The Effect Of Nec-1 On Cognitive Ability In Aluminum-induced Dementia Model And Its Mechanism

Objective:Through use apoptosis inhibitor zVAD-fmk as a parallel control, to study the effect of Nec-1 on changes about pathohistology and neurobehavior of the mice induced by aluminium and the effect on cell death style.Methods:120 maleness kongming mice,adult and health,were divided into 3 three batches randomly,40 in each batch.4 groups were divided into the every batch,10 in each group.4 groups in first batch were:aluminum control group(0.5%AlCl3),2mM,4mM,8mM Nec-1 group. 4 groups in second batch were:aluminum control group (0.5%Al C 13),75ng/2μl,150ng/2μl, 300ng/2μl zVAD group.4 groups in third batch were:aluminum control group(0.5%AlCl3), 4mM Nec-1 group,300ng/2μl zVAD group, Nec-1+zVAD group.Every mouse was treated by lateral cerebral ventricle micro-injection with the corresponding liquid 5 days,l times per day.And aluminum solution was 3μl/day,and Nec-l,zVAD were 2μl/day. On the 15th day after exposure,the Morris water maze test were performed to test the learning and memory abilities of mice. Then on the 20th day after exposure,mice were sacrificed.The cerebral cortex hippocampus was removed. Half of the brain is soaked in 10%formalin 24h which morphological changes were observed by optical microscope. The last half was placed in EP tube at-70℃to save for the Western-blot to detect the protein expression of NF-κB, LC3-Ⅱ, Caspase3 and RIPl.Results:①learning and memory capability test in mice:In the Morris water maze test, different concentrations Nec-1 acted on the mice of aluminum model.To compared with aluminum solvent control group, the latency of 4mM group and 8mM groups Were significantly shortened in place navigation experiment(P<0.01). In the spatial probe test on the 5th day, residence time of the original platform quadrant in 2mM,4mM,8mM group was significantly increased (P <0.01); different concentrations Z-VAD-FMK acted on the mice of aluminum model.To compared with aluminum solvent control group, the latency of the high-dose group escape latency was significantly shortened in place navigation experiment(P<0.01). In the spatial probe test on the 5th day, residence time of the original platform quadrant in low, middle and high-dose group of the original platform quadrant was significantly increased (P<0.01); Individual and combination therapy of Nec-1, zVAD acted on the mice of aluminum model. To compared with aluminum solvent control group, the latency of Nec-1 group and Nec-1+zVAD group was significantly shortened in place navigation experiment (P<0.05). In the spatial probe test on the 5th day, residence time of the original platform quadrant in Nec-1+zVAD group was significantly increased (P<0.05)②Pathohistology results:After different concentrations of Nec-1, zVAD acting on the aluminum model mice, pathological changes in cerebral cortex and hippocampus CA3 area showed that vacuolar degeneration around the nuclear and cytoplasmic,destruction of the individual cell structure, blurred cell outlines. In the hippocampus a small number of cells arranged messy, lysis of cell connections, cell shrinkage, and so on. Some cells were necrotic performance, for example:nucleus aggregation, concentration, dissolution, etc.It can be seen in the minor injuries cells in granular layer that the nucleus was stained deep blue and the cytoplasm deep red, blurred degeneration around nuclear in cortex of the aluminum solvent control group. With Nec-1, zVAD-dose increased, the morphological damage of the cell was gradual Weakened; Combination therapy of Nec-1, zVAD in mice lead to cerebral cortex and hippocampal CA3 area showed pathological changes:Nec-1 group and zVAD group alleviated the above-mentioned morphological changes.Nec-1+zVAD group can be seen rare abnormal cell morphology, the cells significantly Increase and regular arrangement.③death-associated protein detection:Use Western blot to detect expression of caspase3 protein in cerebral cortex which was treated by different concentrations of Nec-1. It has a downward trend.And the expression of caspase3 in the middle and high-dose group was significantly lower than the control group(P<0.01); The expression of LC3-Ⅱalso showed a downward trend, and the low, middle and high-dose group significantly decreased than control group (P<0.01); The expression of NF-κB has been a downward trend, but the difference was not statistically significant (P> 0.05); The expression of RIP has been a downward trend, and the middle dose group was significantly decreased than control group, the difference was statistically significant (P<0.05). The expression of protein in cerebral cortex which was treated by different concentrations of ZVAD.fmk.Caspase3 in the high-dose group was significantly lower than the control group(P<0.01); The expression of LC3-Ⅱalso showed a increasing trend, and the middle and high-dose group significantly increased than control group (P<0.01); The expression of NF-κB has been a downward trend, the low, middle and high-dose group decreased significantly than control group (P<0.01); The expression of RIP has been a increasing trend, and the middle and high dose group was significantly increasing than control group (P<0.01). Combination therapy of Nec-1, zVAD showed that:the expression of caspase3 has been a downward trend, Nec-1 group, zVAD group and Nec-1+zVAD group was significantly decreased than control group (P<0.01), the expression of LC3-II in Nec-1 group has decreasing tendency compared to solvent control group, zVAD group has an increasing trend than control group, Nec-1+zVAD has an increasing trend group than the control, but there was no statistically significant difference (P> 0.05). zVAD group was increased significantly than Nec-1 group (P<0.05). The expression of NF-κB in Nec-1, zVAD group was increased significantly than the control group(P<0.01), zVAD group was decreased significantly than Nec-1 group (P<0.01), Nec-1+zVAD groups was decreased significantly than control group(P<0.05), The expression of RIP 1 in Nec-1 group was decreased significantly than control group (P<0.01), zVAD group was increased significantly than the control group(P<0.01),Nec-1+zVAD group has an increasing trend group than the control, but there was no statistically significant difference (P> 0.05).Conclusion:Nec-1, zVAD can repair the injury of learning and memory in mice which were induced by aluminum.And Nec-1 was more obvious to repair the injury of learning and memory function compared with zVAD. Nec-1, zVAD can repair the injury of cell morphology and amounts which were induced by aluminum and repair more significantly with higher dose. Combination therapy of Nec-1, zVAD can repair more significantly than Individual therapy. The pathological mechanism of Nec-1 may be related to inhibiting the protein expression of RIP 1 and then lead to the loss of the related neurons, which can repair the injury of learning and memory in aluminum-induced mice.