Effects And Mechanism Of Dihydromyricetin Ammonium On Behavioral Changes In Alcohol-drinking Mice

Objective:To explore the effect and mechanism of newly-synthesized dihydromyricetin ammonium（ADHM）on behavioral changes in alcohol-drinking mice.Method:1.Behavioral testings:Adult（6-8 weeks old,body weight 20±2g）male C57BL/6J mice（n=80）were divided into five groups:control,ethyl alcohol（EtOH）,ethyl alcohol+5 mg/kg Dihydromyricetin（DHM）,ethyl alcohol+5 mg/kg dihydromyricetin ammonium（ADHM1）,and ethyl alcohol+10 mg/kg dihydromyricetin ammonium（ADHM2）.The mice of the control group had free access to food and water while the others were treated as follows:（1）56%（v/v）ethyl alcohol was given to the mice of different groups by gavage and the corresponding drugs were then treated on each group.Finally,the loss of righting reflex（LORR）test was performed to observe the onset time and duration of drunkenness in mice.（2）56%（v/v）ethyl alcohol was given to the mice of different groups by gavage to establish the model of chronic intermittent ethanol exposure（CIE）and after 24 hours of withdrawal,the corresponding drugs were given to conduct tests as follows:（1）The Elevated Plus-Maze（EPM）was performed to observe the time of the mouse spent in the open arm and enclosed arm within 5 minutes and calculate the percentage of the time entering into the open arm and enclosed arm over the total time.（2）Alcohol tolerance test was performed on the groups of control,EtOH,and ADHM1 after withdrawal for 24 hours,which were given 56%（v/v）ethyl alcohol by gavage and to observe the onset time and duration of drunkenness in mice.The groups above were recorded as Control/E,CIE/E,and ADHM1/E,respectively.（3）All mice were given free access to 28%（v/v）ethyl alcohol to establish a model of intermittent voluntary drinking and EtOH and water consumption（gram per kilogram per 24 hours）and EtOH preference were calculated to measure the accurate EtOH amount consumed by each mouse.2.In vitro experiments:5 newborn Sprague-Dawley rats were used to extract primary hippocampal neurons and the cells were maintained in culture for 10-14 days to conduct the experiments as follows:（1）Immunofluorescence was carried out to identify hippocampal neurons by staining for microtubule associated protein 2（MAP2）,which was widely expressed in the neuron cell body and dendrites.（2）A laser confocal microscope was used to observe the hippocampal neurons loaded with a new chloride ion fluorescence probe（MQAE）.The fluorescence value was measured by ZEISS ZEN microscopic imaging software,calculate the maximum quenchable fluorescence F0 and the fluorescence change value F_Cl after Cl^-increasing.Furthermore,according to the equation of Stern-Volmer:F₀/F_Cl=1+KSV[Cl^-],quenching constant KSV was calculated and the standard curve of MQAE was plotted.（3）（1）Hippocampal neurons loaded with MQAE were divided into 5 groups:EtOH group（60mM）,A10 group（ADHM 10μM）,A100 group（ADHM 100μM）,EtOH+A10 group,and EtOH+A100Group.The fluorescence value was measured to produce F₀ and F_Cl and the Cl^-concentration（[Cl^-]_i）entering the cell was calculated according to the standard equation to clarify the effect of ADHM on the EtOH-induced Cl^-influx after the corresponding drugs treatments.（2）Hippocampal neurons loaded with MQAE were divided into 8 groups:EtOH group,Bicuculline（Bic）group,Flumazenil（Flu）group,EtOH+Bic group,EtOH+A100+Bic group,A100+Bic,EtOH+A100+Flu,and A100+Flu Group.The fluorescence value was measured to produce F₀ and F_Cl and the Cl^-concentration（[Cl^-]_i）entering the cell was calculated according to the standard equation to clarify the target of ADHM on GABA_AR after the corresponding drugs treatments.（4）After loading with MQAE,GABA_AR was activated by different concentrations of GABA（1uM,3 uM,10 uM,30 uM,100 uM,300 uM）and the fluorescence value was measured to calculate Ft,F_Cl.Finally,the GABA concentration-response curve of hippocampus neurons was drawn,and the same curve was drawn after adding 20uM ADHM（final concentration 10uM）as well to clarify the effect of ADHM on GABA concentration-response curve.Results:1.Animal behavior experiment:LORR test:Compared with the EtOH group,the duration of drunk in ADHM1,ADHM2,and DHM group was significantly shortened（p<0.0001）.There was no statistical difference between ADHM1 group and ADHM2 group compared with DHM group.EPM:Compared with the EtOH group,the time entering the open arm for the ADHM2 group was significantly longer（p<0.001）,the time for the enclosed arm was shortened（p<0.0001）,and the time for the ADHM1 and DHM group to enter the enclosed arm was shorter（p<0.001）.Compared with the DHM group,the entry time of the open arm and the enclosed arm of the ADHM2 group was not statistically different.There was no statistically significant difference in the time to enter the enclosed arm between ADHM1 group,ADHM2 group,and DHM group.Alcohol tolerance test:Compared with the Control/E group,the tolerance of mice in the CIE/E group was significantly increased（p<0.0001）.Compared with the Control/E group,the ADHM/E group had no significant difference in the duration of drunkenness.TBC:Compared with EtOH group,mice in ADHM1 group,ADHM2 group and DHM group consumed less alcohol per kilogram per day（p<0.0001）,but there was no statistical significance between the three groups at the end of week 7.Compared with the end of week 1,the preference for alcohol in EtOH group was significantly increased（p<0.05）,and that in ADHM1 group was slightly increased,but it was not statistically significant at the end of week 7.2.In vitro experiments（1）Immunofluorescence identification:After the hippocampal neurons were cultured in vitro to day 10,the neuron cell body is full and the neural fiber network is dense.Under the fluorescence microscope,microtubule-associated protein 2（MAP2）can be seen to express in the hippocampal neuron cell body and dendrites and the positive rate of neuronal cells is more than 95%.（2）The standard curve of MQAE fluorescence quenching:According to the experimental result,the standard equation was:y=0.078x+1.0987,R2=0.9931(y=F₀/F_Cl,x=[Cl^-]).（3）Compared with the Control group,Cl^-influx occurred in the EtOH group（p<0.0001）.The EtOH+A10 group and the EtOH+A100 group can offset the Cl^-influx caused by EtOH（p<0.0001）.After adding ADHM10μM/100μM alone,there was no statistical difference compared with the control group.Cl^-influx occurred in the EtOH+A100+Flu group,which was not statistically significant compared with the EtOH group.A large amount of Cl^-inflow did not occur in the groups of EtOH+Bic,EtOH+A100+Bic,A100+Bic,A100+Flu,Flu,Bic（p<0.0001）.There was no statistical difference between the groups of A100+Bic,Flu,and Bic.There was no statistical difference between the A100+Flu group and the Bic group.（4）As the concentration of GABA increases,the amount of Cl^-influx increases.However,adding 20uM ADHM low chloride buffer（final concentration 10uM）to the solution above led to the GABA concentration-response curve moved to the left.Conclusions:1.Animal studies have found that like DHM,ADHM has the effects of antialcoholism and sober up,which can relieve anxiety caused by alcohol withdrawal and prevent alcohol tolerance and alcohol dependence.2.ADHM acts on the benzodiazepine site of GABA_AR,and it can target on synaptic and extrasynaptic GABA_AR to stop the Cl^-influx caused by alcohol-activated GABA_AR.