Immunohistochemical Studies Of Gandoufumutang Towards Hepatic Fibrosis In TX Mice,which Based On Clinical Syndrome Features Of Hepatic Fibrosis Of Wilson Disease

Objectives:(1)As the research object with WD liver fibrosis patients, on the basis of fully collected clinical data, using epidemiological and clinical analysis of mathematical statistics method, carry out the WD study of syndromes of liver fibrosis, in order to reveal the basic syndrome characteristics of traditional Chinese medicine of WD liver fibrosis, in order to further put forward quantitative WD liver fibrosis, which provide the basis for deepening traditional Chinese treatment of WD liver fibrosis.(2)To observe how GDFMT at different concentrations and different time intervention can show the effect of anti-hepatibrosis by using TX mice as the research object.Use the immunohistochemical method to detect the TGF-β1,TβRⅠ,TβRⅡ,Smad2,Smad3,Smad4, Smad7 protein expression of liver,and revealing the possible molecular mechanisms of GDFMT towards the effect of anti-hepatibrosis.Methods:(1)Clinical study:To conform to the inclusion criteria of 118 cases of patients with WD of liver fibrosis according to the design of WD liver fibrosis, questionnaires on common TCM symptoms, signs and demographic information,and using cluster analysis and discriminant analysis of mathematical statistics analysis method, observing clinical symptoms and syndrome distribution regular and characteristics of the WD liver fibrosis.(2)Laboratory experiments:96 male TX mice are randomly allocated to model group(physiological saline),penicillamine group, Danshen granules group, group of low dose GDFMT, group of middle dose GDFMT and group of high dose GDFMT.Besides, another 16 DL mice are used as normal control group.Totally seven groups of mice are given corresponding drug perfusion treatment one times a day and 28 days in a row.The mice were sacrificed eight of a group each time at the end of 2,4 weeks after drug perfusion treatment.Use the immunohistochemical method to detect the TGF-β1,TβR Ⅰ,TβRⅡ,Smad2,Smad3, Smad4,Smad7 protein expression of liver tissue of mice.Results:(1)The clinical part:The WD liver fibrosis involves four visceral:tliver, kidney, spleen, heart.The lesions in the four visceral in order for the liver, kidney, spleen, heart,among them liver involving highest frequency. For the factors of the pathological change nature is deficiency and excess,virtual mainly for liver and kidney deficiency, spleen deficiency,firm mainly for phlegm turbidity implication, blood stasis resistance.What to see more is aspected in liver and kidney deficiency, phlegm and blood stasis syndrome. The syndromes distribution of WD liver fibrosis is associated with patients’ age and course of the disease, has nothing to do with gender.(2) Experimental section:1) Both 2 and 4 weeks of treatment show the same results as follows.The expression of TGF-β1,TβRI,TβRII,Smad2,Smad3 and Smad4 significantly increase in model group while the Smad 7 decrease(P<0.01) when compared to normal group. While in comparison to model group, TGF-β1, TβRI, TβRII, Smad2, Smad3 and Smad4 decrease and the Smad7 increase in each dose of GDFMT group, Danshen granules group and penicillamine group(P<0.05, P<0.01). The quantity of TGF-β1, TβRI, TβRII,Smad2, Smad3, Smad4 in high dose group are much lower than Danshen granules group, penicillamine group,lower and moderate dose group,and Smad 7 is much higher(P<0.01). The expression of TGF-β1,TβRI,TβRII,Smad2,Smad3 and Smad4 significantly decrease in Danshen granules group and moderate dose group while the Smad 7 increase(P<0.05, P<0.01) when compared to penicillamine group and lower dose group. There is no significant difference of these proteins in moderate dose group and Danshen granules group(P>0.05).While in comparison to low dose group, TGF-β1,TβRI, TβRII, Smad2, Smad3 and Smad4 decrease and the Smad7 increase in penicillamine group(P<0.05, P<0.01).2) Compared with the results of treatment for 2 weeks, the expression of TGF-β1,TβRI, TβRII, Smad2, Smad3 and Smad4 are down-regulated by the end of 4 weeks,but Smad7 is significantly up-regulated(P<0.05,P<0.01).Conclusion:(1) The WD liver fibrosis is characterized by liver and kidney deficiency, phlegm and blood stasis. The lesions in the four visceral in order for the liver, kidney, spleen,heart.The distribution of syndromes associated with WD liver fibrosis in patients with age and course of the disease, has nothing to do with gender.(2) GDFMT can decrease the expression of TGF-β1,TβRI,TβRII,Smad2,Smad3,Smad4 and increase the expression of Smad7, significantly depending on dose and time of treatment.Thereby, it may reduce the activation of hepatic stellate cells and the secretion of extracellular matrix, and play the role of anti hepatic fibrosis.