| Objectives:(1)Through the hepatolenticular degeneration(WD) patients with different treatment,observation of Gandoufumutang(GDFMT) in patients with WD TCM syndrome integral value, liver function and hepatic fibrosis serological index, serum ceruloplasmin, and the influence of serum copper, copper oxidase.Evaluating the effects of GDFMT when it is used to curing the patients which suffering from Wilson's disease with liver symptoms. Exploreing the molecular mechanism of Chinese medicine in GDFMT.(2)By serology and Western blot method to observe GDFMT under different Concentration, treatment cycle length of the WD model TX treatment of liver fibrosis in mice, confirmed GDFMT have reverse effect on TX mice with hepatic fibrosis of WD model, clarify GDFMT how molecular mechanism of reverse action influence on TX mice with hepatic fibrosis of WD model; Look for ways to reverse WD hepatic fibrosis treatment, lay a foundation for the creation of a reverse WD effective formula of hepatic fibrosis.Methods:Clinical part:Sixty patients who are suffering from Wilson's disease are divided into two groups randomly, half of them for the treatment group(GDFMT group) and the other people for the control group(Fuzhenghuayu capsule group).Using GDFMT for the treatment group.Fuzhenghuayu capsule is used to the control group. Two groups are both given foundation treatment with DMPS.Eight days is counted a course,in which six days to be a copper vein row, two days to be calcium, there are four sessions in total. Contrast TCM syndrome integral scores before and after treatment in WD patients, hepaticfibrosis index(serum HA, LN,PC- ?, C-IV), liver function(ALT, AST, TP, ALB),serum copper and serumceruloplasmin, serum copper oxidase and 24 h urine copper and other index changes.Experiment part:Ninety-six TX mice are randomly divided into model group, penicillamine group,danshen group, group of low dose GDFMT, group of middle dose GDFMT and group of hige dose GDFMT.Besides, there are sixteen DL mice used as normal control group.There are seven groups totally in this experiment.(1)Reitman method is used to detect serum ALT, AST and coomassie brilliant blue method to detect serum TP, bromocresol green method to detect serum ALB;(2) Using the method of competitive set free detection of serum HA, LN, PC-?,C-IV content;(3)Using Western blot detection of liver tissue TGF-?1,T?R?,T?R?, Smad2, Smad3,Smad4 and Smad7 protein content.Results:(1)The clinical part:1) clinical curative effect evaluation: After four courses of treatment, the treatment group that DMPS joint GDFMT clinical total curative effect is better than control group DMPS is with Fuzhenghuayu capsule group;2)symptom score values:There are no difference between two groups of patients in TCM syndrome integral value before the treatment(P > 0.05); Compared with group before treatment, TCM syndrome integral values are lower both the two groups after the treatment, with statistical significance(P < 0.01, P < 0.05); Compared with two groups after treatment, the treatment group improved integral value more significantly than the control group(P < 0.01).3)serum HA, LN, C- IV, PC- ?: Before treatment, two groups of HA, LN, C- IV, PC- ? value had no significant difference(P > 0.05); Within group after treatment, thevalue of HA, LN, the C- IV, PC- ?of the treatment group was significantly lower(P <0.01).There is a decrease in the control group before the treatment, and was statistically significant(P < 0.01, P < 0.05); Comparison between groups after treatment, the treatment group improved more obviously than the control group, two groups compare difference was statistically significant(P < 0.01). So GDFMT has good effect on anting hepatic fibrosis.4)liver function: 2 group serum ALT, AST, TP, ALB value treatment differences were not statistically significant(P> 0.05); After treatment, serum ALT, AST decreased significantly, TP, ALB increased significantly, indicating that the treatment group liver functional indexes before treatment comparison was a significant difference(P <0.01);there was significant difference(P <0.05) the control group liver function Yijiao treatment; between the two groups after the treatment group, treatment group, liver function(ALT, AST, TP, ALB) to improve the situation significantly more than the control group(P <0.01)5)serum ceruloplasmin, serum copper and copper oxidase:There is no significant difference of serum ceruloplasmin, serum copper and copper oxidase the treatment group and control group before and after treatment(P > 0.05); Comparison between the treatment group and the control group, two groups had no significant difference after treatment(P > 0.05), showed that GDFMT had no obvious effect on the three indexes in WD patients.6)24h urine copper: Compared with the same group before treatment, the 24 h urine copper in both two groups after treatment significantly increases, there are significant difference statistically significant( P < 0.01); Between the treatment groups, each course at the end of 24 h urinary copper treatment group were significantly higher than the corresponding control group(P < 0.01). That is to say GDFMT can significantly improve patients with WD copper content.7)electrocardiogram, blood, urine, stool and renal function: 2 WD group of patients before and after treatment ECG, blood, urine, stool and renal function showed no abnormal changes.(2)Experiment part:1)serum HA, LN, PC ?, C- IV:(1) Model group two weeks, four weeks, six weeks with the corresponding normal four weeks, 6 weeks group, model group TX mice serum HA, LN, PC ?, C- IV content increased significantly(P < 0.01), suggesting the WD model TX mice obvious liver damage.(2) with the corresponding model group two weeks, four weeks, six weeks, GDFMT high, middle dose group, danshen group and penicillamine group groups of HA, LN, PC ?, C- IV decreased(P < 0.05, P < 0.01),serum HA, LN, PC ? GDFMT two weeks, C- IV have fallen, but there was no statistically significant difference(P > 0.05).(3) with the corresponding salvia miltiorrhiza group two weeks, four weeks, six weeks group comparison, GDFMT high and low dose groups, serum HA, LN, PC ?, C- IV compared with salvia miltiorrhiza group each group difference was statistically significant(P < 0.05, P < 0.01); GDFMT in each dose group compared with salvia miltiorrhiza group, each group has no statistical significance(P > 0.05).(4) and penicillamine group two weeks, four weeks,six weeks, GDFMT groups of high, middle dose group compared with the corresponding penicillamine group of HA, LN, PC ?, C- IV significantly decreased(P< 0.05, P < 0.01).(5) GDFMT high-dose group two weeks, four weeks, six weeks with the corresponding middle and low dose groups between two groups of comparison, the difference was statistically significant(P < 0.05, P < 0.01).(6)two weeks corresponding GDFMT compare high, medium and low dose group, 4 weeks between groups of serum HA, LN, PC- ?, C- IV were significantly lower(P < 0.05).(7) with the corresponding GDFMT high, medium and low dose groups of four weeks, six weeks between groups of serum HA, LN, PC- ?, C- IV were significantly lower(P < 0.05).2) serum AST, ALT vitality:(1) Model group two weeks, four weeks, six weeks with the corresponding normal two weeks, four weeks, six weeks group, model group TX mouse serum ALT, AST activity increased significantly(P < 0.01), suggesting the WD model TX obvious liver damage in mice.(2) model group two weeks, four weeks, six weeks,and the corresponding GDFMT high, medium and low dose group, danshen group and penicillamine group two weeks, four weeks, six weeks, GDFMT high, medium and low dose group, danshen group ALT, AST and penicillamine group were decreased(P < 0.05,P < 0.01).(3) with the corresponding salvia miltiorrhiza group two weeks, four weeks,six weeks, GDFMT groups of serum ALT, AST energy high dose group were significantly lower in the corresponding salvia miltiorrhiza group(P < 0.05); GDFMT dose groups and the corresponding salvia miltiorrhiza group in each group comparison difference has no statistical significance(P > 0.05).(4) Corresponding penicillamine group two weeks, four weeks comparison, six weeks, GDFMT high dose group in each group was statistically significant(P <0.05, P <0.01) and the difference between groups penicillamine group; GDFMT low differences between the groups for each dose group with the corresponding penicillamine group was not statistically significant(P> 0.05).(5) GDFMT high-dose group two weeks, four weeks, six weeks with the corresponding middle dose group and low dose group between two groups of comparison, the difference was statistically significant(P < 0.01, P < 0.05). four and six weeks GDFMT high, medium and low dose group and the corresponding GDFMT two weeks was high,medium and low dose group, four weeks between groups of serum ALT, AST vitality,though to some extent to drop, but there was no statistically significant difference(P >0.05). Cruelly GDFMT six weeks of high, medium and low dose group and the corresponding GDFMT high, medium and low dose group four weeks, 6 weeks between groups of serum ALT, AST activity were significantly lower(P < 0.05).3) the content of serum TP, ALB:(1) model group two weeks, four weeks, 6 weeks with the corresponding normal two weeks, four weeks, six weeks group, model group TX mice serum TP, propagated content significantly decreased(P < 0.01), suggesting the WD model TX mice liver protein synthesis ability to drop, obvious phenomenon of liverdamage.(2) with the corresponding model group two weeks, four weeks, 6 weeks,GDFMT TP groups in terms of high, middle dose group and salvia miltiorrhiza group,propagated content were significantly increased(P < 0.01), GDFMT low dose group and penicillamine group two weeks of TP, although have propagated content increases,but no statistical significance(P > 0.05).(3) with the corresponding salvia miltiorrhiza group two weeks, four weeks, six weeks, GDFMT group serum TP, propagated content high dose group were between groups increased significantly in the corresponding salvia miltiorrhiza group(P < 0.01); GDFMT dose groups and the corresponding salvia miltiorrhiza group in each group comparison difference has no statistical significance(P> 0.05).(4) with the corresponding penicillamine group two weeks, four weeks, six weeks, GDFMT high dose group, middle dose group two weeks, four weeks, six weeks from the corresponding TP and propagated penicillamine group were significantly higher(P < 0.01, P < 0.05). GDFMT high dose group and middle dose group, low dose group between two comparison, the difference was statistically significant(P <0.01, P < 0.05).(6) with the corresponding GDFMT two weeks was high, medium and low dose group, serum TP GDFMT four weeks each, propagated content were significantly higher(P < 0.05).(7)with the corresponding GDFMT four weeks was high,medium and low dose group, GDFMT six weeks group serum TP, propagated content were significantly increased(P < 0.05).4) TGF-?1,T?R?,T?R?, Smad2, Smad3,Smad4 and Smad7 protein expression: With the corresponding normal group two weeks, four weeks, six weeks, TGF-?1,T?R?,T?R?, Smad2, Smad3 and Smad4 protein expression of each group of model group significantly increased, Smad7 protein expression significantly decreased(P < 0.01),suggesting the WD model TX obvious liver fibrosis in mice.(2) with the corresponding model group two weeks, four weeks, six weeks, GDFMT high, medium and low dose group, danshen group and penicillamine group each group TGF-?1,T?R ?,T?R ?,Smad2 and Smad3 and Smad4 protein expression decreased, Smad7 protein expression is higher, the difference was statistically significant(P < 0.05, P < 0.01).(3) salvia miltiorrhiza group two weeks, four weeks, six weeks with the corresponding GDFMTgroups of high and low dose group, the difference was statistically significant(P < 0.01);Salvia miltiorrhiza group two weeks, four weeks, six weeks with the corresponding GDFMT dose group, there was no statistically significant difference(P > 0.05).(4)penicillamine group two weeks, four weeks, six weeks with the corresponding GDFMT high, middle dose group of comparison difference was statistically significant(P < 0.05,P < 0.01); Penicillamine group two weeks, four weeks, six weeks with the corresponding GDFMT low dose group of comparison difference has no statistical significance(P > 0.05). GDFMT high-dose group two weeks, four weeks, six weeks with the corresponding in the comparison group, low dose group, high dose groups,TGF-?1,T?R ?,T?R ?, Smad2, Smad3 and Smad4 protein expression were significantly lower, Smad7 protein expression was significantly increased(P < 0.01).Four weeks GDFMT high, medium and low dose group and the corresponding GDFMT two weeks was high, medium and low dose group, TGF-?1,T?R?,T?R?, Smad2 and Smad3 and Smad4 in four weeks each protein expression were significantly lower,Smad7 protein expression was significantly increased(P < 0.05, P < 0.01). All landowners GDFMT six weeks of high, medium and low dose group and the corresponding GDFMT four weeks was high, medium and low dose group,TGF-?1,T?R ?,T?R ?, Smad2 and Smad3 and Smad4 in six weeks each protein expression were significantly lower, Smad7 protein expression was significantly higher(P < 0.05, P < 0.01).Conclusion:(1)GDFMT can effectively improve the clinical symptoms of WD patients with liver fibrosis and improve liver function(ALT, AST, TP, ALB), increasing the 24 h urinary copper excretion.(2)WD GDFMT can effectively reduce serum fibrosis patients with liver fibrosis four indicators(HA, LN, PC-III, C-IV), which play the role of anti-fibrosis.(3)GDFMT can effectively improve liver function WD TX model mice; and can reduce TX liver fibrosis serum markers(HA, LN, IV-C, PCIII), it has a good explanation GDFMT hepatic fibrosis; and showed a dose- dependent aging.(4) GDFMT can regulate the expression of TGF-?1 and Smads, thereby inhibiting TGF-?1 / Smad signaling activated, preventing the occurrence and development of hepatic fibrosis, and on the TGF-?1 / Smad signaling in the regulation of a dose- aging dependence. |
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