纳米氧化铝致小鼠学习记忆损伤的粒径效应、时间效应及其机制探讨

发布时间：2018-05-17 04:13

  本文选题：纳米氧化铝 + 氧化应激　； 参考：《山西医科大学》2014年硕士论文

【摘要】：目的通过采用不同粒径的纳米氧化铝在不同时点对ICR小鼠进行急性、亚慢性染毒，来探讨纳米氧化铝对小鼠学习记忆能力损伤的粒径效应、时间效应以及其可能机制。 方法 Ⅰ.选取72只健康成年ICR小鼠，分为4组（对照组(生理盐水)、10μm Al2O3组、50nmAl2O3组、13nm Al2O3组），每组18只，染毒后分为3个观察期（3天、7天、14天），每个观察期、每组小鼠6只。用灌胃法进行急性染毒，染毒剂量均为5g/kg，每个观察期结束后处死小鼠，取脑，测脑体比，分离皮质，检测炎症因子（TNF-α、IL-1β）和氧化应激水平（MDA、SOD），其中观察期为14天小鼠，观察结束后先用Morris水迷宫对小鼠进行学习记忆能力检测，而后同上。 Ⅱ.选取120只健康成年ICR小鼠，分为4组(对照组(生理盐水)、10μmAl2O3组（50mg/ml）、50nm Al2O3组（50mg/ml）、13nmAl2O3组（50mg/ml）)，每组30只，经滴鼻染毒法染毒，连续染毒时间分为3个时间段（30天、60天、90天），其中，每个时间段每个处理组平均10只小鼠。各时间段组染毒结束后用Morris水迷宫对小鼠进行学习记忆能力检测。行为学检测结束后每组取两只用4%多聚甲醛灌注，取脑做病理切片，进一步做HE染色、硫堇染色，剩余小鼠处死后取脑组织，测定炎症因子反应（TNF-α、IL-1β）和氧化应激水平（MDA、SOD、GSH、GSH-PX），Western-blot法检测溶酶体相关蛋白（Cathepsin-B、Cathepsin-L），凋亡相关蛋白（C-Caspase3），自噬相关蛋白（LC3- Ⅱ、Beclin-1），坏死相关蛋白（RIP）的蛋白表达水平。 结果 Ⅰ.急性染毒结果显示：水迷宫结果显示：13nmAl2O3组与其余各组相比，学习记忆能力显著下降，差异有统计学意义（P0.05）。炎症因子结果显示：与对照组相比，炎症因子首先在第3天（TNF-α）、第7天（IL-1β）显著升高，各组差异有统计学意义（P0.05），但在14天各染毒组与对照组相比，差异无统计学意义（P0.05）。氧化应激结果显示：与对照组相比，各染毒组的MDA含量在第3天差异无统计学意义（P0.05），从第7天开始显著增加，差异有统计学意义（P0.05），到第14天达到峰值；与对照组相比，各染毒组SOD活性从第3天开始随时间延长逐渐降低，到第14天降到最低水平，差异有统计学意义（P0.05），且有随粒径减小其活性显著降低（P0.05）。 Ⅱ.亚慢性染毒结果显示：水迷宫结果显示：与对照组相比，各染毒组逃避潜伏期差别有统计学意义（P0.05），50nm Al2O3组、13nm Al2O3组逃避潜伏期显著高于10μm Al2O3组和对照组，差异有统计学意义（P0.05）；各组不同时间段逃避潜伏期差别有统计学意义（P0.05），随时间的延长，逃避潜伏期时间逐渐延长。 病理结果显示：HE染色光学显微镜下观察显示：对照组海马CA3区神经细胞形态正常，排列整齐，连接紧密；10μm Al2O3组海马CA3区神经细胞数量减少；50nm Al2O3组小鼠海马CA3区神经细胞数量进一步减少，胞浆出现肿大，染色质比较疏松；13nm Al2O3组小鼠海马CA3区神经细胞数量明显减少，胞核固缩；随着时间的延长，各染毒组小鼠海马CA3区神经细胞数量减少，锥体层细胞稀疏、松散且排列无序，层次不清晰，连接不紧密。硫堇染色光学显微镜下观察显示：对照组海马CA3区细胞数量多排列规则，普遍染色较深，细胞形态清晰，可见尼氏体颗粒；10μm Al2O3组海马CA3区细胞数量减少，部分细胞肿胀呈空泡样，胞浆染色深浅不一，50nmAl2O3组海马CA3区细胞排列杂乱，细胞空泡样改变，染色更浅，13nm Al2O3组海马CA3区空泡现象更严重，神经元呈圆形；随着时间延长，各染毒组小鼠海马CA3区神经细胞数量减少，染色变浅，尼氏体逐渐溶解并减少。 炎症因子结果显示：与对照组相比，各染毒组TNF-a含量，差异有统计学意义（P0.05），随粒径的减小TNF-a含量逐渐升高；随时间的延长各染毒组TNF-a含量差别有统计学意义（P0.05），30天与60天、90天相比差异有统计学意义（P0.05）；60天与90天相比差异无统计学意义（P0.05）；各组IL-1β表达水平在粒径水平、时间水平均无显著差异（P0.05）。氧化应激结果显示：与对照组相比，各染毒组MDA含量差异均有统计学意义（P0.05），随粒径减小含量增加，随时间延长含量增加；与对照组相比，各染毒组SOD活性差异有统计学意义（P0.05），随粒径减小活性降低，随时间延长活性降低；与对照组相比，各染毒组GSH含量在粒径水平、时间水平均无显著差异（P0.05）；与对照组相比，各染毒组GSH-PX活性差异有统计学意义（P0.05），随粒径减小活性降低，随时间的延长活性降低。 Western blot法检测氧化铝染毒小鼠大脑皮质内相关蛋白：溶酶体的相关蛋白（Cathepsin-B、Cathepsin-L）结果显示：与对照组相比，各染毒组蛋白表达水平，差异有统计学意义（P0.05），随粒径减小蛋白表达水平增高，随时间延长表达水平增高。凋亡相关蛋白（C-Caspase3）结果显示：与对照组相比，各染毒组蛋白表达水平差异有统计学意义（P0.05），随粒径减小蛋白表达水平增高，随时间延长表达水平增高。自噬相关蛋白（LC3-Ⅱ、Beclin-1）结果显示：与对照组相比，各染毒组蛋白表达水平差异有统计学意义（P0.05），随粒径减小蛋白表达水平增高，随时间延长表达水平增高。坏死相关蛋白（RIP）结果显示：与对照组相比，各染毒组蛋白变化率差异有统计学意义（P0.05），随粒径减小蛋白表达水平增高，随时间延长表达水平增高。 结论 Ⅰ.纳米氧化铝经灌胃法一次急性染毒可以造成脑组织氧化应激损伤和炎症因子反应，降低小鼠的学习记忆能力，随粒径的减小，时间的延长损伤增大，，存在粒径效应、时间效应。炎症因子是这一过程中的早期事件，而氧化应激水平的升高可能是学习记忆能力下降的主要原因之一。 Ⅱ.纳米氧化铝经滴鼻法亚慢性染毒，可以导致海马神经元的结构和功能损伤，造成氧化应激损伤和炎症因子反应，诱发了溶酶体相关蛋白表达的升高，进而导致细胞死亡，凋亡、自噬、坏死均参与了这一过程。神经细胞的死亡是造成学习记忆能力死亡的主要原因之一。 Ⅲ.纳米氧化铝染毒提示存在慢性毒性，其中50nm Al2O3组在30天到60天毒性较大，13nmAl2O3组在不同时间点毒性一直呈上升状态。
[Abstract]:Objective To investigate the effect of nano - alumina on the effects of nano - alumina on learning and memory ability of mice and its possible mechanism by using nano - alumina with different particle sizes to study the effects of nano - alumina on the damage of learning and memory ability of mice .

method

I . 72 healthy adult ICR mice were divided into 4 groups ( control group ( normal saline ) , 10 渭m Al2O3 group , 50 nm Al2O3 group and 13 nm Al2O3 group . Each group was divided into 3 observation periods ( 3 days , 7 days , 14 days ) . Each group of mice was divided into 3 observation periods ( 3 days , 7 days , 14 days ) .

Methods 120 healthy adult ICR mice were divided into 4 groups ( control group ( normal saline ) , 10 渭m Al2O3 group ( 50 mg / ml ) , 50 nm Al2O3 group ( 50 mg / ml ) and 13 nm Al2O3 group ( 50 mg / ml ) .

II . Beclin - 1 ) , protein expression level of necrosis - related protein ( RIP ) .

Results

Compared with the control group , there was no significant difference in the levels of MDA in the groups compared with the control group ( P0.05 ) . The results showed that the MDA content of each group increased significantly in the third day ( P0.05 ) , and the difference was statistically significant ( P0.05 ) .
Compared with the control group , SOD activity decreased gradually from the third day to the lowest level on day 14 , and the difference was statistically significant ( P0.05 ) .

The results of sub - chronic toxicity showed that the water maze showed that the difference of escape latency was statistically significant ( P0.05 ) , 50 nm Al2O3 group and 13 nm Al2O3 group were significantly higher than that in control group ( P0.05 ) .
The escape latency of each group was statistically significant ( P0.05 ) , and the escape latency gradually increased with the extension of time .

The pathological results showed that the morphology of nerve cells was normal in the CA 3 region of the control group , the arrangement was regular and the connection was close ;
The number of nerve cells decreased in the hippocampus of 10 渭m Al _ 2O _ 3 group .
The number of nerve cells in hippocampus CA 3 of 50 nm Al _ 2O _ 3 group decreased further , and the cytoplasm appeared enlarged and chromatin was loose ;
The number of nerve cells in the hippocampal CA 3 region of the 13nm Al2O3 group was significantly decreased , and the nuclei were pyknosis .
With the extension of time , the number of nerve cells decreased and the pyramidal layer cells were sparse , loose and disordered , the level was not clear , and the linkage was not compact . The results showed that the number of cells in the hippocampus of the control group was more regular , the general dyeing was deeper , the morphology of the cells was clear , and the microstructure of the cells was visible .
The number of cells in hippocampal CA 3 area of 10 渭m Al _ 2O _ 3 group was decreased , some cells swelled in vacuoles , and the cells in the hippocampus of 50 nm Al2O3 group were disordered . The cells in the CA 3 area of 50 nm Al _ 2O _ 3 group were disrupted and the cells were stained more light . The vacuoles in the CA 3 area of the 13nm Al2O3 group were more serious , and the neurons were circular ;
As the time was prolonged , the number of nerve cells in the hippocampal CA 3 area of each group decreased , the staining became shallow , and the bainite gradually dissolved and decreased .

Compared with the control group , the TNF - a content of each group was significantly higher than that of the control group ( P0.05 ) , and the content of TNF - a gradually increased with the particle size .
The content of TNF - a in each group was significantly different with time ( P0.05 ) , and the difference was statistically significant ( P0.05 ) in 30 days and 60 days .
There was no significant difference between 60 and 90 days ( P0.05 ) .
The levels of IL - 1尾 in each group were not significantly different from those in control group ( P0.05 ) .
Compared with the control group , the activity of SOD in each group had significant difference ( P0.05 ) , the activity of SOD decreased with the decrease of particle size , and the activity of SOD decreased with time .
Compared with the control group , the GSH content of each group had no significant difference ( P0.05 ) .
Compared with the control group , the activity of GSH - PX in each group was statistically significant ( P0.05 ) . The activity of GSH - PX decreased with the decrease of particle size , and the activity of GSH - PX decreased with time .

Compared with the control group , there was significant difference in the expression level of the protein in the infected mice ( P0.05 ) . The results showed that compared with the control group , the difference of the expression level of the protein of each group increased . The results showed that compared with the control group , the difference of the expression level of the protein of each group increased . The results of the necrosis - related protein ( RIP ) showed that the expression level of the protein of the infected group increased with the increase of the expression level of the protein .

Conclusion

I . The acute toxicity of nano - alumina by gavage can cause oxidative stress injury and inflammatory factor reaction in brain tissue , decrease the learning and memory ability of mice , increase with the decrease of particle size , increase the time , and have particle size effect and time effect . The inflammatory factor is an early event in this process , and the increase of oxidative stress level may be one of the main reasons for the decline of learning and memory ability .

The results showed that the cell death , apoptosis , autophagy and necrosis were one of the main causes of death in learning and memory .

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