| Part Ⅰ Effects of manganese exposure on scavenging Aβ in APP/PS1 miceObjective: To investigate the effect of manganese exposure on Aβ scavenging in APP/PS1 mice cerebral cortex.Methods: The research group established an animal model of cognitive impairment in APP/PS1 mice caused by manganese exposure.By random grouping,twenty transgenic mice were divided into manganese exposure group（APP/PS1-Mn）and control group（APP/PS1-Control）,and 20 co-litter wild-type mice were divided into manganese exposure group（WT-Mn）and control group（WT-Control）.The manganese contaminated solution was prepared with normal saline,and the dose of manganese contaminated group was 60 mg/kg.The mice in the control group were given the same normal saline in the same way,once a day,five times a week.The manganese exposure period was about 4 months.When the mice in the manganese exposure group showed cognitive impairment and memory loss,the mice were killed and collected the cerebral cortex.Elisa was used to measure Aβ40 and Aβ42 in cortex,and the number of astrocytes in the cerebral cortex of the mice was determined by immunofluorescence.Real-time-PCR was used to measure the mRNA expressions of P53,Bcl-2,Bax,caspase-9,caspase-3 in each group of cortices.Western Blot was used to measure the expression levels of P53,Bcl-2,Bax,caspase-9 and caspase-3 proteins in each group’s cortices.Results: 1.The contents of Aβ40 and Aβ42 in each group’s cortices: the content of Aβ40 in WT-Mn mice’s cerebral cortex was obviously higher than WT-Control group（3.04±0.78 vs 2.27±0.30）,and the content of Aβ42 was significantly higher than that of WT-Control group（2.49±0.84 vs 2.06±0.36）,the comparison was statistically significant（all P<0.05）;the content of Aβ40 in cerebral cortex of APP/PS1-Mn was significantly higher than that of APP/PS1-Control（3.27±0.22 vs 2.51±0.44）,and the content of Aβ42 was significantly higher than that of APP/PS1-Control（2.78±0.26 vs 2.45±0.25）,the comparison was statistically significant（all P<0.05）.2.Changes in the number of astrocytes in cerebral cortex of each group: the number of astrocytes in cerebral cortex of WT-Mn mice was significantly lower than that of WTControl group（14.52% vs 0.75%）,and the number of astrocytes in cerebral cortex of APP/PS1-Mn mice was significantly lower than that of APP/PS1-Control group（19.58% vs 1.39%）,the comparison was statistically significant（all P<0.05）.3.The expression levels of apoptosis-related genes in each group’s cortices: the content of P53 in APP/PS1-Mn group’s cerebral cortex was significantly higher than APP/PS1-Control（2.91±0.13 vs 1.06±0.46）,the expression level of Bax was obviously higher than APP/PS1-Control（1.34±0.14 vs 1.00±0.04）,the expression level of caspase-9 was obviously higher than of APP/PS1-Control（1.49±0.22 vs 1.00±0.09）,the comparison was statistically significant（P<0.05）,the expression levels of Bcl-2 and caspase-3 didn’t change obviously compared with the control group（P>0.05）;the expression level of P53 in WT-Mn group’s cerebral cortex was significantly higher than that of WT-Control（3.79±1.27 vs 1.46±0.79）,the expression level of caspase-9 was obviously higher than WT-Control（1.00±0.12 vs 0.50±0.05）,these comparisons were statistically significant（all P<0.05）,Bax,Bcl-2 and caspase-3 were not significantly changed（P>0.05）.4.The expression levels of apoptosis-related proteins in each group’s cortices: the content of P53 in the cerebral cortex of APP/PS1-Mn was obviously higher than APP/PS1-Control（1.02±0.02 vs 0.60±0.01）,the content of Bax was significantly higher than that of APP/PS1-Control（0.34±0.01 vs 0.19±0.00）,the content of caspase-9 was significantly higher than that of APP/PS1-Control（0.27±0.02 vs 0.12±0.01）and the content of caspase-9 was significantly higher than that of APP/PS1-Control（0.56±0.02 vs 0.27±0.02）,the content of Bcl-2 was significantly lower than that of APP/PS1-Control（0.25±0.01 vs 0.64±0.02）,these comparisons were statistically significant（all P<0.05）;the expression level of P53 in WT-Mn group’s cerebral cortex was obviously higher than WT-Control（0.67±0.01 vs 0.22±0.01）,the expression level of Bax was obviously higher than WTControl（0.49±0.02 vs 0.20±0.02）,the content of caspase-9 was significantly higher than that of WT-Control mice（0.29±0.02 vs 0.16±0.01）,the content of caspase-3 was significantly higher than that of WT-Control mice（0.61±0.02 vs 0.29±0.02）,the content of Bcl-2 was significantly lower than that of WT-Control mice（0.08±0.01 vs 0.49±0.02）,these comparisons were statistically significant（all P<0.05）.Conclusion: Manganese exposure can affect the clearance of Aβ in mice brain,which may be related to the manganese-induced apoptosis of astrocytes.Part Ⅱ Effects of manganese exposure on Aβ scavenging in astrocytesObjective: To investigate the effects of manganese exposure on Aβ scavenging and apoptosis-related proteins mediated by mitochondria in astrocytes.Methods: CCK8 method was used to determine the survival rate of astrocytes exposed to different contents of manganese.The cells were divided into control group,manganese treatment group（50 μmol/L）,Aβ42 treatment group（2 μg/mL）,50 μmol/L MnCl2+2 μg/mL Aβ42 group.Apoptosis rate of each treatment group was determined by flow cytometry.Elisa was used to measure the contents of intracellular and extracellular Aβ for each group.Transmission electron microscopy was used to observe the ultrastructure of cells for each group.Real-time PCR was used to detecte the expression levels of P53,Bcl-2,Bax,caspase-9 and caspase-3 for each group.Western Blot was used to detecte the contents of P53,Bcl-2,Bax,caspase-9 and caspase-3 for each group.Results: 1.Apoptosis rate of each treatment group was determined by flow cytometry: compared with the control group,apoptosis was significantly increased in the manganese treated group and the Mn+Aβ42 group,and the difference was statistically significant（P<0.05）;compared with the control group,apoptosis was slightly increased in the Aβ42 treated group,but it was not significant and the difference was not statistically significant（P>0.05）.The above results showed that manganese exposure could increase the occurrence of apoptosis in astrocytes.2.The contents of intracellular and extracellular Aβ in each group: the contents of intracellular Aβ in each group were compared,manganese treatment group was significantly higher than control group（96.71±3.01 vs 38.00±1.38）,the Aβ42 treatment group was significantly higher than the control group（80.80±0.66 vs 38.00±1.38）,Mn+Aβ42 group was significantly higher than control group（110.52±1.62 vs 38.00±1.38）,these comparisons were statistically significant（all P<0.05）;manganese treatment group was significantly higher than Aβ42 treatment group（96.71±3.01 vs 80.80±0.66）,Mn+Aβ42 treatment group was significantly higher than Aβ42 treatment group（110.52±1.62 vs 80.80±0.66）,these comparisons were statistically significant（all P<0.05）;Mn+Aβ42 treatment group was significantly higher than that of manganese treatment group（110.52±1.62 vs 96.71±3.01）,the difference was statistically significant（P<0.05）;comparing the extracellular Aβ content of each group,manganese treatment group was obvious higher than the control group（90.99±2.56 vs 35.21±0.36）,the Aβ42 treatment group was obvious higher than the control group（72.49±0.35 vs 35.21±0.36）,Mn+Aβ42 group was obvious higher than control group（103.92±0.57 vs 35.21±0.36）,these comparisons were statistically significant（all P<0.05）;manganese treatment group was significantly higher than Aβ42 treatment group（90.99±2.56 vs 72.49±0.35）,Mn+Aβ42 treatment group was significantly higher than Aβ42 treatment group（103.92±0.57 vs 72.49±0.35）,these comparisons were all statistically significant（P<0.05）;Mn+Aβ42 treatment group was significantly higher than that of manganese treatment group（103.92±0.57 vs 90.99±2.56）,the difference was statistically significant（P<0.05）.3.The ultrastructural changes of cells in each group were observed by transmission electron microscopy: in the control group,no swelling of mitochondria and no abnormality of mitochondrial matrix and cristae were observed;moderate mitochondrial edema,weak mitochondrial matrix,fuzzy crista and disappearance were observed in manganese treatment group;in Aβ42 treatment group,mitochondria were severely swollen,the space of mitochondrial matrix was reduced,and the number of cristae was reduced,presenting vacuolous shape;the mitochondria of Mn+Aβ42 treatment group were slightly swollen and the bilayer membrane structure was fuzzy.4.mRNA expression of apoptosis-related genes in each group: the expression level of P53 in the Mn-treated group was lower than that in the control group（0.86±0.08 vs 1.00±0.07）,the expression level of P53 in Mn+Aβ42-treated group was lower than that in the control group（0.82±0.05 vs 1.00±0.07）,the comparison was all statistically significant（P<0.05）;the expression level of P53 in the Mn+Aβ42 group was significantly lower than that in the Aβ42-treated group（0.82±0.05 vs 0.97±0.07）,the difference was statistically significant（P<0.05）;the expression level of Bax in the Aβ42-treated group was obvious lower than that in the control group（0.71±0.02 vs 1.00±0.07）and Mn-treated group（0.71±0.02 vs 0.98±0.08）,and the difference was statistically significant（both P<0.05）;the expression level of caspase-9 in Mn+Aβ42 treatment group was significantly higher than that in the control group（1.78±0.20 vs 1.00±0.12）,manganese treatment group（1.78±0.20 vs 1.05±0.12）and Aβ42 treatment group（1.78±0.20 vs 1.25±0.06）,all differences were statistically significant（P<0.05）;the expression level of caspase-3 in Aβ42 group was obvious higher than that in the control group（1.70±0.59 vs 1.00±0.08）,and the difference was statistically significant（P<0.05）.5.Expression levels of apoptosis-related proteins in each group: the expression level of p53 in Mn-treated group was obvious higher than that in the control group（1.33±0.38 vs 0.30±0.20）,the expression level of p53 in Aβ42-treated group was obvious higher than that in the control group（1.94±0.57 vs 0.30±0.20）,the expression level of p53 in Mn+Aβ42 group was obvious higher than that in the control group（1.40±0.57 vs 0.30±0.20）,all were statistically significant（P<0.05）;the expression level of Bcl-2 in Aβ42-treated group was obvious higher than that in the control group（1.01±0.17 vs 0.21±0.18）and Mn-treated group（1.01±0.17 vs 0.54±0.07）,the expression level of Bcl-2 in Mn+Aβ42 group was obvious higher than that in the control group（0.96±0.31 vs 0.21±0.18）and Mn treatment group（0.96±0.31 vs 0.54±0.07）,and all differences were statistically significant（P<0.05）;the expression level of Bax in Mn-treated group was significantly higher than that in the control group（1.64±0.27 vs 0.92±0.22）,and that in Aβ42 treatment group was significantly higher than that in control group（2.28±0.11 vs 0.92±0.22）,the expression level of Bax in Mn+Aβ42 group was obvious higher than that in the control group（1.47±0.15 vs 0.92±0.22）,and all differences were statistically significant（all P<0.05）,the expression level of Bax in Mn+Aβ42 group was obvious lower than that in Aβ42 group（1.47±0.15 vs 2.28±0.11）,and the difference was statistically significant（P<0.05）,the expression level of Bax in Aβ42-treated group was obvious higher than that in Mn-treated group（2.28±0.11 vs 1.64±0.27）,and the difference was statistically significant（P<0.05）;the expression level of caspase-9 in Mn-treated group was obvious higher than that in the control group（1.33±0.38 vs 0.30±0.20）,and the expression level of caspase-9 in Aβ42 treatment group was obvious higher than that in the control group（1.94±0.57 vs 0.30±0.20）,the expression level of caspase-9 in Mn+Aβ42 group was obvious higher than that in the control group（1.40±0.57 vs 0.30±0.20）,and all differences were statistically significant（P<0.05）;the expression level of caspase-3 in Mn-treated group was obvious higher than that in the control group（2.87±0.84 vs 0.75±0.23）,and the expression level of caspase-3 in Aβ42-treated group was obvious higher than that in the control group（3.79±1.01 vs 0.75±0.23）,the expression level of caspase-3 in Mn+Aβ42 group was obvious higher than that in the control group（2.54±0.79 vs 0.75±0.23）,and all differences were statistically significant（all P<0.05）.Conclusion: Manganese exposure can reduce the clearance of Aβ in astrocytes,which may be related to mitochondrial damage and apoptosis induced by manganese. |
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