| Research backgroundWilson’s disease is an autosomal recessive inherited disorder of copper metabolism. At present, the patients with the combination of WD and hypersplenia are more than a few. This kind of patients are suffering an immune dysfunction in their bodies, and presumably it may have a close connection with changes in the state of the splenic immune function. But its internal mechanism is not clear yet, and now there are very little relative literature being reported.As one of the few treatable neurogenetic diseases, Wilson’s disease is now mainly treated with copper ion chelating agent for removing copper, but the defect is having many side effects. As a traditional Chinese medicine, hepatic bean soup is proved to be effective clinically in treating Wilson’s disease with features of fewer adverse reactions,effect of lasting and moderate. According to dialectics and theoretical study of traditional Chinese medicine and clinical experience of several years, professor Hu Wenbin concludes that the clinical treatment by applying hepatic bean tablet made from the original prescription of hepatic bean decoction to Wilson’s disease has obtained a more satisfactory curative effect in terms of the copper displacement effect and the improvement of clinical symptoms. At present, the pharmacological mechanism of hepatic bean soup is still in the research stage. It is necessary to carry out the related research about whether it has treatment effect through regulating immune function of ailing spleen, namely Wilson’s disease.TX mice’s genetic background, copper biochemical characteristics and pathology features are similar to that of Wilson’s disease. Therefore, it is a relatively ideal animal model for the research of Wilson’s disease.Researches showed that the disorders of immune function of Wilson’s disease are related to several factors, such as the reduction of the ability of processing antigens caused by liver damage, intestinal endotoxemia, the disorders of cellular factors, portal hypertension with hypersplenia, the metabolism of microelementand biological conversion disorders, etc; spleen is the center of cell-mediated immunity and humoral immunity of human and other kinds of mammals(such as mice). It participates in the immunoregulation of T lymphocytes, adjusting the quantity and distribution of peripheral blood T cells; it participates in mediating the presenting response of B lymphocyte; participates in making immune substance such as immunoglobulin and alexin, accelerating phagocytosis, cleaning up the internal and external antigen; At the same time, splenic sinusoid also has the functions of storage of blood, adding blood volume if necessary, and englobing aging erythrocyte, causative agent and foreign matter. The disorder of the splenic immune function is the main reason of the occurrence and development of many diseases, and there is no relative report at present about whether it also has some important relation with the abnormal immune function of Wilson’s disease.The first part: Professor Yang Ren-ming’s theoretical basis and summary of clinical experience for applying hepatic bean soup(pill) to treat Wilson’s disease1. Summarize Professor Hu Wenbin’s basic viewpoints and methods in syndrome differentiation and treatment in TCM for Wilson’s disease;2. Summarize Professor Hu Wenbin’s clinical experience and research achievements of applying hepatic bean soup(pill) to Wilson’s disease;3. Summarize the improvements and treatment aspects of related immune index that hepatic bean soup has made on Wilson’s diseasePart 2: Experimental Research on the Influence of Hepatic Bean Soup to immune function of ailing spleen, namely Wilson’ DiseasePurpose:Understanding the splenic pathology, expressions of immune index and status of the metabolism of microelement of TX mice with Wilson’s disease model, and the effects and improvement of related index hepatic bean soup has on TX mice’s spleen.And also discussing the status of TX mice’s splenic immunity and related mechanism of therapeutical effect of hepatic bean soup with related documents.Method:1. The treatment group of hepatic bean soup: choose 40 TX mice at random and put them in the treatment group of hepatic bean soup, and two times per day, give them Chinese medicine of 0.2ml per time by gavage for 30 days on end; Model group:randomly select 40 TX mice as the TX model group, and feed them saline by gavage twice per day with 0.2ml per time, for 30 successive days. Normal reference group:randomly select 40 normal DL mice, and feed them normally for 30 successive days. In30 days, randomly select 20 mice from each group and kill them by pulling their necks,get and save their spleens as samples.10g10g2. Observe the histopathologic changes on the spleens of the TX mice in the Wilson’s disease model group, by doing HE staining for the frozen sections of spleens.3. Apply flow cytometry to detect the mice T lymphocyte subsets of splenic tissue CD4+, CD8+, CD4+/CD8+, and apply Western-Blot to detect the expression of interleukin-8(IL-8), interleukin-18(IL-18), to understand the influence and the improvement of the hepatic bean soup to the state of the mice’s splenic immunity ofthe model of Wilson’s disease TX.4. Detecting contents of microelement of Cu, Zn, Fe in splenic tissues of TX mice in each group of Wilson’s disease model by inductively coupled plasma atomic emission spectrometry, understanding the impact of hepatic bean soup on the metabolism of microelement of spleen of TX mice in Wilson’s disease model.Results:1. The pathological features and change of tissue of spleen of TX miceIn the TX group of hepatic bean soup, the dividing line of the white pulp and the red pulp in the HE staining sections of the splenic organs of mice is relatively clear.Compared with the normal control group, the number of the white pulp does not increase or decrease obviously and a lot of lymphocytes cluster in the white pulp; their splenic cords in red pulp area are thinner and less than those of the control group and the splenic sinusoids are indistinguishable from the cracks of slices; the morphological structure of trabeculae lienis is incomplete. In the 400 times of visual field, there are cells whose nucleuses are deeply dyed on the edge of white pulp, the number of macrophages seemingly increases, and several macrophages with abnormal shapes appear. On the HE dyed slice of TX mice’s spleen in the model group, it can be seen that the dividing line between white pulp and red pulp is still distinct, and several white pulp’s shape has been destroyed by crazes, and there is a huge amount of lymphocytes gathering in the white pulp; The number of splenic cords in the red pulp area is less than the former two groups, the shape is thinner, the number of splenic sinuses increases and obviously enlarges; unbroken spleen trabecula structure has not been found. In the 400 times of visual field, cells whose nucleus are deeply dyed can be seen around the white pulp, and the number of macrophages seemingly increases.2. The FCM detection of the expression of CD4+, CD8+and CD4+/CD8+inside the TXmice’s splenic tissues after the treatment of Gandou DecoctionThe T lymphocyte subpopulation expressed inside the splenic tissues of the three groups of mice CD4+, CD8+and CD4+/CD8+all have obvious statistical differences(the p values are 0, 0, 0.016 respectively, p<0.01). In splenic tissues, the model group’s TX mice are the highest in the expression of both CD4+and CD8+, and the normal control group’s DL mice are the lowest and the differences are statistically significant. While in the Gandou Decoction group’s and the model group’s TX mice’s spleens, CD4+/CD8+is obviously lower than the normal control group’s DL mice, and the differences are statistically significant; the CD4+/CD8+of Gandou Decoction group is slightly higher than the model group, but there is no statistical difference.3. The Western-Blot detection of the quantitative expression of proteins in IL-8and IL-18 in the TX inside the TX mice’s splenic tissues after the treatment of Gandou DecoctionAmong these three groups, the relative expression amounts of IL-18 in spleen tissues have some differences, which has statistic meanings(the value of p is 0.045, p<0.05); among which the IL-18 relative expression amounts of spleen tissues in the model group are higher than normal control groups, differences have statistic meanings; while the relative expression of the spleen tissue IL-18 between the model group and the Gandou Decoction group, and that between the Gandou Decoction group and the normal control group there is no statistical difference. Among the three groups, the relative expression quantity of IL-8 in the spleen tissues has remarkable differences, which has which has statistic meanings(p value is 0, p<0.01); among which splenic tissue IL-8 of the model group’s TX mice has obviously higher relative expression amount than other two groups(p values are respectively 0, 0; p<0.01),which has extremely obvious statistical differences; the relative quantitative expression of IL-8 in TX mice’s spleen tissues of the Gandou Decoction group is are higher than normal control groups, and the differences are statistically significant(p value is 0.04,p<0.05).4. The changes of trace elements in TX mice’s spleens after the Gandou Decoction treatmentSplenic Zn contents of three groups have no obvious statistical differences(F value is 0.24, p value is 0.787, p>0.05). In comparison, splenic Cu contents of three groups have extremely obvious statistical differences(H value is 15.53, p value is 0, p<0.01);among them, contents of Cu in the spleens of the model group’s TX mice are much higher than that in the other two groups, the difference has statistical significance(with values of p respectively 0.02, 0, p<0.05); the Cu contents in the spleens of the Gandou Decoction group’s TX mice are slightly are higher than normal control groups,but it has there is no statistical difference(p value is 0.6, p>0.05). There is relatively apparent difference among the contents of Fe in the three groups of spleens, and which has statistic meanings(H value is 7.088, p value is 0.029, p<0.05); the splenic Fe of the TX mice in the model group is are higher than normal control groups, and the difference has statistical significance(p value is 0.04, p<0.05); The splenic Fe of the TX mice in the Gandou Decoction group has there is no statistical difference when compared with the other two groups(p values are respectively 0.89, 0.13, p>0.05).Conclusion:1. The spleens of TX mice of WD model have abnormalities of red pulp structure,including splenic cord thinning, the number reducing, splenic sinusoids expansion; the the number of macrophages increases and some shapes are abnormal; some cells undergo deep dye in the nucleuses and other pathologic changes. A medium-short term treatment of Gandou Decoction does not have an obvious effect on TX mice’s spleen shape.2. The function of T lymphocytes in the spleens of TX mice is active and there exists the cellular immunity hyperfunction, but it’s mainly dominated by the negativeimmunoloregulation. The Gandou Decoction has improvement and regulation functions to the cellular immunity hyperfunctional state of TX mice, but it’s difficult to change the negative immune regulation of TX mice only relying on Gandou Decoction.3. The high expression of TX mice’s spleen cytokines IL-8, IL-18 further proves that the immunologic derangement state of cellular immunity hyperfunction exits. It may be relate to TX mice’s damaged tissue caused by copper deposition and related immunization injury mechanism of hepatic fibrosis. At the same time, the high expression of IL-18 also maybe a compensatory mechanism or negative feedback mechanism of the TX mice’s immune disorder, and it requires further study to confirm.Gandou Decoction has the effect on adjusting the immune dysfunction of TX mice cytokines; however, there is a limited adjusting capacity in a middle or short treatment for TX mice IL-8 and IL-18.4. The spleen of TX mice is one of the target organ where the copper is deposited which is caused by copper cycle disorders; the metabolic disorder of microelements like Cu and Zn of splenic tissues exists in TX mice, but the experimental data are still insufficient whether metabolic disorder of spleen Fe exists or not. It has been proved in the experiment that the Gandou Decoction indeed has relatively obvious therapeutic action of removing copper from TX mice; and maybe the Gandou Decoction also has the effect of flushing out Zn when flushing out copper; the impact of the Gandou Decoction on the metabolism of spleen Zn and spleen Fe of TX mice is insignificant. |
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