The Clinical Effect Of Gandoufumutang On Hepatolenticular Degeneration Hepatic Fibrosis And The Impact On TGF-β1/Smad Signaling

Objectives:(1) To observe the influence of serum markers of hepatic fibrosis and liver function of patients with hepatolenticular degeneration when using Gandoufumutang(GDFMT). Evaluating the effects of GDFMT when it is used to curing the patients which suffering from Wilson’s disease with liver symptoms. Discussing the molecular mechanism of Chinese medicine in GDFMT.(2)To observe how GDFMT at different concentrations can prevent and treat TX mices with hepatic fibrosis of Wilson’s disease through serum markers, pathomorphological targets and molecular biology targets. To research how GDFMT can influence the TGF-β1 / Smad signal pathway and revealing its clinical efficacy mechanism of TX mices with hepatic fibrosis in hepatolenticular degeneration.Methods:Clinical study:Sixty patients who are suffering from Wilson’s disease are divided into two groups randomly, half of them for the treatment group and the other people for the control group. There are also twenty healthy volunteers as parts to be the healthy group.Using GDFMT and DMPS for the treatment group.Liver-protecting tablets and DMPS are using for the control group. Eight days for a course of six days in which to be a copper vein row, two days to be calcium, a total of four treatments.To evaluate the effects of GDFMT and observe the integral of syndrome of Traditional Chinese Medicine, TNF‐α, TGF-β1, Serum copper, serum ceruloplasmin and serum copper oxidase, 24 h urinary copper, blood(RBC, WBC,PLT)liver function(ALT, AST) and the safety index.Laboratory experiments:Fourty-eight TX mices are randomly allocate to model group(physiological saline), salvia miltiorrhiza group, penicillamine group, group of high dose GDFMT, group of middle dose GDFMT and group of low dose GDFMT. Besides, separate eight DL mices were used as normal control group. There are totally seven groups in this experiment.(1) Serum markers:1) Using Reitman- Frankel method to detect the serum ALT and AST level.(the variation of liver function); 2) Using competitive radioimmunoassay to meature the serum level of HA, LN, I V – C and PCⅢ(the variation of serum markers of hepatic fibrosis).(2) Pathomorphological targets:The samples of HE and Masson in the liver are taken out for section and staining respectively(to observe the variation of pathological changes of hepatictissue).(3) Molecular biology targets:1)Using RT-PCR to detect the expression of hepatic tissue level, which includes TGF-β1, Smad2, Smad3, Smad4, Smad7 m RNA;2)Using the Western blot to detect the hepatic tissue level of TGF-β1, Smad2, Smad3, Smad4, Smad7, TβRⅠ, TβRⅡ(to observe the variation of TGF-β1 / Smad signal pathway).Results:(1)The clinicalpart: 1) There is no significant difference of patient’s TCM system score in GDFMT group and Hugan tablets group before treatment. Both GDFMT and Hugan tablets group a could significantly down-regulate the TCM score of these two group patients(P<0.05), and the decreased level in GDFMT group is much lower than Hugan tablets group(P<0.01). 2) Compared with healthy control group, serum level of TNF-α、TGF-β1 significantly increased(P<0.01) in GDFMT group and Hugan tablets group; Meanwhile, serum level of TNF-α、TGF-β1 obviously decreased after GDFMT therapy(P<0.01). Even there is a little decline in control group, but without statistical differences(P>0.05). GDFMT group compared to Hugan tablets group, GDFMT has an significant preponderance on decreasing serum TNF-α、TGF-β1. 3) Before any treatments were given, there is no significant difference of CER, TYHM and serum copper between GDFMT group and Hugan tablets group(P>0.05). We may concluded that GDFMT might not influence the serum level of ceruloplasmin, copper oxidase and copper of WD patients. 4) Tweenty-four hour urine cooper of patients significantly increased in GDFMT group and Hugan tablets group after treatment(P<0.01). And the increase of urine cooper in GDFMT has a significant difference in contrast to Hugan tablets group(P<0.01). 5) Before treatment,no significant difference of the level of RBC, WBC and PLT was found in GDFMT group and Hugan tablets group(P>0.05). However, after treatment, the level of RBC, WBC and PLT slightly decreased in GDFMT group, But not statistically significant(P>0.05),and significantly decreased in Hugan tablets group(P<0.01). After treatment, and there is a significant difference between GDFMT group and Hugan tablets group(P<0.05). 6) Before treatment, there is no difference of ALT, AST, BUN and Scr between GDFMT group and Hugan tablets group(P>0.05). After treatment with GDFMT, ALT and AST significantly declined(P<0.01), compared sligltly decreased in Hugan tablets group(P<0.05). But both Bun and Scr haven’t change too much and no significant difference(P>0.05). Therefore, after treatment, GDFMT could significantly change ALT and AST than Hugan tablets group(P>0.05).(2) Experimental section: 1) ALT and AST significantly increased in model group in contrast to normal group(P<0.01). Compared with model group, ALT significantly declined in each treatment group(P <0.05,P<0.01), however, there is no significant changes about AST after treatment with low dose of GDFMT(P<0.05). When compared with group of high does of GDFMT, both ALT and AST significantly decreased in each treatment group(P<0.05,P<0.01). 2) Compared to normal group, HA, LN,IV-C, and PCⅢ significantly increased in model group(P<0.01). There is a significant difference of the level of HA,LN,IV-C and PCⅢ in each treatment group(P<0.05,P<0.01),except for low dose of GDFMT when compared with model group(P<0.05). High dose of GDFMT can obviously decreased the level of HA, LN, IV-C and PCⅢ in comparison to penicillamine group(P<0.05). 3) HE staining: hepatic lobule is intact in normal group, liver cells arranged in order. There is no cell degeneration and necrosis in hepatic lobule, no inflammatory cell infiltration and no hyperemia, hemorrhage or edema in hepatic sinusoid position; Fibrous hyperplasia was not found in the area of hepatic lobules adjacent to the portal. No pathological changes of liver fibrosis on the whole. However, in TX model group, showed most visible normal lobular structure had been destroyed, and the number decreased. Typical pathological features of liver fibrosis appeared. Importantly, some specimens existed a significant pathological features of liver cirrhosis as the formation of false lobules. Liver cells have obvious edema and present as extensive fatty changes. Partial liver cells have been necrotized. The level of liver fibrosis in each treatment group has been alleviated. Even there are cell necrosis or degeneration in penicillamine group and GDFMT group. Although high-dose group had liver cell necrosis or degeneration, but hepatic cord structure still remained. There are fibrous tissue stretched to the portal area toward the portal area or lobular central vein, but did not completely surrounding the formation of the lobule. 4) Masson staining: hepatic lobular architecture is intact in normal group,no fibrous connective tissue hyperplasia. The hepatic lobular structure was destroyed in model group. Portal area was expanded significantly and there were significant fibrosis and significant fibrosis around central veins. Widened fibrous septum resulted to thickened of the central vein wall. Fibrous tissues, since the portal area and hyperplasia central vein extending to surround the pseudolobule. The fibrosis in high dose of GDFMT, moderate dose of GDFMT and penicillamine group is thin, blonder and significantly alleviated compared with the model group. Thin discontinuous fibers could be seen in periportal and hepatic lobular around the central vein area and extending to the surrounding, not completely split lobule. The color of fiber in periportal and lobular central vein was significantly lighter compared with the model group. 5) Compared to the normal group, the expression of TGF-β1、Smad2、Smad3、Smad4 m RNA was up-regulated in model group(P<0.01), but Smad7 m RNA is significantly down-regulated(P<0.01). While in comparsion to model group, TGF-β1、Smad2、Smad3、Smad4 m RNA were all down-regulated in high and moderate dose of GDFMT group, penicillamine group and salvia miltiorrhiza group. Simultaneously, Smad7 m RNA was upregulated(P<0.01). There was no significant difference of the expression level of all of these genes between lower dose of GDFMT group and model group(P>0.05). Compared to the high dose of GDFMT group, the expression of TGF-β1、Smad2、Smad3、Smad4 m RNA was up-regulated in moderate dose and lower dose group(P<0.01), but Smad7 m RNA is significantly down-regulated(P<0.01). The expression level of TGF-β1、Smad2、Smad3、Smad4 m RNA in salvia miltiorrhiza group and penicillamine group were much higher than high dose GDFMT group(P<0.01) while Smad7 is lower in both salvia miltiorrhiza group and penicillamine group(P<0.01). 6)TGF-β1、Smad2、Smad3、Smad4、TβRⅠ、TβRⅡ protein were enhanced in model group accompanied with lower Smad 7 expression(P<0.01) when compared to normal group. While in comparison to model group, TGF-β1、Smad2、Smad3、Smad4、 TβRⅠ、TβRⅡprotein were all decreased in each dose of GDFMT group, salvia miltiorrhiza group and penicillamine group(P<0.05, P<0.01). The quantity of these protein in lower and moderate dose group and salvia miltiorrhiza group were much higher than high dose group. And Smad 7 is much lower(P<0.01). There was no significant difference of these protein when compared to penicillamine group(P>0.05).Conclusion:(1) GDFMT can improve the clinical symptoms and signs of patients with WD effectively through perfect liver function(ALT, AST) and promote urinary copper excretion.(2) GDFMT can concentration-dependently decreases the expression of TGF-β1、TNF-α,thereby inhibit the occurrence and development of hepatic fibrosis.(3) GDFMT can improve liver function(ALT, AST) of TX mices with liver fibrosis and decreases the expression of HA、LN、IV-C、PCIII in TX mices serum.(4) GDFMT can improving the pathological state of liver tissue of TX mices with liver fibrosis.(5) GDFMT can inhibit the activation of TGF-β1/Smad pathway through decrease the expression of TGF-β1/Smad in liver tissue of TX mices. The result may reduce the activation of hepatic stellate cells and the secretion of extracellular matrix, thereby inhibit the occurrence and development of hepatic fibrosis.