| I. Research Background and ObjectiveSpleen-asthenia syndrome refers to the synthetic pathological condition of the whole body caused mainly by such functional disorder of digestive system as assimilating disorder, hypofunction of gastroenterological barrier. Spleen-asthenia syndrome should be treated mostly with the therapy of regulating Qi and strengthening spleen with such representative Chinese medicines as Radix Codonopsis, Rhizoma Atractylodis Macrocephalae, Radix Astragali, Poria Radix Glycyrrhizae, and with such classic prescriptions as Sijunzhi Decoction, Buzhongyiqi Decoction, Shenlingbaizhu Powder, Huangqijianzhong Decoctionas. Many studies have shown that both sufferers and animals of sleen-asthenia have the morphological and functional pathological changes of enterocyte, both of whom can be evidently improved by treatment with prescription for regulating Qi and strengthening spleen. It is indicated that the above-mentioned Chinese medicines can maintain the integrity of enterocyte, improve the function of enterocyte and protect intestinal mucosa, few reseaches on the mechanism of repairing intestinal mucosa with Chinese medicine for regulating Qi and strengthening spleen have been done.It was extracted recently by our laboratory that 18 components were extracted from Radix Codonopsis, Rhizoma Atractylodis Macrocephalae, Radix Astragali and Radix Glycyrrhizae, some of which were found able to promote the multiplication, migration and differentiation of small intestine IEC-6 cell, to facilitate the expression of ODC(ornithine decarboxylase), Villin and DMT 1 (divalent metal transporter 1), to increase the polyamine(putrescine) and so on. From our experiments, we choose AMPS-II of Rhizoma Atractylodis Macrocephalae, total saponin of Radix Astragali and total flavones of Radix Glycyrrhizae which presented relatively better pharmacological activity, studied theimprovement of the extraction technics of the 3 components to meet the requirements of devoloping new Chinese medicine, and found the optimum compatibility formula of medicines by animal experiments with uniform design. In the light of Baizhu-Huangqi Decoction set by Doctor Liu Wansu in the Jin-Yuan Dynasty, we created the Baizhu-Huangqi Prescription which is composed of AMPS-II of Rhizoma Atractylodis Macrocephalae, total saponin of Radix Astragali and total flavones of Radix Glycyrrhizae. As is pointed out by the book named plain questions on diarrhea "Baizhu-Huangqi Decoction should be administered when diarrhea has cured to achieve the intention of regulation and cooperation between spleen and stomach 10 tael Rhizoma Atractylodis Macrocephalae, 7 tael Radix Astragali, .3 tael Radix Glycyrrhizae." The therapy is similar to that in both convalescence of diarrhea and relaxation of ulcerative colitis, and the curative effect is possibly related to promoting repair of intestinal mucosa and protecting the barrier in intestinal mucosa. Therefore, besides the ODC and the polyamine, the enteric epithelium cell junction, the important part of intestinal mucosa barrier, is also possibly one of the targets acted on by the Chinese medicine for regulating Qi and strengthening spleen. Recently, the effects of Baizhu-Huangqi Prescription to the tight junction of cells and the relative protein ZO-1 were observed in our laboratory, and the results indicated that the prescription can promote multiplication of enteric epithelium Caco-2 cell, facilitate the expresstion of ZO-1 protain and that both the AMPS-II of Rhizoma Atractylodis Macrocephalae and the saponin of Radix Astragali in the prescription can promote the differentiation of enterocyte by way of promoting the connection of the cell (The data has not been published yet) .On the basis of the above-mentioned experiments and according to the clinical function of Baizhu-Huangqi Decoction, ulcerative colitis from the trauma of intestinal mucosa, i.e.dysentery in TCM, was chosen as the object of this study, and comparison was made in the aspects of indication, pharmacodynamics and toxicity and the like between the original Baizhu-Huangqi Decoction prescription and the Baizhu-Huangqi prescription which is created through the extraction, screening and formulation of the components of the medicines. This research aims at making sure by observation whether or not the Baizhu-Huangqi prescription is able to enhance the curative effect but the toxicity, to retain its therapeutic function in the treatment of spleen-stomach asthenia during the convalescence of dysentery and to keep the characteristic of enhancing therapeutic function by the compatibility of Chinese medicines, besides, whether or not there is relationship between the action mechanism and both closely connection of enteric epithelium and therelative proteins claudin-1 and occludin which mainly contain the tight junction. This research involves the establishment of animal model which is suited to the evolution of ulcerative colitis during treatment with Chinese medicines, and the comparison of both the curative effect and the toxicity of the Baizhu-Huangqi prescription for ulcerative colitis with that of Baizhu-Huangqi Decoction, and the observation of the curative effect of the Baizhu-Huangqi prescription in each pathological phases of ulcerative colitis, and of the effect of the prescription to claudin-1 and occludin protains, so as to study the therapeutic function in the convalescence of ulcerative colitis of the Baizhu-Huangqi prescription for regulating Qi and strengthening spleen, and the possible mechanism of acting on the intestinal mucosal barrier of the prescription. Under the guidance of Traditional Chinese Medicine theories and with the application of modern scientific methods, the research findings would help to expound the action mechanism and the material base of Chinese medicines for regulating Qi and strengthening spleen in the treatment of intestine disease.II. Research Methods1. Improvement of the Rat Model with Ulcerative colitis Caused by Trinitro-benzene-sulfonic acid (TNBS) .Through imitation of the syndrome according with "Baizhu-Huangqi Decoction should be administered when diarrhea has cured to achieve the intention of regulation and cooperation between spleen and stomach", the animal model, by which observation of both the curative effect and the action mechanism of the Baizhu-Huangqi prescription was made, was improved to near the clinical condition during relaxation of ulcerative colitis still further. Chosen in the experiment were 5 schemes of the model-building to observe the effect of TNBS for the formation of the ulcerative colitis of large mouse. Scheme 1: rat of 180-200g each, TNBS lOOmg/kg;Scheme 2: rat of 210-250g each, TNBS 90mg/kg;Scheme 3: rat of 220-290g each, TNBS 85mg/kg;Scheme 4: rat of 200-220g each, TNBS 50mg/kg. All the rats in the above 4 Schemes were SD rat, and all the experiments lasted for 15 days. Scheme 5: rat of 210-260g each;Wistar rat, TNBS 90mg/kg, and the experiment lasted for 10 days, 20 and 30 respectively. Method of operation: the rat were kept away from food but water for 24 hours, anaesthetized with ether, and infused with the reagent for the model-building in the stipulated dose through anus of the rat into the colon for 8 cm or so, then placed upside down by the tail for 30 seconds to let the reagent stay in the colon as much as possible. The rat models were raised as usual, and then killed after the experiments. The abdomen of the body was cut open rapidly, and then the segment ofintestines was taken out and rinsed. After observing the general pathological changes with naked eyes, the colon segments were fixed with formaldehyde, 24 hours later, embedded in paraffine, sliced up and dyed with HE, and then observed with microscope for the pathological changes of the tissue.2. Comparison between the Curative Effect of the Baizhu-Huangqi Prescription for Ulcerative colitis of Rat and That of Baizhu-Huangqi Decoction.After model building of rat TNBS (50mg/kg), they were divided into three groups, namely, model group, the Baizhu-Huangqi Prescription (0.3g/kg) group, and the Baizhu-Huangqi Decoction (15g/kg) group, with 16 mice in each group. They were infused via mouth with medicine and killed 14days later. The general condition of rat was observed, pathological changes of the colon was appraised with naked-eyes, tissue pathology, together with ultrastructure was appraised too. A comparison of curative effects of the Baizhu-Huangqi Prescription for Ulcerative colitis of rat and that of Baizhu-Huangqi Decoction, under the condition that the doses of the medicinal materials were nearly the same.3. Influence on Peroxidase in the Marrow of Mice with Colonitis Exerted by the Baizhu-Huangqi Prescription or its components.Influence on myeloperxidase (MPO) of mice by AMPS-II of Rhizoma Atractylodis Macrocephalae, total saponin of Radix Astragali, total flavones of Radix Glycyrrhizae, the AMPS-II + total saponin of Radix Astragali, the Baizhu-Huangqi Prescription, with 0.2g/kg each, was observed by building the model of TNBS resulting in colonitis of mice. Besides, model group, control group treated with Prednisone, and normal group were built. The mice in each group were infused via mouth with medicine and were killed ten days later, and MPO in colon was extracted and determined.4. Influence of the Baizhu-Huangqi Prescription on each Pathological Phases of Ulcerative Colitis.Rat with TNBS ulcerative colitis were divided into two groups, namely, model group and Baizhu-Huangqi Prescription group, 48 in each group. They were infused via mouth with medicine after the model was built, and were killed at 3 times in three periods after the model was built, 10 days, 20 days and 30 days later respectively. In addition, a normal group was built to observe the general condition, to appraise the colon pathological changes as well as the pathological changes of the tissue of the rat, so that the influences on the acute period, relieved period and the recovery period of mice ulcerative colitis by the the Baizhu-Huangqi Prescription were investigated.5. Influence of the Baizhu-Huangqi Prescription on the Relative Protein Claudin-1 > Occludinof Rat Ulcerative Colitis.The specimens of mice ulcerative colitis in Experiment 4 were examined the manifestation of claudin-1,occluding with immunofluorescence, so as to check the manifestation of claudin-K occludin of mice ulcerative colitis and find out whether the influence of Baizhu-Huangqi Prescription existed.6. Acute Toxicity Experiment of the Baizhu-Huangqi Prescription and Baizhu-Huangqi Decoction.Mice infused via mouth only once with Baizhu-Huangqi Prescription or Baizhu-Huangqi Decoction would stay alive, which enables the experiment of maximum dose that they could stand to be possible. Mice, infused via mouth with the largest amount of medicine of highest concentration, raised for 7 days under normal condition in order to observe their reaction to toxicity.DI.Research Findings1. Improved Model of Rat Ulcerative colitis Caused by TNBS Was Built.Scheme?: Death rate: 90% (9/10);appraisal of colon with naked eyes: 5.9+0.3. Scheme?: Death rate: 66.7% (6/9);appraisal of colon with naked eyes: 5.0± 1.7. Scheme (3): Death rate: 18.2% (2/11);appraisal of colon with naked eyes: 4.3 + 1.3. Scheme?: Death rate: 37.5% (6/16);appraisal of colon with naked eyes: 4.0+1.8. The model-building scheme can choose SD rat of 220-240g and TNBS dose of 50-60mg/kg, besides, the death rate shall be controlled within 20%.To search the pathological changes of mice ulcerative colitis at different phases, Wistar rat in same batch would be chosen in Scheme 5. They were killed in the first period (ten days), second period (20days) and the last period (30days) respectively. The findings for the first period: Death rate: 18.8% (3/16), appraisal of colon with naked eyes: 5.2+1.0, appraisal of tissue pathology: 7.8+0.6. The findings for the second period: Death rate: 12.5% (2/16), appraisal of colon with naked eyes: 4.3 + 1.3, appraisal of tissue pathology: 6.5+2.5. The findings for the third period: Death rate: 18.8% (3/16), appraisal of colon with naked eyes: 3.5 + 1.6, appraisal of tissue pathology: 5.6+1.7. It has shown that the pathological changes of mice ulcerative colitis appeared to be the most serious in the early days after the model-building, finding expression in such changes as acute inflammation and tissue putrescence. It was alleviated in the second period and it entered the recovery term in the last period. The model-building scheme can choose Wistar rat of 220-250g and TNBS dose of 50-60mg/kg, besides, and the death rate shall be controlled within 20%.2. Comparison between the Curative Effect of the Baizhu-Huangqi Prescription for Ulcerative colitis of Rat and That of Baizhu-Huangqi DecoctionThe appraisal of colon with naked eyes after the experiment: model group: 4.0+1.8;the Baizhu-Huangqi Prescription group: 2.8 + 1.5;the Baizhu-Huangqi Decoction: 3.4 + 1.7. Compared the Baizhu-Huangqi group with the model group: P<0.05;appraisal of tissue pathology: model group: 4.7+1.2, the Baizhu-Huangqi Prescription group: 3.1 + 1.6, the Baizhu-Huangqi Decoction group: 4.6+1.4, all the three group: PO.05. The statistics mentioned-above have indicated that the curative effects of Baizhu-Huangqi Prescription for ulcerative colitis of rat is better than that of Baizhu-Huangqi Decoction. The observance of colon ultrastructure has shown that the mice colonitis lead to the damage of closely interrelated structure, which could be ameliorated by the treatment of the Baizhu-Huangqi Prescription.3. Influence of Components of the Baizhu-Huangqi Prescription on MPO, be it Single Use or Compatibility Formula of the Parts.The MPO of the model group is evidently higher than the normal group, indicating that the model-building was successful. The fact that AMPS-II of Rhizoma Atractylodis Macrocephalae, AMPS-II of Rhizoma Atractylodis Macrocephalae+ total saponin of Radix Astragali, and the Baizhu-Huangqi Precription can lower the amount of MPO has shown that all the medicins is of anti-inflammation for the mice colonitis. The effect of AMPS-II of Rhizoma Atractylodis Macrocephalae+ total saponin of Radix Astragali is better than AMPS-II of Rhizoma Atractylodis Macrocephalae, while the effect of the Baizhu-Huangqi Prescription is the best. To use total saponin of Radix Astragali or total flavones of Radix Glycyrrhizae shows no evident effect on MPO.4 Influence of the Baizhu-Huangqi Prescription on each Pathological Phases of Ulcerative Colitis.The appraisal, respectively, of colon with naked eyes in the first, second, and last period by Baizhu-Huangqi Prescription group: 4.5 + 1.5, 3.8 + 1.7, 2.6+1.5. Compared the Baizhu-Huangqi Prescription in the last period, with the model group of the same period: PO.05;appraisal of tissue pathology: 8.0 + 0.0, 6.4+1.3, 4.9 + 2.0;Compared the Baizhu-Huangqi Prescription with the model group of the same period P>0.05. The mice ulcerative colitis went through putrescence of acute inflammation to recovery of chronicinflammation, but the Baizhu-Huangqi Prescription takes effect on merely the recovery of chronic inflammation and ulcer.5. Influence of the Baizhu-Huangqi prescription on relative protein claudin-l % occludin of rat ulcerative colitis.The tissue pathology of colon specimen in Experiment 4 includes normal tissue, ulcerative colitis domain (the original tissues were damaged, evident cells were invaded and soaked, leaked, putrescent or flesh shoots have grown, ulcer recovered, cicatrization and so on), the marginal tissues of ulcerative colitis domain (parts of such tissues as intestinal mucosa, the back of mucosa, the muscle layer could be seen, but they were invaded and soaked by inflammation cells), parts relatively far away from ulcerative colitis domain (the original tissues remained the same, relatively fewer inflammation cells invaded and soaked). The findings have shown that the fluorescence signs of occludin and claudin-l in normal colon tissue is weak to medium manifestation (comparatively speaking, the fluorescence signs of occludin is stronger than that of claudin-l). There were no fluorescence signs of occludin and claudin-l in the domain of ulcerative colitis. Stronger fluorescence signs lie in marginal tissues of ulcerative colitis and parts that are invaded and soaked by inflammation cells. It is the same with model group and the Baizhu-Huangqi Description. It is suggested that this phenomenon is related to the characteristics of tissue pathology of ulcerative colitis but with the medical treatment.6. Acute Toxicity Experiment of the Baizhu-Huangqi Prescription and Baizhu-Huangqi Decoction.Mice infused via mouth with the medicines were in normal condition, alive. The maximum dose of Baizhu-Huangqi Decoction that mice could stand once is 60g medicine substances/kg, equal to 180 times of clinical use;the maximum dose of Baizhu-Huangqi Description that mice could stand once is 4g extracted drug /kg, equal to 180g medicine substances/kg. It is suggested that the dose of the Baizhu-Huangqi Prescription or Baizhu-Huangqi Decoction be in safe scope.IV. Conclusions1. The procedures of rat ulcerative colitis caused by TNBS is similar to clinical ulcerative colitis as well as the etiology evolution of syndrome type of TCM, from acute inflammation, tissues putrescence to chronic inflammation and ulcer recovery. However, factors influencing the degrees of pathological changes are varied, including the dose ofmodel-building, the experimental circle, the animal weight and the species. As a result, the same animals need to be used in the trial experiment to determine the model-building schemes of the formal experiment. The effects of four model-building schemes were compared in this experiment. Animals with the right death rate were selected for the research of Chinese medicine treating syndrome according with Baizhu-Huangqi Description. In addition, the effect of the medicine was observed in different phases of pathological changes of ulcerative colitis, making the studies of pharmacology of TCM become closer to the clinical situation.2. The curative effects of Baizhu-Huangqi Prescription for ulcerative colitis is better than that of Baizhu-Huangqi Decoction. After the effective parts of the Baizhu-Huangqi Prescription is put into compatibility prescription of the parts, the effect of reducing colonitis mouse MPO is much better. Effective parts of various medicines in hinted prescription is put into compatibility description of the parts, and hence the aim of increasing effect is achieved. The toxicity of the Baizhu-Huangqi Prescription is not enhanced after the process of distilling effective parts.3. From the ulcerative colitis, the structure of tight junction of enterocyte have been damaged, which have been improved by the Baizhu-Huangqi Description. It is deduced that its function is likely to have got something to do with influence of the Prescription on the relevant claudin-1, occludin. The findings of Experiment 5 have only suggested that the claudin-1, occludin are index related to inflammation of ulcerative colitis. Not enough evidence has been found to prove that the Baizhu-Huangqi Description has direct influence on the protein claudin-1, occludin.4. As Chinese medicines for regulating Qi and strengthening spleen, the Baizhu-Huangqi Prescription is scientific, reasonable and safe after extracted, selected, and prescribed.
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