Study On The Interaction Between HDAC1 And P21^WAF1/CIP1 In Cognitive Changes Induced By Ethanol At Different Concentration In Female Mice

ObjectiveAt present,the social and medical problems caused by alcoholism have become a major public health challenge.The hippocampal structure of the limbic system plays an important role in higher nervous activities such as learning and memory.Histone deacetylases（HDACs）are tissue-modifying enzymes that can regulate gene transcription through the modification of histone and chromatin structure,affect gene expression,and play an important regulatory role in the process of learning and memory.Among them,histone deacetylase 1（HDAC1）can down-regulate the expression of p21^WAF1/CIP1 in mammals.The mechanism of HDAC1 and p21^WAF1/CIP1interaction in the effects of ethanol on learning and memory is unclear.Many studies have shown that excessive alcohol consumption can lead to cognitive loss,but because the relationship between ethanol and the brain is very complicated,some studies have also pointed out that low-concentration alcohol intake does not damage the ability of learners to learn and remember.The purpose of this study was to investigate the changes of HDAC1 and p21^WAF1/CIP1 protein expression in the hippocampal structure of female mice after ethanol administration at different concentrations,and to explore its mechanism of action at the molecular level.Methods1.8-week-old female Kunming mice were randomly divided into control group,2%ethanol group,10%ethanol group and 50%ethanol group.Mice were given the drug by gavage（0.1 ml/10 g）every day and the control group was given the same concentration of normal saline for 10 consecutive days.2.Five days after ethanol administration,the Morris water maze experiment was used for behavioral test to evaluate the learning and memory ability of the animals.Then the animals were killed and hippocampal tissue was taken on ice.3.The expression levels of HDAC1 and p21^WAF1/CIP1AF1/CIP1 proteins in the hippocampus of mice were detected by Western blot.4.The expression levels of HDAC1 and p21^WAF1/CIP1 proteins in the hippocampal region of mice after ethanol administration were detected by immunohistochemical staining.5.The high-affinity binding sites of HDAC1 in p21^WAF1/CIP1 promoter region was screened by using ChIP-qPCR.Results1.Morris water maze results:The escape latency of female mice of each group which in the navigation experiment of ethanol poisoning in mice,from the third day,compared with the control group,the escape latency of the 50%ethanol group was significantly longer（P<0.05;P<0.05;P<0.001）.However,from the fourth day,the escape latency of the 2%ethanol group was significantly shortened（P<0.01;P<0.001）.In the space exploration experiment,compared with the control group,the swimming speed of the female mice in each group of the experimental group was stable without significant changes（P>0.05）.In addition,compared with the control group,the effective area staying distance in the 50%ethanol group was shortened（P<0.05）,and the number of crossing platforms was significantly reduced（P<0.01）.However,compared with the control group,the effective area stayed longer in the 2%ethanol group（P<0.05）,and the number of crossing the platform was increased（P<0.05）.There was no significant change in the staying distance in the effective area of the 10%ethanol group（P>0.05）,but the number of crossing the platform was more than that of the control group（P<0.05）.2.Western blotting test results:After administration of different concentrations of ethanol to female mice,compared with the control group,the expression level of HDAC1 protein in the hippocampus of female mice in the 50%ethanol group was significantly reduced（P<0.01）,and the expression level of p21^WAF1/CIP1 protein was increased（P<0.05）.In contrast,the expression of HDAC1 protein in the hippocampus of female mice in the 2%ethanol group was significantly increased（P<0.01）,and the expression level of p21^WAF1/CIP1protein was significantly reduced（P<0.01）.There was no significant change in HDAC1 and p21^WAF1/CIP1 protein expression in hippocampus of female mice in the 10%ethanol group（P>0.05）.3.Immunohistochemical test results:Compared with the control group,the expression of HDAC1 protein in the hippocampal CA1 region of female mice in the50%ethanol group was significantly reduced（P<0.001）,and the expression of p21^WAF1/CIP1 protein was significantly increased（P<0.01）.The expression of HDAC1 protein in the hippocampal CA1 region of female mice in the 2%ethanol group was increased,and the expression of p21^WAF1/CIP1 protein showed a downward trend,but the difference was not statistically significant（P>0.05;P>0.05）.The expression of HDAC1 protein in the hippocampal CA1 area of female mice in the 10%ethanol group was decreased,and the expression of p21^WAF1/CIP1 protein was increased,but the differences were not statistically significant（P>0.05;P>0.05）.4.ChIP-qPCR test results:The relative expression of DNA in f1 to f10 fragments of p21 ^WAF1/CIP1AF1/CIP1 promoter region and HDAC1 antibody were:0.16±0.04,4.53±1.17,5.87±0.27,0.28±0.12,0.37±0.17,0.36±0.11,3.84±1.08,0.34±0.09,0.64±0.02,0.92±0.07.It can be seen that the relative expression of DNA in f2,f3,and f7fragments was significantly higher than the corresponding fragments in the control group（1±0）（P<0.01）.ConclusionsAlthough high concentrations of ethanol can cause learning and memory impairment in adult female mice,low concentrations of ethanol may improve cognitive function in adult female mice.The mechanism underlying these changes may be related to the participation of HDAC1 and p21^WAFI/CIP1.Among them,the p21^WAF1/CIP1promoter region（-400bp^-800bp）,（-800bp^-1200bp）and（-2400bp^-2800bp）may be the functionally active regions combined with HDAC1.