| ObjectiveAluminum is the third most abundant metal element in the crust.With many excellent physical and chemical properties,it is widely used in medical,food,industry,agriculture and other fields.There are many opportunities for the body to absorb aluminium.Besides the absorption of aluminium caused by medicines containing aluminium and occupational exposure to aluminium,water,food additives,food packaging materials and aluminium dissolved from aluminium utensils are also the main ways for the body to absorb aluminium.However,there are few ways for the body to excrete aluminium,usually through urine and feces,so the absorbed aluminium is very easy to accumulate in the body.Aluminum was once considered to be an element with low absorptivity and harmless to human body,but in recent decades,studies have shown that the accumulation of aluminium can cause toxicity to many organs and tissues.In particular,the toxicity of aluminium on nervous system has attracted much attention in recent years.The harm of aluminium on nervous system is mainly manifested in its influence on learning and memory function,and leads to apoptosis.Flavonoids have been used as food supplements,and their effects on improving cognitive function and preventing neurodegenerative diseases in humans have been emphasized.However,there are many kinds of neurodegenerative diseases,and there are many subtypes.There are few reports about the effects of different subtypes on the prevention and treatment of neurodegenerative diseases.In this experiment,Wistar rats were used as the research object,and the model was established by intragastric administration of aluminum salt.Then,three representative flavonoids,namely rutin(flavonols),silymarin(dihydroflavonols),and puerarin(isoflavones)were given for detoxification.Behavioral experiments were conducted to evaluate the effects of three different types of flavonoids on learning and memory in rats exposed to aluminum.Molecular experiments were conducted to detect the overall situation of apoptosis,the expression levels of apoptotic genes and proteins,and the expression levels of autophagy-related proteins.Thus to study and explore the effects of flavonoids on learning and memory impairment and cell apoptosis in rats exposed to aluminum.Methods1.Animal modelingTwo rats were randomly selected from each group.1%sodium pentobarbital was used as anesthetic and anesthetized by intraperitoneal injection.After gradually replacing the blood of rats with normal saline,perfusion with 4%paraformaldehyde was carried out at a faster rate and then slower rate.The time was about 30 to 40 minutes.When it was found that the liver of rats began to harden and whiten,cardiac perfusion was complete.Brain tissue was immediately separated and immersed in 4%polyformaldehyde to fix the shape for paraffin section.The remaining rats were fasting for 24 hours after the end of the water maze experiment.After weighing,they were decapitated and killed.Brain tissues and hippocampus were quickly separated from the ice,washed with cold saline water,then frozen rapidly in liquid nitrogen.Finally,they were stored at-80 C to prepare for the follow-up experiment.2.Assessment and Measurement of Learning and Memory Ability in Aluminum-exposed RatsMorris water maze test was used to evaluate the effects of flavonoids on learning and memory function in rats exposed to aluminum.The experiment was divided into two stages:training and testing,lasting for 5 days.During the training period from day 1 to 4,the improvement of learning function of rats exposed to aluminum was evaluated by measuring the latency and total swimming distance of rats.On day 5,the improvement of memory function of rats exposed to aluminum was evaluated by measuring the swimming time of target quadrant and the frequency of times of crossing the platform.3.Determination of apoptotic and autophagic levels in hippocampus of rats exposed to aluminiumAfter the end of the whole feeding period,2 rats in each group were anesthetized and then perfused.After decapitation,brain tissue was quickly removed and fixed in 4%paraformaldehyde for at least 24 h to prepare paraffin sections.The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay(TUNEL)was used to detect the overall apoptosis of hippocampus cells in each group.Expression of Bcl-2 and Bax mRNA was detected by RT-PCR.Western blot was used to detect Bcl-2,Bax,Beclin-1 and LC3 protein expression level,to study on the antagonism of flavonoids on apoptosis of hippocampus cells in rats exposed to aluminum.Results1.Effects of flavonoid intervention on learning and memory function in rats exposed to aluminumDuring the training period,tlatency(s)and the total swimming distance showed a downward trend with the increase of training days.Compared with other groups,the aluminum exposure group decreased slowly,and there was no statistical difference between the groups(P>0.05).During the test period,compared with the negative control group,the target quadrant swimming time and the frequency to reach the platform in the aluminum-treated group were significantly decreased,and the difference was statistically significant(P<0.05).The target quadrant swimming time and the frequency to reach the platform in the detoxification group showed different degrees of increase compared with the aluminum exposure group.The swimming time of the target quadrant in the high dose group of rutin and the high dose group of puerarin increased significantly(P<0.05);the frequency to reach the platform in the high dose rutin group and puerarin groups increased significantly,the difference was statistically significant(P<0.05).The rutin and puerarin high dose groups had longer swimming time in the target quadrant than in the low-dose group,and more frequency to reach the platform.While in the silymarin group,the opposite was true,it is the low dose group that had longer target quadrant swimming time than the high dose group,and more frequency to reach the platform.2.Effect of Flavonoid on Apoptosis of Hippocampus Cells in Aluminum-exposed RatsIn the negative control group,almost no apoptotic cells were observed;compared with the negative control group,brain tissue sections of rats exposed to aluminum showed hippocampal neuronal degeneration accompanied by apoptotic neurons(brown-yellow neurons);Brain tissue sections in rutin and puerarin groups showed that although some apoptotic neurons still exist,the number of apoptotic cells induced by aluminum has been significantly reduced,and in the high dose of rutin and puerarin groups there were fewer apoptotic cells than the low dose group of rutin and puerarin.However,compared with the aluminum-treated group,the number of apoptotic cells was decreased in the low dose group of silymarin compared with the aluminum-treated group,but a large number of apoptotic cells were still observed in the brain tissue sections of the high-dose silymarin group.Compared with AI-exposed group,there was no significant reduction in the number of apoptotic cells.Compared with the negative control group,the expression of Bax mRNA and Bax protein in the aluminum-induced group was significantly increased,and the difference was statistically significant(P<0.05).Compared with the aluminum-treated group,Bax mRNA and Bax protein expression were significantly decreased in the detoxification group except for the high dose of silymarin(P<0.05).Moreover,the reduction of Bax mRNA and Bax protein expression in the high-dose rutin group and the high-dose puerarin group were more than that in the low-dose group.There was no significant difference in the expression of Bcl-2 mRNA and Bcl-2 protein between the groups(P>0.05),but the expression of Bcl-2 protein was less in the aluminum-treated group than in the control group.3.Effect of flavonoids on autophagy in rats exposed to aluminumCompared with the negative control group,the expression of Beclinl protein in the aluminum-treated group was significantly decreased,and the difference was statistically significant(P<0.05).Compared with the rats exposed to aluminum,the expression of Beclinl protein in the low dose rutin group,the high dose puerarin group and silymarin groups increased significantly(P<0.05).Compared with the negative control group,the expression of LC3 protein in the aluminum-induced group was significantly decreased(P<0.05).Compared with the rats in the aluminum-treated group,the expression of LC3 protein in the detoxification group was significantly increased,and the difference was statistically significant(P<0.05).Conclusion1.Flavonoids can significantly improve the learning and memory ability of rats exposed to aluminum.2.Rutin,puerarin and low concentration of silymarin can effectively regulate apoptosis and autophagy-related protein expression in hippocampus,inhibit apoptosis and increase to normal autophagy level,thus protecting the nervous system of rats exposed to aluminum.3.Higher concentration of silymarin antagonized the apoptosis of hippocampal cells induced by aluminum by increasing the level of autophagy of hippocampal cells.4.Flavonoids have a protective effect on the morphological damage of brain tissue in rats exposed to aluminium. |
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