| BackgroundsWilson’s disease(WD) is one of a small number of neurological genetic diseases canbe treated. At present, penicillamine(PCA) as the representative of the metal chelatingagent, although having a high copper content in urine discharge, and symptomsimproved significantly, etc., but it has more adverse reactions. Zinc as therepresentative of reduces copper absorption agent, has little adverse drug reactions, butits capability to drive copper is weak. So it usually used for presymptomatic patients ormaintenance treatment. Therefore, the treatment of WD urgent need appear effectivetherapeutic drugs with fewer side effects.Tetrathiomolybdate is a chelator of copper ions, absorbed into the bloodstream toform a group of the three non-toxic complexes with albumin and serum copper, andinhibited competitively the copper in intestinal absorption, which can effectivelypromote copper excretion, and reduce the concentration of free copper. Currently,Tetrathiomolybdate has been reported successful in treatment of WD, and found thatTetrathiomolybdate have a certain effect in treatment of liver and brain damage. Thedrug is still in clinical trials, so the specific mechanism of efficacy and adversereactions is unclear.The liver and kidney tissues of copper load model rat can produce copper depositionsimilar to WD. And the copper load model rat has a low price, good reproducibility,easy to obtain, etc., is a good animal model to study pathology, pathogenesis andanimal Therapeutics of WD.Studies have shown that liver damage is the most common clinical manifestations ofWD patients, and liver fibrosis is the most common pathological changes. P38, ERKand JNK as leading member of mitogen-activated protein kinase pathway have beenfound that play an important role in development of liver fibrosis. Another study foundthat excess copper can lead to damage free radical of nerve cells, activate the MAPKpathway and other intracellular signaling cascade of proteins and enzymes, regulate nuclear transcription factors and gene expression, intracellular calcium overload, andresult in nerve cell degeneration, necrosis, and on this basis to activate c-fos, c-jun,c-myc gene expression, etc., ultimum induced neuronal apoptosis. Whethertetrathiomolybdate adjust the MAPK signaling pathways on the base of chelatingcopper ions, to play the role of repairing the liver and brain tissue has not beenreportedObjectives1. To observe the changes of copper and other trace elements of copper overloadedrats influenced bytetrathiomolybdate, and to explore the mechanism of influence ofmolybdenum on other trace elements;2. To observe the changes of blood routine, liver function and kidney function,influenced by tetrathiomolybdate, and to observe the adverse reactions oftetrathiomolybdate;3. To check out the expression level of p38, ERK, JNK protein in liver and braintissue of copper overload rats before and after treatments, in order to explore the roleof P38, ERK, JNK in the process of WD liver damage and central nervous systemdamage and their regulation mechanisms; To analysis the impact of tetrathiomolybdateon the expression of p38, ERK, JNK in the liver, brain tissues of copper overload rats,and to provide experimental and theoretical basis to drug treatment for Wilson′sdisease.Methods1. The140Sprague-Dawley rats were randomly divided into5groups：normalcontrol group, copper load model group, Tetrathiomolybdate group, Penicillaminegroup and gandou decoction group., etc. Penicillamine and gandou decoction used aspositive control drug. According to pre-establish the copper loaded rats model byfeeding containing copper sulfate a1g/kg feed and0.185%copper sulfate water. Therearing environment of Normal control group was the same to all treatment groups,and ordinary normal feeding, gavaged saline0.2ml/10g once daily for4weeks. Copper load model group gavaged0.185%copper sulfate0.2ml/10g once daily andfed feed containing copper sulfate1g/kg for4weeks. Tetrathiomolybdate groupgavaged Tetrathiomolybdate once daily by2mg/kg for4weeks; Penicillamine groupgavaged Penicillamine once daily by0.09g/kg for4weeks; Gandou decoction groupgavaged. gandou decoction once daily by0.2ml/10g for4weeks, and behavior andother changes.of all of rats were observed.2. The trace elements in serum, liver tissue and brain tissue were detected byinductively coupled plasma-atomic emission spectrometry and inductively coupledplasma-mass spectrometry, to analysis the correlation between molybdenum andcopper and other trace elements.3. The blood routine, liver function and kidney function of copper overloadedrats were detected before and after treatments, to explore the adverse reactions oftetrathiomolybdate.4. Expression of p38, ERK, JNK protein in liver and brain tissue of copperoverload rats before and after treatments were observed with immunohistochemicaland western-blot methods. To explore the mechanism of the tetrathiomolybdate inthe repair of liver and brain damage of copper overload rats.Results1. The general status of model group，including body weight, fur, mental state and soon, were poorer than that of the control group, but it was improved after treatments.2. The serum copper level of copper overload rats of12weeks was significantlyhigh compared with the4,8,16weeks′copper overload rats（P＜0.05）, up to nearly2times.3. Serum trace elements: the serum Cu of tetrathiomolybdate group was significantlylower than the model group（P<0.01）, but there were no significant changes comparedwith penieillamine group and gandou decoction group（P>0.05）；The serum Zn oftetrathiomolybdate group increased as compared with the model group (P<0.05); Theserum Fe of tetrathiomolybdate group was significantly lower than penieillamine group (P<0.01); Levels of serum Mo of model group content with the normal group and thewere significantly lower (P<0.01); The serum Se of tetrathiomolybdate group wassignificantly higher than that of normal group and model group(P<0.01); The serumNi of tetrathiomolybdate group were significantly higher than the normal group, modelgroup, gandou decoction group (P<0.05).4. Trace elements in liver tissue: The content of Liver Cu of tetrathiomolybdategroup compared with the model group were significantly lower (P<0.01), comparedwith penieillamine group, gandou decoction group was lightly lower (P>0.05); Thecontent of Liver Zn of tetrathiomolybdate group was no significant change comparedwith the other groups (P>0.05); The content of Liver Fe of tetrathiomolybdate groupwere significantly increased compared with the other groups (P <0.01); The content ofLiver Mo of tetrathiomolybdate group were significantly increased than the othergroups (P<0.01); The content of Liver Al of tetrathiomolybdate group compared withthe model group were significantly lower (P<0.01); The content of Liver I oftetrathiomolybdate group compared to the other groups were significantly increased(P<0.01); The content of Liver Pb of tetrathiomolybdate group compared with themodel group was significantly lower (P<0.01).5. Trace elements in brain tissue: The content of brain Cu of tetrathiomolybdategroup compared with the model group, gandou decoction group were significantlylower (P<0.05); The content of brain Zn of tetrathiomolybdate group compared withthe other groups were significantly lower (P<0.05); The content of brain Fe oftetrathiomolybdate group compared with the model group were significantly lower (P<0.01); The content of brain Al of tetrathiomolybdate group were significantly lowerthan model group (P<0.01); The content of brain Pb of tetrathiomolybdate groupcompared with the model group and gandou decoction group were significantly lower(P<0.01).6. All of blood routine, liver function and kidney function of tetrathiomolybdategroup were within normal range, compared with the normal group had no significant changes.7. The expressions of p38, ERK, JNK signaling pathway were detected withimmunohistochemical method: The positive signal of each proteins were tan-yellow incolor, and there were visible positive expression in model group and each treatmentgroups. The datas of all of p38, ERK, JNK protein expression in liver tissue oftetrathiomolybdate group were significantly enhanced than penieillamine group(P<0.05); But the datas of all of p38, ERK, JNK protein expression in brain tissue oftetrathiomolybdate group were decreased more than penieillamine group(P<0.05); TheJNK protein had no significant positive cells in the brain tissue.8. The expressions of p38, ERK, JNK signaling pathway were detected withwestern-blot method: There were significant differences in all of p38, ERK, JNKprotein levels between the groups. Compareed with the other groups, there were visiblespecific protein strip expressions of p38, ERK, JNK in model group（P<0.01）.Compareed with the model group, specific protein strip expressions of p38, ERK, JNKin tetrathiomolybdate group, penieillamine group and gandou decoction group weresignificantly depressed（P<0.01）. All of p38, ERK, JNK protein strip were expressedslightly increase in tetrathiomolybdate group, compared to penieillamine group（P>0.05）. There was hardly any strip expressions of JNK in the brain tissue of anygroups.Conclusions1. Copper load causes excessive accumulation of copper in liver and brain tissues ofrats, and can lead to appearance liver and brain tissue damage performance, such asliver dysfunction and behavioral abnormalities.2. The P38, ERK, JNK and other signaling pathways of rats liver and brain tissueswere activated by copper load, and causing liver and brain cell damage.3. The adverse drug reactions, such as blood routine, liver function andkidney function, had no obvious abnormalities after treatment of tetrathiomolybdate.4. The content of copper in the serum, liver and brain of copper overload rats were reduced by tetrathiomolybdate(Compared with penieillamine, the effectiveness oftetrathiomolybdate to drive the copper was no significant difference), which was themain reason for improve the damage of liver and brain tissue.5. T There were positively correlations between molybdenum and copper, zinc,ferrum, etc other trace elements in serum of copper overload rats after treatment bytetrathiomolybdate; The antagonistic impact of molybdenum on these trace elementsmay be involved in the mechanisms of liver cell injury and cell damage of the centralnervous.6. The levels of phosphorylation of P38, ERK, JNK proteins in liver and basalganglia of the brain of copper overload rats were significantly increased treated bytetrathiomolybdate, which was the important reason for improve the damage of liverand brain tissue. |
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