause of disease and the pathogenesis not yet expounded until now.At present we thought that the enhancement of internal organs sensitivity is one of IBS most important pathology physiology characteristics, which is he foundation and symptom diversification reason which the IBS patient symptom produces.In recent years many studies tell us that the mast cell play a critical role during the the pathology and physiological process of intestinal irritable disease. After being activated, the mast cell might release many biological activity different mediums, which release may further enlarge each physiology and pathological reaction, which thus can caused various symptoms.Therefore, suppressing activation of the intestinal mast cell, causing its reduced medium, including histamine, P material quantities, and so on, is the key to treats the intestinal irritable disease.The pathogenesis of IBS is complex, involving many aspects, including the spirit, the diet, individual quality, and so on. The sole medicine clinical function is usually limit, which cannot satisfy the clinical request.The Chinese Traditional Medicine also believed that, the origin reasons the abdominal pain and diarrhea are complex, while the the laws of treating are also many. Jianpijieduhuazhuofang(JPJDHZF) is a classics Chinese Traditional Medical prescription by famous Chinese Traditional Medical expert Professor Li Diangui, which summarizes after many year clinical practices, is composed of pulsatilla root, goldthread root, scutellaria root, ginseng, astragalus root, white peony root, bighead, tangerine peel, ledebouriella root, ash bark, flavescent sophora root, sanguisorba root, aucklandia root, coix seed, chinese angelica root, liquorice, etc, has the may effections, including removing the poisonous quality of any substance, relieving the depressed liver and invigorating the spleen, reducing fever and remove dampness. Through the long-term clinical observation, it has been proved to have good curative effect on IBS. In order to further probe into the pharmacological action mechanism of JPJDHZF in treating IBS, we embarks from the Pharmacology research, taking the mast cell function research as the key point, applying the verious medhods, including blood biochemistry, the medicine serology, the immunity histochemistry, extriinsic cell culture, as well as the molecular biology method, separately observe IBS rat model behavior, the electricity physiology target, and the blood biochemistry, the phenomenon of degranulation of abnorminal mast cell taken from the rat, as well as the surface antibodyon the abdominal mast cell of the rat, attempts discussesing the effect and action mechanism of JPJDHZfang on IBS from three aspects, including the organ level, the cell level and the gene level, as well as the clinical practical application curative effect research, to provide scientfic basis for giving play to superiority of multi-ways, the multi-targets and the whole treatment of Chinese troditional Medicine, and to exploit and utilize scitificly Chinese troditional Medicine, simultaneously to build the foundation for the new medicine development.MethodPartⅠ: The Experimental Study on Effect and Mechanism of JPJDHZF on visceral hypersensitivity Rat Model of IBSTests one: To apply the acetic acid chronic stimulation in colon to manufacture visceral hypersensitivity Rat Model of IBS. The rats were divided into three groups: the childhood rat acetic acid chronic stimulation group (the NA group), the grown-up rat colon acetic acid chronic stimulation group (the AA group) and the blank control group (the NS group).After modeiiing, the visceral sensitivity measurement were carried on separately to each group of IBS rat model, which conclude the capability limens of the sacculus that caused abdomen-uplifting and back-arching and the time of contract of abdomen muscle in rats, to confirm the succeeds of model.Tests two: To discuss the effect and Mechanism of JPJDHZF on visceral hypersensitivity rat model of IBS. After have maken the rat model successfull, the rats were divided into the normal control group (A group), the model control group (B group), the dicetel control group (Group C), the low dosage of JPJDHZF group (Group D), the high dosage of JPJDHZF group (Group E). Different groups were received oral administration of different treatment for one month. The capability limens of the sacculus that caused abdomen-uplifting and back-arching and the time of contract of abdomen muscle in rats were observed. The levels of 5-hydroxytryptamine (5-HT), substance P (SP), and calcitonin gene related peptide (CGRP) were evaluated. PartⅡ: The Influence of Drug Serum of JPJDHZF on Degranulation of Peritoneal Mast Cell in Rats Activated by AntigenAt first, the peritoneal mast cell suspension was prepared and was carried on the appraisal.Simultaneously the drug serum of JPJDHZF, the drug serum of cromolyn sodium and the drug serum of bland control group were prepared separately. The experiment was divided into four groups: the high dosage of drug serum of JPJDHZF group, the low dosage of drug serum of JPJDHZF group, the drug serum of cromolyn sodium group, positive control group, and normal control group. The fluorescent Ca2+ indicator Fluo-3 was used to observe and quantitate the effect of JPJDHZF on degranulation percentage, intracellsula[rCa2+]of peritoneal mast cells and histamine release in sensitized rats by confocal laser scan microscope. Simultaneously ,the Influence of Drug Serum of every group on appearance of mast cell, releasing histamine and TNF-α, which degranulated actived by antigen.PartⅢ: The molecular biology mechanism of JPJDHZF on Restrain Peritoneal Mast Cells from Degranulation At first, the peritoneal mast cell suspension was prepared and was carried on the appraisal.Simultaneously the drug serum of JPJDHZF, the drug serum of cromolyn sodium and the drug serum of bland control group were prepared separately. The experiment was divided into three groups: the high dosage of drug serum of JPJDHZF group, the low dosage of drug serum of JPJDHZ group, and normal control group. The expressions of TLR2 and the Fas were examines separately in each group of rat abdominal mast cell using the RT-PCR method and the histochemistry colored method. PartⅣ: The Clinical Research About the Effect JPJDHZF on Patients With D-IBSThe prospective, randomized, placebo-controlled, double-blind clinical trial was used. The training was carried on to the participation researcher, about the diagnosis, the integration, and the elimination standards, as well as implementation plan. 137 D-IBS patients were enrolled in this clinical trail from December, 2004 to December, 2006, entering the JPJDHZF treatment group and the western medicine treatment group by 1:1 proportion according to the random digit.The JPJDHZF treatment group takes orally JPJDHZF, the western medicine treatment group takes orally dictel.The observation projects include the situation of abdominal pain and the excrement, the other symptoms which had to be observed still include the felling of urgent or gravistatic defecation, the chest coerced or the lower abdomen abdominal bloating, the mood tight or despondent angry when flows swiftly releases the aggravation situation, the mucus then, belches as well as the feed quantity and so on; Symptom examination including colon area tenderness and tongue and pulse condition.Before the treatment and after 3 weeks of stopping the the treatment, the urine and the feces convention, hepatorenal inspections and electrocardiogram were examined. The close observation included bad event and bad respondence, recording the time when it occured, the performance which it takes, and the processing which we takes, and the result. We should pays attention to the serious bad event and bad respondence.Result PartⅠ: The Experimental Study on Effect and Mechanism of JPJDHZF on visceral hypersensitivity Rat Model of IBS Tests one: In the 6th week of modelling, The capability limens of theAWR that cause abdomen-uplifting and back-arching of NS group are (0.70±0.10)ml and (0.94±0.16)ml, The capability limens of the AWR that cause abdomen-uplifting and back-arching of AA group are (0.76±0.06)ml and (1.02±0.88)ml, There is no difference between them (P>0.05). Those of NA group are respectively (0.43±0.06)ml and (0.61±0.08)ml, which is significant lower than other two groups (P<0.01); When being tested in capacity of 0.5ml, the phenomenon of contract of abdomen muscle in rats of NS group and the AA group is not obvious, but the times of contract of abdomen muscle in rats of the NA group rat reached (6.63±1.06) time /5min, There is significantly different among them (P<0.01). When being tested in capacity of 0.8ml, the times of contract of abdomen muscle in rats of the NA group were more than other two groups obviously (P<0.05). When being tested in capacity of 0.8ml, the times of contract of abdomen muscle in rats of the NA group were more than NS group obviously (P<0.05), while there is no difference does not have the significance between the NA group and the AA group (P>0.05).Tests two: In the aspect of behavior test, there is significantly different between the B group and A group in the capability limens of the AWR that cause abdomen-uplifting and back-arching after the medication (P<0.01, P<0.05), and there is no difference between the A group and other three groups (P>0.05); there is no remarkable difference among D group, E group and C group (P>0.05); The result of abdominal myo-electricity showed that, when being tested in capacity of 1.0ml and 1.5ml, the times of contract of abdomen muscle in rats of the B group were more than A groups obviously (P<0.01) after the medication. While there is no difference among D group, E group and C group and A group (P>0.05); D, E group compare with C group, not remarkable difference (P>0.05); Comparing with the model control group, every group have distinct improvement in behavior and the electricity physiology target; 5-HT and SP drops obviously in each treatment group; The content of CGRP increases than before.PartⅡ: The Influence of Drug Serum of JPJDHZF on Degranulation of Peritoneal Mast Cell in Rats Activated by AntigenThe degranulation percentage of peritoneal mast cells in high dosage of JPJDHZ group was 13%, which was significantly lower than that of the model control group (65%, p<0.05); There was also significant difference between the low dosage of JPJDHZF group and the model control group(23% vs 65%, p<0.05). The phenomenon of degranulation of mast cells in model control group is more obvious than that of treatment groups. Intracellsular[Ca2+]of peritoneal mast cells of high dosage of JPJDHZF group was significantly lower than that of control group (48.74±5.43nmol/L vs 436.16±12.30nmol/L, p<0.05); The histamine concentration of high dosage of JPJDHZF group was significantly lower than that of control group (33.23±21.50nmol/L vs 98.41±7.23nmol/L , p<0.05).PartⅢ: The molecular biology mechanism of JPJDHZF on Restrain Peritoneal Mast Cells from DegranulationThe result showed that, the ratio of TLR2mRNA/βactinmRNA and the ratio of FasmRNA/βactinmRNA of peritoneal mast cells in both the high-dosage of JPJDHZF group and the low-dosage of JPJDHZF group were higher than those of normal control group after they are stimulated by drug serum of JPJDHZF for 30min (P<0.05), which is confirmed that the drug blood serum can stimulate the expression of TLR2mRNA and FasmRNA; The effect of high-dosage of JPJDHZF group was most obvious, with the significance difference with the control group (P<0.05).The result of the immunohistochemical experiment showed that TSP-2 could combine with peritoneal mast cells of rats; While rabbit IgG of control group cannot combine with peritoneal mast cells of rats. The membrane of peritoneal mast cells were stainned, which were explained that the expression of Fas is positive..PartⅣ: The Clinical Research About the Effect JPJDHZF on Patients With D-IBSThe cure rate of JPJDHZF on abdominal pain in patients with D-IBS is better than that of dicetel [57.7% vs 16.0%(PP), 31.0% vs 7.1%(ITT)]; The effective rate of JPJDHZF on abdominal in patients with D-IBS is also better than that of dicetel [92.3% vs 44.0%(PP)与82.7% vs 39.3%(ITT)], which shows that there is significant difference between them (P<0.05). The cure rate of JPJDHZF on diarrhea in patients with D-IBS is better than that of dicetel [46.2% vs 20.0% (PP), 41.4% vs 17.2 (ITT)]; The effective rate of JPJDHZF on abdominal in patients with D-IBS is also better than that of dicetel [96.2% vs 48.0%(PP)与86.2% vs 42.9% (ITT)], which shows that there is significant difference between them (P<0.05). There were no difference in both the antidiarrhea time and analgesic time between the two groups (P>0.05).The cure rate of JPJDHZF group on syndrome of TCM in patients with D-IBS is better than that of dicetel group [30.8% vs 4.0%(PP), 27.6% vs 3.6%(ITT)]; The effective rate of JPJDHZF on abdominal in patients with D-IBS is also better than that of dicetel [92.3% vs 48.0%(PP), 82.7% vs 42.9%(ITT)], which shows that there is significant difference between them (P<0.05). There are more development in JPJDHZF group with the degree of abdominal pain, the frequence of abdominal pain, the time of abdominal pain, etc, which are better than those of dicetel group (P<0.05). There are more development in JPJDHZF group with concomitant symptom of syndrome of TCM, which including eructation, anorexia, abnorminal distension, tenesmus, than those of dicetel group (P<0.05).Conclusions⒈Visceral hypersensitivity Rat Model of IBS which was manufactured by the acetic acid chronic stimulation in colon is steady and practicable.⒉JPJDHZF could lower the level of 5-HT and SP, and increase CGRP of IBS model rats, especially high dosage of JPJDHZfang.⒊The mechanism of JPJDHZF may be increasing the limen of pain of bowel, eliminating hypersusceptibility of bowel, and decreasing excitability of nerve cells by decreasing the contents of 5-HT and SP in IBS model rats. ⒋JPJDHZF can avoid being activated of mast cells by inhibiting entering of Ca2+, thus it has obvious inhibitory effect on degranulation and histamine releasing of peritoneal mast cells in sensitized rats, which can reduce the content of histamine in the serum, raise threshold of pain, and eliminate the symptom of intestinal irritability.⒌The molecular biology mechanism of JPJDHZF on restrain peritoneal mast cells from degranulation is promote and induce the depression of mast cell directly by the combination of TLR2 and TSP-2 and the expression of Fas on the surface of peritoneal mast cells of rats.⒍JPJDHZF has the may effections, including not only removing the poisonous quality of any substance, relieving the depressed liver and invigorating the spleen, reducing fever and remove dampness, but also improving the function of immunity and microcirculation, elimilating the free radical, inhibiting the inflammatory reaction, and relieving pain. It has superiority of multi-ways, the multi-targets and the whole treatment of Chinese troditional Medicine to treat with IBS.