The Study On Mechanism Of Apoptosis Of Copper-induced Neuronal Damage By Traditional Chinese Medicine On TX Mice

BackgroundWD（Wilson’s disease） is one of the few remediable nervous system geneticdisease. If patients could be timely given diagnosis and treatment, most of them couldbe obtained good curative effect. Currently, it mainly treated by Penicillamine, PCAmetal complexing agent which is the representative of removing copper therapy. it wasstill ineffective to those WD patients who have serious neurological symptoms;although the copper metabolism can be corrected by removing copper therapy,neuronaldamage induced by copper can’t be terminated. The main treatment of WD is stilllifetime drug therapy. The therapy for cerebral WD is still a challenge for clinicians.Since the1970s, the topic of this unit is good curative effect on WD from TCMGanDouTang.For the patients with cerebral WD, it adds peony, dogwood notoginsengon the basis of the original TCM GDT. It has obtained some curative effect with themethod of "relieving and raising pulp". Although TCM GDT has obtained significanteffect on neuroprotective, such as antioxidant and radical scavenging, it is still notclear to neuronal damage induced by copper on the regulation mechanism of signaltransduction. This research intends to adopt the molecular regulatory mechanism ofGDT on the regulation mechanism of signal transduction of Wilson’s disease model-txmouse by immunohistochemistry, western blot and Real-time PCR, and clarify itsmechanism of improvement of neuronal damage induced by copper at the molecularlevel.Objectives1. To observe the changes of copper and zinc elements of tx mice before and aftertreatment of GDT, and to explore the mechanism of influence of GDT on tx mice.2. To observe the changes of liver function of tx mice before and after treatmentof GDT, and to explore the therapeutic effect of GDT. 3. To check out the expression level of XIAP, Caspase-9and Caspase-3in braintissue in tx mouse before and after treatments with GDT. Explore its mechanism ofsignaling pathways of WD in brain damage and the protection mechanism of adjustingthe brain damages of WD. Explore its protection mechanism of the caspase signalingpathways of GDT in the neuronal damage induced by copper at the molecular level.Methods1.Selecting1-month-old tx mice (140mice) and dividing into3groups randomly:GDT treatment group (40mice), butylphthalide group (40mice), model group(60mice). For GDT treatment group, feed0.2ml/10g GDT by gastric perfusion every dayand twice a day for2-4months. For model group, feed the same amount of saline bygastric perfusion every day and twice a day for2-4months. For butylphthalide group,put every0.1g drug dissolved in12.5ml of sesame oil and then feed0.2ml/10gbutylphthalide by gastric perfusion every day and twice a day for2-4months.2. Detecting the copper and zine elements content of tx mice between surem andbrain tissue by atomic absorption spectrophotometer.3.Testing the liver function of tx mice before and after treatment, observing thedifference and the curative effect of GDT.4. By researching immunohistochemical, western-blot and Real-time PCRmethods, it is to observe the expression of XIAP,Caspase-3and Caspase-9in braintissue of tx mice before and after treatments.Results1.The effect of GDT in copper related trace elements of tx mice1.1The serum copper: The serum copper level of GDT treatment group wassignificantly high compared with model group tx mice at the same mouths of age(P<0.01).The serum copper level of butylphthalide group has no significant change（P>0.05）. The serum copper level of model group tx mice was significantly lower compared with normal group DL mice at the same mouths of age (P<0.01).1.2The serum Zine: The serum Zine level of GDT treatment group was slightlylower compared with model group tx mice at the same mouths of age (P>0.05). Theserum Zine level of butylphthalide group has no significant change（P>0.05）.Theserum Zine level of model group tx mice was significantly lower compared withnormal group DL mice at the same mouths of age(P<0.01),1.3Copper in brain tissue: The copper level of GDT treatment group wassignificantly lower compared with model group tx mice in brain tissue at the samemouths of age (P<0.01). The copper level of butylphthalide group has no significantchange（P>0.05）.The copper level of model group tx mice was significantly highcompared with normal group DL mice in brain tissue at the same mouths ofage(P<0.01),1.4Zine in brain tissue: The Zine level of GDT treatment group was slightly highcompared with model group tx mice in brain tissue at the same mouths of age (P>0.05).The zine level of butylphthalide group has no significant change（P>0.05）. The Zinelevel of model group tx mice was significantly high compared with normal group DLmice in brain tissue at the same mouths of age(P<0.01),2The effect of GDT on serum liver function of tx mice. AST: The serum ASTlevel of model group tx mice was significantly high compared with normal group DLmice at the same mouths of age(P<0.01), The serum AST level of GDT treatmentgroup was significantly lower compared with model group tx mice at the same mouthsof age (P<0.01).The serum TBA level of model group tx mice was significantly highcompared with normal group DL mice at the same mouths of age(P<0.01), The serumTBA level of GDT treatment group was significantly lower compared with modelgroup tx mice at the same mouths of age (P<0.01).3The effect of GDT in related signaling pathway of tx mice3.1The expressions of XIAP, Caspase-3and Caspase-9protein in brain withimmunohistochemical method: The positive signals of each protein were tan-yellow in color, and there were visible positive expression in model group and each treatmentgroups. In brain, the XIAP protein positive expression are visible in the cytoplasm, theCaspase-3and Caspase-9protein positive expression are visible in the cellplasma.XIAP protein positive expression of GDT group tx mice was more than modelgroup tx mice at the same mouths of age, Caspase-3and Caspase-9protein positiveexpression of GDT group tx mice was less than model group tx mice at the samemouths of age.3.2The expressions of XIAP, Caspase-3and Caspase-9mRNA in brain withRT-PCR method: The expressions of XIAP, Caspase-3and Caspase-9mRNA in brainof normal group DL mice had no significant change between the groups (P>0.05). Theexpressions of XIAP mRNA in brain of model group tx (3months age) mice wassignificantly lower compared with mouse at one mouth of age (P<0.01). Theexpressions of XIAP mRNA in brain of model group tx (5months age) mice had nosignificant change compared with model group at3mouths age(P<0.05). With theincrease of month age of model group tx mice, the expressions of Caspase-3andCaspase-9mRNA in brain was increasing(P<0.05); With the increase of month age ofbutylphthalide group tx mice, the expressions of XIAP,Caspase-3and Caspase-9mRNA in brain of GDT group tx mice had no significant change between the groups(P>0.05); With the increase of month age of GDT group tx mice, the expressions ofXIAP,Caspase-3and Caspase-9mRNA in brain of GDT group tx mice had nosignificant change between the groups (P>0.05); The expressions of XIAP mRNA inbrain of model group tx (3months age) mice was significantly lower compared withnormal group DL mice at one mouth age(P<0.01). the expressions of XIAP mRNAwas declining, and The expressions of Caspase-3and Caspase-9mRNA in brain ofmodel group tx mice was significantly high compared with normal group DL mice atthe same mouths of age(P<0.01); The expressions of Caspase-3and Caspase-9mRNAin brain of GDT group tx mice was significantly lower compared with model group txmice at the same mouths of age(P<0.01);The expressions of XIAP mRNA in brain of GDT group tx mice had no significant change compared with butylphthalid group txmice at the same mouths of age(P<0.05);3.3The expressions of XIAP, Caspase-3and Caspase-9protein in brain withWestern-Blot method: The expressions of XIAP,Caspase-3and Caspase-9mRNA inbrain of normal group DL mice had no significant change between the groups (P>0.05).The expressions of XIAP mRNA in brain of model group tx (3months age) mice wassignificantly lower compared with mouse at one mouth of age(P<0.01). Theexpressions of XIAP mRNA in brain of model group tx (5months age) mice had nosignificant change compared with model group at3mouths age(P<0.05). With theincrease of month age of model group tx mice, the expressions of Caspase-3andCaspase-9mRNA in brain was increasing(P<0.05); With the increase of month age ofbutylphthalide group tx mice, the expressions of XIAP,Caspase-3and Caspase-9mRNA in brain of GDT group tx mice had no significant change between the groups(P>0.05); With the increase of month age of GDT group tx mice, the expressions ofXIAP,Caspase-3and Caspase-9mRNA in brain of GDT group tx mice had nosignificant change between the groups (P>0.05); The expressions of XIAP mRNA inbrain of model group tx (3months age) mice was significantly lower compared withnormal group DL mice at one mouth age(P<0.01). the expressions of XIAP mRNAwas declining, and The expressions of Caspase-3and Caspase-9mRNA in brain ofmodel group tx mice was significantly high compared with normal group DL mice atthe same mouths of age(P<0.01); The expressions of Caspase-3and Caspase-9mRNAin brain of GDT group tx mice was significantly lower compared with model group txmice at the same mouths of age(P<0.01);The expressions of XIAP mRNA in brain ofGDT group tx mice had no significant change compared with butylphthalid group txmice at the same mouths of age(P<0.05).Conclusions1. GDT could reduce the copper level in brain and adjust the metabolism of the zinc level of tx mice. This might be the most important reason for improving cerebralinjury.2. GDT could improve the liver function of tx mice, it indicates that GDT will notcause adverse effect on liver.3. The deposit of copper could induce the protein activation such as Caspase-3and Caspase-9protein in brain tissue. It indicates that neuronal damage induced bycopper associated with Caspase depended on apoptosis signaling pathways.4. GDT could reduce the expression level of XIAP protein in brain tissue, itindicates that copper could combine with XIAP protein and reduces the expression ofXIAP protein and thus it leads to neuronal damage.5. GDT could reduce the expression level of Caspase-3and Caspase-9protein inbrain tissue. It indicates that GDT could inhibit Caspase depended on apoptosissignaling pathways and thus inhibit neuronal damage.6. GDT can regulate the expression of XIAP protein in brain tissue of tx mice. Itindicates that GDT could reduce the content of copper in the brain tissue and reducethe combination of copper and XIAP protein and thus inhibit neuronal damage.