| Brackground:Hepatolenticular degeneration also called Wilson’s disease, is a kind of autosomalrecessive disorder of copper metabolism, and clinically its main symptoms includeliver damage, neuropsychiatric symptoms, extrapyramidal syndrome, corneal K-Fring and so on, and it will also be life-threatening under worse conditions. However,Wilson’s disease is one of few nerve genetic disorders which can be cured. Currently,the metal chelator, mainly represented by penicillamine has certain curative effects,but it has too much toxic and side effect, which can worsen patients’ state andprognosis, so lots of patients can’t stand it. As a result, it is badly in need of newprescriptions and medicine to relieve the suffering of the patients with WD.The workunit where the research group belongs find out that gandou decoction has a certaineffect on curing WD through almost30-year research, and the improved prescriptionbased on this has a better effect on curing patients with WD. Although it is found thatthe improved prescription has a prominent role on wiping off copper, yet the exactmechanism for producing curative effect is still not clear. With the use of RT-PCR,immunohistochemistry and Western blot in this study, we aim at observing the signaltransduction mechanism of how hepatic bean soup protects neuronal injury in mice ofWD mode-TX, and clarifying preliminarily the effect mechanism of how the beansoup improves clinical symptoms of patients with WD.Objective：1. To observe how the modified Gandou decoction cures TX mice before and afterserum in Cu, brain tissue Cu, the nerve behavior levels, brain tissue morphology,andthen discuss the possible mechanism of the change.2. To observe expressional level of how the modified gandou decoction cures TXbrain tissue of the mice before and after, like ASM, CER, JNK and P38albumen, and then discuss the functional mechanism about how ceramide signal transductionpathways get involved in cerebral injury and discuss the defense mechanism abouthow the modified gandou decoction adjust the signal pathway above, in order toprovide the theoretical foundation for how the modified gandou decoction cures WD.Methods:1. Divide180TX mice into TX model group, gandou decoction treatment group,and butylphthalide control group in random, and choose60DL mice as normalcontrol group at the same time. with butylphthalide positive control drug, DL controlgroup and TX model group are the same as the gandou decoction group in feedingenvironment and food. The mice are given normal saline0.2ml/10g/each time twice aday in gavage, for2—4months successionally. In butylphthalide control group, TXmice are fed with pellet feed, dissolving every0.1g butylphthalide in12.5ml ediblesesame oil to make suspension, and then feeding mice with80mg/kg in butylphthalidein gavage. According to the weight of the mice, they are given0.2ml/10g each time,and it will be given twice a day for2to4months successionally. And the gandoudecoction group is just the same as the control group above.2. Observe the nerve behavior levels score before and after treatment in each groupof mice.3. Test the content of serum Cu and brain tissue Cu in the mice before and after thetreatment, and observe the therapeutic effect of the improved treatment.4. Test the brain tissue ASM, Cer, P38of TX mice and the expressional level ofJNK albumen by immunohistochemical-DAB staining method and Western-Blot andtest ASMnRNA, CermRNA, JNKmRNA, and expressional level of P38Mrna, and atlast discuss the signal transduction mechanism of how the improved prescriptionadjusts how much the above albumen protects brain injury. Results:1. The modified gandou decoction has an influence on the neuronal injury of TXmice.1.1The nerve behavior levels score of TX mice are in unnormal, while those of DLmice are under normal,but improved after treatment.1.2After treatment, copper content in serum is higher than that in TX model group,3-month treatment (P<0.05),5-month treatment (P<0.01); comparing with3ages ofthe moon in DL control group, copper content in serum is low, after5months’treatment, it’s even lower, but it has no statistical significance; so goes the same withbutylphthalide control group.1.3After treatment, the content of brain tissue Cu in TX mice is lower than that inTX model group,3-month treatment (P<0.05),5-month treatment (P<0.05);comparing with DL control group, the content of brain tissue Cu rises, the3and5ages of the moon both have remarkable statistical significance; while comparing withbutylphthalide control group, the content reduces, it still has no statisticalsignificance.1.4TX HE staining of mouse brain tissue at high magnification observation:1-month TX model of brain tissue seen among a small group of nerve cells andinterstitial edema, nuclear stain, shrinkage,3-month and5-month old boundaries arenot nerve cells in mice Ching, cell spacing increased, a large number of deeply stainedkaryopyknosis neurons, a small amount of scattered neuronal degeneration andnecrosis; treatment of modified gandou decoction, liver neuronal injury treatmentgroup improved compared with the model TX group.2. The mechanism of how the modified gandou decoction regulates the ceramidecascade signal transduction pathways inside and outside cells of copper inducedneuronal damage.2.1Test ASM, Cer, JNK, P38expression by immunohistochemistry:1) positiveexpression of each albumen is claybank, ASM, Cer and P38in TX model group andevery treatment are all positive cellular expression. Positive expression in TX modelgroup enhances notably comparing with the other groups (P <0.01); with the same age of moon, positive expression in treatment is a little higher than that in butylphthalidecontrol group, but it has no statistical differences.2) JNK in DL control group, TXmodel group and each treatment is always positive cellular expression, yet positiveexpression of JNK in TX model has no statistical significance comparing with theother groups.2.2RT-PCR tests ASMmRNA、CermRNA、JNKmRNA、and P38Mrna expressionin the brain tissue of TX mice in each group; ASMmRNA、CermRNA、JNKmRNA、and P38mRNA in TX model group are much more than those in other groups,3and5ages of moon both have statistical significance (P<0.05); they in TX are a little higherthan those in other groups, but it has no statistical significance.2.3Using Western-Blot method, test ASM, Cer,P38and proteic expression of JNK:the proteic expression in TX model group enhances notably comparing with normalgroups (P<0.01); After3-month treatment, positive albumen relative transcript levelin treatment group and butylphthalide control group reduces (P<0.05) comparing withTX mode group, after5-month treatment, the relative transcript level declinesdramatically (P<0.01); the relative transcript level of positive albumen in treatmentgroup is a little higher than that in butylphthalide control group, but still it has nostatistical differences.Conclusion1. The excessive accumulation in copper in brain tissue and in vivo of TX mice cancause abnormity in mice’s behaviors and liver function and brain injury.2. Copper load can activate ceramide signaling pathways of brain tissue, and thencause cell damage.3. The modified gandou decoction could improve the behaviors of TX mice.4. The modified gandou decoction can reduce the content of serum and brain tissue Cuin TX mice, which is the main reason for improving brain tissue damage.5. The modified gandou decoction can lower proteic phosphorylation level of ASM,Cer,P38and JNK in brain tissue, which is the main reason for improving brain tissue damage.6. The modified gandou decoction has a similar curative effect on improving braintissue damage to butylphthalide, but the functional mechanism is different, whichprovides theoretic foundation for curing WD clinically. |
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