| Aims:Irritable bowel syndrome(IBS) is one of common functional bowel disorders, and is characterized by abdominal discomfort, pain and alterations of defecation in absence of biochemical or structural abnormalities that could normally explain the symptoms. This disease has significantly negative impact on the quality of life and relevant health care costs. Though IBS is prevalence in populations, its pathogenesis remains uncertain, many scientists want to seek the mechanisms, but there are various opinions about it, and up to now, many scientists are devoting themselves to developing new drugs to treat this disorder. The Wuji Wan is a famous prescription in traditional Chinese medicine, which is included in the china pharmacopoeia (2005 edition). It is composed of huanglian, wuzhuyu and baishao. Wuji Wan, which has the effects of mitigating stomach and antiemetic, could be used in the diseases of bellyache, gastralgia and stomach-hot-ache caused by the maladjustment of the liver and the stomach. In the clinic, the Wuji Wan prescription is used to treat IBS, gastric ulcer and other digestive disorders.On the basis of our previous experiments, making the two compatibilities as therapeutic medcine, and a model of chronic visceral hypersensitivity in adult rats induceded by colon Irritation(CI) during postnatal development as an animal model, we want to make preliminary experimental studies on the therapeutic effects and its relevant mechanisms of Wuji Wan extracts on IBS, through carrying out the isolated colon experimemts in guinea pig and animal experiments in vivo, viewing the neuroendocrine-immuuno-net as a drug target, and from the perspectives of the gut motility and visceral perception, and on the one hand, we could test our former work, on the other hand we could provide the cue for the investigation of compatibilities of traditional Chinese medicine prescription.Methods and Results:1. Effects of Wuji Wan on motility of isolated colon in guinea pigTo investigate the effects of Wuji Wan extracts with two different compatibilities (the NO.1 and NO.2) on motility of isolated colon in guinea pig, and compare the therapeutic effects with each other, we performed this experiment, and calculate the ED50 with the method of Bliss with different concentrations on the contraction induced by acetylcholine(Ach) and inhibitioninduced by adrenalin(Ad) respectively.Results as the follows: the NO.l and NO.2 Wuji Wan extracts can significantly inhibit the contraction of colon, and in our observing 15 minutes, there is significant time-dependent and concentration-dependent, and ED50 of the NO.l Wuji Wan is lower than the NO.2,but when the colonic is inhibit by E, at the time of 9mhK 15min,the NO.l at the level of 300 mg-L"1, and at the time of 15min,the NO.2 at the level of 30 mg-L-1, can both excite the colon significantly (PO.05) .2. Animal experiments2.1 Animal modelExperiments were performed using Sprague-Dawley(SD) female rats obtained as preweanling neonates(younger than 8 days), 90 neonates rats(8-21 days), were divided into 2 groups(10 controls,80 models),named the CI team and the control respectively), exposed to mechanical irritation with PTC A balloons, twice a day, separated by 30 min within an hour, exerting a pressure of 60mmHg for 1 min,(as measured with sphygmomanometer),lasting 2 weeks, weighing the rats once a week.2.2 Therapeutic effects experimentsNeonates model rats of 8 days old, according to the above method, when it came to the end of the seventh week, were divided into 8 groups in random, named CI group> the NO.l and the NO.2Wuji Wan Wuji Wan of large-dosage-treated> middle-dosage-treated^ small-dosage-treated group ^ Dicetel-treated group respectively, when the femal rats 8-weeks old, behavioral response to colorectal distention(CRD) were assessed in all the groups by measuring the threshold of abdominal withdrawal reflex(AWR),that is: abdominal-lifting(0.2 cm to the level) and the pelvic structure-elevating(lcm to the level), and when it is lh after the CRD is over, the adult females were anesthetized with vrethan (1000mg/kg,intraperitoneally),the distal descending colon(above 2cm to the anus) were removed, placed in 4% paraformaldehyde solution, and cut serially and to be stained, then to calculate the density of MCs.Results as the follows: to compare with the control, the threshold of abdominal-uplifting and the back-elevating of CI group are significantly low(P<0.05), at the same time, the density of MCs are obviously higher(P<0.05). the NO.l and the NO.2 Wuji Wan can both lift the threshold of abdominal-uplifting and the back-elevating(P<0.05), and significantly lower the density of MCs(P<0.05).2.3 Studies on the therapeutic mechanisms of Wuji WanThe blood of All the groups are obtained to measure the content of 5-HT(ELISA)> SS> SP(RIA),and the colon were stained by immunohistochemistry method for 5-HT>. VIP> SS-, SP, and the result were investigated by qualitatively and quantitatively by means of color image analyzer through analyzing the opacity density(OD)> area and integrated OD (IOD) of our interests in muscular layer of the colon, selecting 5 fields in random in the microscope, to becalculated with mean.Results as the follows: For the plasma concentrations of SP> SS and serum concentrations of 5-HT, the CI group are obvious higher than the control(P<0.05), and for the every parameter of 5-HT ? SP^ SS and the area and IOD of VIP immuoreactive of colon, it is also upregulated(P<0.01);For the large and middle-dosage of the NO.l, the concentrations of serum 5-HT and plasm SP and SS are significantly low (PO.01), the small-dosage can decrease the concentration of serum 5-HT and plasm SP(P<0.01, P<0.05,respectively), comparatively, the large and middle-dosage of NO.2 can markedly decrease the concentration of serum 5-HT and plasm SP and SS. For the NO.l, at the large-dosage, the OD of 5-HT> SS and the area and IOD of immuoreactive all significantly downregulate(P<0.01);the area and IOD of positive expression at the middle-dosage of 5-HTn SS^ VIP is remarkable differ from the CI group, and for the small-dosage, the area of 5-HT> VIP and the IOD of 5-HT > SS is significantly low;For the NO.2, at the large-dosage, the OD of 5-HT> SP positive expression markedly downregulate, and the area and IOD of 5-HT> SS together with the positive area of VIP are all significantly lower than the CI group(P<0.01);when it is at the middle-dosage, the OD of SP is significant low(P<0.01), the area and IOD of 5-HT, SS together with the positive area of VIP are also all significantly lower than the CI group;there is remarkable difference between the small-dosage of the NO.2 group and the CI group in positive area and IOD at small-dosage of the NO.2. For serum concentrations of 5-HT, every dosage of the NO.l have significant difference with the NO.2(P<0.01), but for the the plasma concentrations of SP> SS ,there is no remarkable difference between them, P>0.05;There is no significant difference between the NO.l and the NO.2 in all parameters of the expression of colon 5-HT(P>0.05);For the OD of SP expression, the NO.l is significantly higher than the NO.2 at large and middle-dosage, and when it comes to the expression area and IOD, there is no marked difference amomg each dosage of these two prescriptions;Comparing with the NO.2, the OD of SS expression is significantly low, .P<0.05, and for the area, the NO.l is remarkably larger than the NO.2;For the expression of VIP, except that the area and IOD of the small-dosage of the NO.l are both significantly smaller than the same dosage of the NO.2( PO.05), there is no difference between the same dosage of the two compatibilities.Conclusions:The mouse model is reliable and maintainable, and in accordance with the clinical character of IBS to some extent, this approach has potential for defining the pathophysiology of the visceral allodynia and hyperalgesia in functional bowel disease such as IBS, and studies on the functional mechanisms of drugs.Our experiment indicate that Wuji Wan extracts can significantly inhibit the isolated colon contraction in guinea pig, and the NO.l Wuji Wan extracts is superior to the NO.2.and at thesame time, we can also conclude that Wuji Wan extracts have also an excited effects on inhibited colon, so we can draw a conclusion that Wuji Wan extracts, on some extent, have different effects on isolated colon in guinea pig which are in different states. For the ED50, the NO.l Wuji Wan extracts is superior to the NO.2,but observing from the whole dates, the NO.l and the NO.2 each have its own advantages, the former effects is potent, while the latter is rapid comparatively.The NO.l and NO.2 Wuji Wan extracts can both lower the threshold of abdominal withdraw reflex, relief the visceral hypersensitivity, the NO.l is good at lowering the pain threshold, while, the NO.2 is better in decreasing the density of MC, but there is no marked difference.The NO.l and NO.2 Wuji Wan extracts can both revert the disturbances of gut hormones, and adjust the NIM back to balance, so it can make the body return to normal. With the difference of the combinations of components, there are different advantages to the disorders. Though the NO.2's mean residence time is short and its bioavailability is bad, its therapeutic effects on IBS is believable, based on our results, we speculate that the colon local function of the NO.2 prescription is stronger than its effect on whole body, and also maybe there are new uncertain compounds or metabolisms when and after it is absorbed by the body. |
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