| ObjectiveAlcoholism is a very common medical and social problem.Long-term exposure to high concentration alcohol can cause permanent damage to the central nervous system and produce hippocampal dependent cognitive impairment.The mechanism by which alcohol impairs learning and memory is complex.In recent years,more and more attention has been paid to the correlation between mTOR signaling pathway and autophagy and alcohol-induced cognitive impairment.Activated mTOR signaling pathway can down-regulate autophagy related factors.Puerarin can prevent spatial learning and memory impairment in mice induced by acute and chronic alcoholism.The mechanism of mTOR signaling pathway and autophagy in hippocampal structure has not been clarified in the process of different concentrations of puerarin interfering with the cognitive function of 52% alcohol-infected mice.The purpose of this study was to investigate the effects of different concentrations of puerarin on learning and memory of alcohol-infected mice and the changes in expression of mTOR signaling pathway and autophagy related proteins,so as to provide a new drug target for clinical prevention of alcohol-induced cognitive impairment.Methods1.Forty 8-week-old kunming female mice were randomly divided into 5 groups,including control group,52% alcohol group,25mg/kg puerarin +52% alcohol group,50mg/kg puerarin +52% alcohol group,and 100mg/kg puerarin +52% alcohol group.Mice in the 3 doses of puerarin group were first intraperitoneally injected with puerarin（0.2ml/10g）.After 1h,52% alcohol（0.1ml/10g）was given intragastric administration,while normal saline was applied in the control group at the dose of 0.1 ml/10 g by intragastric administration.The drug was administered at 4 p.m.every day for 28 consecutive days.2.On the 24 th day after administration,animals were subjected to behavioral tests using Morris water maze for 6 consecutive days to assess learning and memory function.From the 24 th day to the 28 th day of the test cycle,the mice in each group were administered after the daily positioning navigation test.On day 29,the space exploration test was carried out.After the test,some animals were immediately killed and their hippocampal tissues were separated on ice.In other animals,brain tissue was isolated on ice after cardiac perfusion and fixed in a 4% paraformaldehyde solution.3.The experimental method of HE staining was applied to observe the differences and similarities in the morphological structure of pyramidal neurons in the hippocampal CA1 area of different groups of mice.4.The expression levels of mTOR,p-mTOR,4EBP1,p-4EBP1,Beclin1,LC3 I and LC3 II in hippocampus of each group of mice were detected by western blotting assay and quantitatively analyzed.5.Immunohistochemistry was applied to detect the localization and expression levels of p-mTOR,p-4EBP1,Beclin1 and LC3 proteins in hippocampus of each group.Results1.Morris water maze results: From day 3 to 5 of the positioning navigation test,compared with the control group,the escape latency of mice in 52% alcohol group was significantly longer（P<0.01）.From day 3 to day 5,the escape latency of mice in the50mg/kg puerarin group was significantly shorter than that in the 52% alcohol group（P<0.01）.From day 4 to 5,the escape latency of mice in the 100mg/kg puerarin group was significantly shorter than that in the 52% alcohol group（P<0.01）.Compared with the 52% alcohol group,the 25mg/kg puerarin group showed significantly shorter escape latency only on day 5（P<0.01）.The results of space exploration test showed that compared with the control group,the ratio of the distance（time）to the total distance（time）in the target quadrant of the escape platform was significantly reduced in the 52% alcohol group（P<0.01）,the frequency of crossing the platform was significantly reduced（P<0.01）.Compared with 52% alcohol group,the ratio of the distance（time）to the total distance（time）in the target quadrant of the escape platform was significantly increased in the medium-high dose puerarin pretreatment group（P<0.01）,but there was no statistical significance in the comparison of the above two parameters between the two groups（P>0.05）.2.HE staining results: Compared with the control group,the number of pyramidal neurons in the hippocampus of mice in 52% alcohol group was reduced,and the arrangement was relatively loose,and the pyramidal neurons were damaged seriously.The ameliorative effect of medium and high dose puerarin on these injury changes was more obvious.The number of pyramidal neurons in hippocampus of mice in medium and high dose puerarin group was significantly higher than that in 52% alcohol group,and the arrangement was denser.However,low dose of puerarin did not significantly improve the pathological damage of pyramidal neurons in hippocampus induced by high concentration of alcohol.3.Western blotting results: Compared with the normal saline control group,the ratio of p-mTOR/mTOR and p-4EBP1/4EBP1 in hippocampus of mice in 52% alcohol group were significantly up-regulated（P<0.01）,while the ratio of Beclin1/β-actin and LC3 II/I was significantly down-regulated（P<0.01;P<0.05）.Compared with 52%alcohol group,the proportion of p-mTOR/mTOR and p-4EBP1/4EBP1 in medium and high dose puerarin groups was down-regulated（P<0.05;P<0.01）,while the ratio of Beclin1/β-actin and LC3 II/I was obviously up-regulated（P<0.05）.In addition,the ratios of Beclin1/β-actin and LC3 II/I in low-dose puerarin group were significantly up-regulated compared with that in 52% alcohol group（P<0.05）.There was no significant difference in the protein expression levels of mice between medium-dose and high-dose puerarin groups（P>0.05）.4.Immunohistochemistry results: The IHC staining results in hippocampal tissue were consistent with western blotting results.The protein expression levels of p-mTOR and p-4EBP1 in hippocampal CA1 region of 52% alcohol group were significantly higher than those in control group（P<0.01）.In contrast,the protein expression levels of Beclin1 and LC3 were significantly lower than those of the control group（P<0.01）.Compared with 52% alcohol group,the expression levels of p-mTOR and p-4EBP1 protein in medium and high dose puerarin groups were significantly decreased（P<0.05;P<0.01）,while Beclin1 and LC3 expression levels were significantly increased（P<0.01;P<0.01）.There was no significant difference in the protein expression levels of mice in medium and high dose puerarin groups（P>0.05）.ConclusionsLong-term consumption of high concentration alcohol（52% alcohol）can lead to impaired learning and memory function in female mice.50mg/kg and 100mg/kg puerarin can significantly improve the cognitive dysfunction caused by high concentration alcohol,which may be related to the inhibition of mTOR-4EBP1 signaling pathway by puerarin and the activation of autophagy related protein expression. |
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