| ObjectiveThis study is aim to explore the effect of EGCG on learning and memory of dementia model mice by observation of the changes of behavioral features of SAMP8mice after the treatment with EGCG. And furher explore the possible active mechanisms, then it can apply a reference methods for AD research.Methods1. Experiments in vivo1.1Morris Water Maze:24male Senescence-accelerated mouse/prone8(SAMP8) of4months old were divided randomly into model group, low dose of EGCG group and high dose of EGCG group.8male Senescence accelerated mouse/resistance1(SAMR1) of the same age were used to be negative control group.The low and high doses of EGCG groups were administered5mg/kg· d and15mg/kg· d respectively. The negative group and mode group were treated with0.9%normal saline. Every group were gave intragastric administration for once per day and administrated60days totally. The learning and memory ability were detected by Morris Water Maze when the treatment ended.1.2Molecular biology experiments:The expression of Aβ42in the cortex of mice were detected by Western Blotting and ELISA. And the expression of APP, NEP, Bcl-2and Bax in the cortex of mice were tested by Western Blotting.1.3Immunology experiments:The neuron damage was detected by Nissl body immunostaining of brain paraffin section of mice.2. Experiments in vitro 2.1The expression of NEP, APP, Aβ42, Bax, and Bcl-2were detected by Western Blotting after M146L cells treated with EGCG.2.2The expression of extracellular Aβ42secreted by M146L cells was tested by ELISA after M146L cells treated with EGCG.2.3NEP was knockout by gene silencing, and then the expression of A β42was detected. The effect on Aβ42clearance of EGCG was analysed, and further demonstrated that EGCG was the critical factor to Aβ42regulation.2.4The effect on cell survival of EGCG was detected by MTT before or after NEP gene silencing respectively.Results1. Experiments in vivo1.1After60days of administration with EGCG, the learning and memory abilities of SAMP8mice were obviously improved in high dose of EGCG group (15mg/kg· d). In the place navigation test, compared with model group, the escape latency of high dose of EGCG group was shorter, and the differences were statistical significance(P<0.01), while there’s no significant differences comparing the model group on the swimming path length(P<0.05). In the spatial probe test, there were no significant differences between groups on the nμMbers crossing plat (P>0.05), while time spent in the target quadrant of high dose of EGCG group was significantly increased compared with model group (P<0.01). However, there was no significant difference between the low dose of EGCG group (5mg/kg· d) and model group on the ability of learning and memory of mice (P>0.06).1.2Compared with SAMR1mice, the expression of A P42were significantly increased in the cortex of SAMP8mice(P<0.05). There were significant differences between low dose of EGCG group and model group on the expression of totalA β42(Western blotting and ELISA) in the cortex of mice (P<0.01, P<0.05), and the expression of Aβ42were significantly decreased in high dose of EGCG group (P<0.01, P<0.01). The trend of APP expression was decreased in the EGCG treatment group comparing with model group, but there were no significant differences among these groups on the expression of APP (P>0.05). 1.3Compared with normal control group, the expression of NEP was downregulation, but there was no significant difference (P>0.05). The expression of NEP in both low and high dose of EGCG groups was remarkably-increased compared withmodel group (P<0.05).1.4The expression of Bcl-2in model group was significantly decreased compared with normal control group (P<0.05) while the expression of Bax was remarkable increased (P<0.05). Compared with model group, the expression of Bcl-2was increased while Bax was decreased in high dose of EGCG group, and there were significant statistic differences (P<0.05, P<0.01). There was no significant difference between low dose of EGCG group and model group on the level of Bcl-2(P>0.05), but the Bax level was remarkably reduced (P<0.01).1.5After Nissl staining using the paraffinsections which were made by brains of mice, neurons were observed by fluorescence microscope. And in model group, the quality of neurons was decreased, arrangement of neurons was disorder, neurons vacuolar degeneration and Nissl bodies reduced even disappeared. All these represent that neurons were damaged remarkably in morphology. However, the neurons damage in the two EGCG groups was alleviate compared with model group.2. Experiments in vitro2.1The cell viability was remarkably increased in high dose of EGCG group (32mmol/L)(P<0.01). But there’s no significant effect in low dose of EGCG group (16mmol/L)(P>0.05). The expressions of intracellular and extracellular Aβ42were significantly reduced in high dose of EGCG group (P<0.05, P<0.01). There was no remarkable effect on the intracellular Aβ42level in low dose of EGCG group (P>0.05) while the extracellular Aβ42level was significantly reduced (P<0.05). The expression of APP in M146L cells was remarkably decreased in low and high dose EGCG groups(p<0.05, p<0.01).2.2Further experiments show that the expressions of NEP and Bcl-2were upregulated in M146L cells in high dose of EGCG group (P<0.01, P<0.01). In low dose of EGCG group, there was no significant difference compared with model group (P>0.05) while the expression of Bcl-2was remarkably increased (P<0.05). And there were no significant decreasing of Bax in low and high dose of EGCG groups (P>0.05). 2.3EGCG can’t down-regulate the level of Aβ42after NEP gene silencing in M146L cells. Comparing with groups which were not transfected with siRNA-NEP, the expression of A β42was remarkably increased (P<0.05).Conelusion1. The ability of spatial learning and memory of SAMP8mice was effectively improved after continuous60days administration with15mg/kg· d dose of EGCG to4months old mice.2. The expression of Aβ42was significantly reduced both in brains of SAMP8mice and M146L cells after using EGCG administration. The trend of APP expression was decreased in animal brains without significant differences. However, the APP expression of EGCG treatment groups was decreased in M146L cells.3. EGCG can up-regulate the expression and activity of NEP in brains of SAMP8and M146L cells, and promoted the clearance of Aβ.4. EGCG can up-regulate level of Bcl-2while down-regulate expression of Bax, sequentially inhibit apoptosis of neurons to some extent.5. The possible mechanism of EGCG which improved ability of spatial learning and memory of SAMP8mice was that increasing Aβ degeneration by up-regulatingNEP expression. And then the aggregation of Aβ was reduced. All these lead to the neurotoxicity of Aβ decreasing and neurons apoptosis inhibiting. EGCG plays a role of neuroprotective and shows a lot of values and meanings in study of treatment of AD. |
PDF Full Text Request