| Metallothionein-3 (MT3) is a low molecular mass and dissoluble protein which is first discovered in 1991 and specific expressed in central nervous system. It has the role of regulating metabolism of trace element, clearing of free radical and inhibiting growth. Later it has been found that MT3 in AD patients'brain obviously decreased, which promoted the study of the relation between MT3 and AD. In recent years, there are many other dementia models which can imitate a part of characteristics of the AD only; there are not any ideal animal models which has all of the AD characteristics completely. Thus the study of mechanism and medicine treatment is limited severely. The SAMP8 is an ideal model whose ethology, histopathology, neuro-biochemistry, enzyme, protein and gene expression are all abnormal. To explore the protective mechanism of MT3 on central nervous system, we carry out a series of tests on behavior, histology, oxidative stress, apoptosis, astrocyte and the change of the zinc and copper ion.1. The study of the variation of the MT3 mRNA expression in hippocampus of the SAMP8The result shows that the content of MT3 is lower in SAMP8 group than in SAMR1, which is the same as the AD. It suggests that the decreasing of MT3 in SAMP8 causes the descent of study and memory ability of the mice. The SAMP8 is an ideal model that can be used to study the relation between MT3 and AD.2. Determination of learning and memory ability of SAMP8 and the influence of MT3The 7 month old SAMP8 were divided into 6 groups: dementia group, saline injection group, 3 different concentrations of MT3 (28 days injection with 75ug/ml, 150ug/ml, 300ug/ml), MT1 injection group. The SAMR1 acted as normal control group. Learning and memory ability of each group rat was determined by Morris Water Maze and the escape latency was recorded. The less time of rats span the platform, the better learning and memory ability they have..The results showed that①t he escape latency prolonged in 8 monthes SAMP8 rats Compared with SAMR1 normal control group;②the escape latency of high concentration of MT3 group was the shortest in the three different concentrations and it was shorter than no injection group;③the escape latency of the high concentration group was longer than SAMR1, and it suggests that the dementia mouse'memory ability does not recover normally yet.It demonstrates that SAMP8 has the obvious characteristic of the descent of study and memory ability. After treatment the memory ability has been improved but can not recover to the state as normal. In the three treatment groups the results of high concentration was the best.3. HE dyeing and electron microscope results in hippocampus of SAMP8 and the influence of MT3①HE staining of 8 monthes SAMR1 showed that frontal lobe and hippocampal pyramidal cell lined up in order, uniformity, cellular structure was integrity. Electron microscope showed that cellular membrane was integrity, and cellular nucleus was large and ellipse. Nucleole was clear, Neuropil was normal, Pro-membrane and post-membrane of synapse was clear. Synaptic vesicle was abundance; HE staining of 8 monthes SAMP8 showed that the hippocamp CA1 pyramidal cell layers were disturbed, diluted, interrupted; the nucleus were pyknosised and over stained; hippocamp neuron were missing, irregular and the amount of colloid cell increased. With the electricity microscope, we can see most neuron turned swelling even cavitation. Neuropil also turned cavitation.②The pyramidal cells of hippocampus CA1 in different concentration MT3 groups lined up in order, cellular structures were more clear, and the amount of colloid cells decreased comparing with the control group; The protective effect is most obvious in high concentration of MT3 group.These results demonstrate that the damage of SAMP8 mouse hippocampal neuron causes the descent of study and memory ability, but MT3 has the improvement function.4. Oxidizing injure in hippocampus of SAMP8 and the influence of MT3 We used biochemical reagent kits and UV spectrophotometer to detect biochemical indicator (T-SOD, CuZn-SOD, GSH-Px, MDA) in SAMP8 and the influencement of MT3.The results showed that:①the MDA content in hippocampus of SAMP8 was obviously higher than the SAMR1 normal group, and the activity of CuZn-SOD and GSH-Px was lower than the SAMR1 normal group.②the MDA content was the lowest in the group of high density MT3, but SOD's, CuZn-SOD's and GSH-Px's activity were the highest. There was the density dependence relation.③the SOD's and GSH-Px's activity in MT3 group were higher than in MT1 control group.These results confirm: The change of oxidative stress in SAMP8 is similar to AD, the degree of over oxidizes to the lipoid is high, and Erzymes of anti-oxidizing decrease; oxidizing injure is the pathogeny of aging and dementia in SAMP8 mouse; MT3 can not only clear free radicals, but also increase the activity of the metabolic enzyme of free radicals. With density increasing, the fuction is more powerful.MT3 is better than MT1.5. Tunel detecion and Bcl-2,Bax protein expression in SAMP8 mouse hippocampus and the influence of MT3 on itWe used method of Tunel to detect apoptosis cells and immunohistochemistry staining to observe Bcl-2,Bax protein expression in hippocampus.The results:①we can scarcely see apoptosis cells in hippocampus of normal control group, Bax seldom expressed and Bcl-2 expression was more; Apoptosis cells in hippocampus of SAMP8 obviously increased. Bax protein expression obviously increased and Bcl-2 protein expression decreased.②Although there were apoptosis cells in various density of MT3, with density increasing apoptosis cells decrease and the high density of MT3 had the most powerful effect.③the expression of Bax in MT3 group was lower than in MT1 group.The experimental result indicates that there is a large amount of apoptosis cells in SAMP8 hippocampus. MT3 can significantly down-regulate the expression of Bax protein and up-regulate the expression of Bcl-2 protein. Therefore,it can inhibit apoptosis and improve the learning and memory ability . There is no such function in MT1. 6. Assaying zinc content in hippocampus of SAMP8 and the influence of MT3 on itWe used atomic absorption spectrophotometer to assay zinc and copper. The results:①z inc content in hippocampus of SAMP8 was less than in SAMR1 group, but there was no difference in the copper.②zinc content increased by MT3 injection, and the middle concentration had the best result.③the content of copper did not change.The results: the change of the zinc content in SAMP8 hippocampus was one of the reasons which gives rise to the abnormal nerve function. MT3 can transport zinc and increase the content of it, but when the content of zinc get balanced it did not rise with the enlarged concentration. MT3 can keep the zinc content in balance.7. GFAP expression in SMAP8 hippocampus and the influence of MT3 on itWe used immunohistochemistry and image analysis system to observe the changes of expression of GFAP in hippocampus.The results:①the expression of GFAP was little in SAMR1, but increased in hippocampus and frontal lobe of dementia model rats.②Along with the increasing of the medicine density, the number of GFAP expression in the SAMP8 reduced consequently.③t he expression of GFAP in MT3 was less than MT1 group。The result demonstrates that MT3 can inhibit the expression of GFAP, and the efficiency increased with the enlarged concentration. MT1 has no such function correspondingly.In conclusion, anti-oxidizing injure of MT3 is the foundation of improving the ability to SAMP8's learning and memory. It is a mechanism of protecting neuron survival by decreasing the expression of Bax, increasing the expression of Bcl-2 in hippocampus, and reducing the rate of cell apoptosis. It is a precondition that body maintains its normal function by transporting the zinc ion and keeping the zinc ion content in balance. Furthermore, reducing the GFAP expression excessively can protect neuron indirectly. We can make a conclusion that the reduction of MT3 can be a reason of invoking AD, and this research affords a target for the treatment of AD.
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