| Objective:To study the effects of Polygala tenuifolia Willd/Acorus tatarinowii have on ethanol-induced memory reappearance disorder in mice,and research its mechanism.Methods:1.The new object recognition method was used to determine the optimal model dose of ethanol-induced learning and memory impairment in mice(gavage single dose 30%ethanol/2 g·kg-1,50%ethanol/2 g·kg-1,30%ethanol/0.1 ml·10 g-1,50%ethanol/0.1ml·10 g-1).Established the model of ethanol-induced memory reappearance disorder in mice with the optimal dose selected.2.The new object recognition method was used to evaluatethe effects of single dose of dedoction of Polygala tenuifolia Willd(0.65 g·kg-1、1.3 g·kg-1、2.6 g·kg-1),Acorus tatarinowii(0.65 g·kg-1、1.3 g·kg-1、2.6 g·kg-1),and the two were combined in different proportions(1:1、1:2、2:1,the dose all were 1.3 g·kg-1)to the model mice,and select the appropriatedose.3.Enzyme-linked immunosorbent assay was used to detect the influence of Pt,At,Pt:At with different propotion on the Glu and GABA content in hippocampus of mice whose memory reappearance disorder induced by ethanol.Explored the mechanism from the perspective of excitement-inhibition nervous system balance.4.Western blot was used to detect hippocampal GAD67（GABA synthase）,GAT1（GABA transporter 1）,VGAT（GABA vesicle transporter）,GABAAR（GABAA receptor）protein expressionin of mice treated with ethanol at optimal concentrations of Polygala tenuifolia Willd,Acorus tatarinowii and their different proportions of drug groups.5.The CCK8 method was used to determine the suitable ethanol concentration damaged PC12 cells,established the ethanol-induced injury model of PC12 cells with the best dose.The CCK8 method was used to evaluate the effects of Polygala saponin,Polygalanone,3,6-disaperoyl sucrose,α-asarone,β-asarone,and the mouse serum containng Pt,At,combination on ethanol-induced injury model of PC12 cells.Results:1.When the ethanol concentration is 30%,50%,and the dose is 2 g·kg-1（just single dose）,the new object recognition index of mice reduced significantly.The new object recognition index of the control group was 57.46±5.14.Compared with the control group,the new object recognition index of the 30%ethanol 2 g·kg-11 model group was 44.21±5.03（P<0.05）,and the new object recognition index of 50%ethanol 2 g·kg-11 model group was 40.69±5.86（P<0.01）.2.After gavaged of 50%ethanol 2 g·kg-1,compared with control mice,the new object recognition index reduceed significantly（P<0.001）.Compared with the model group,the new object recognition index of mice in the Pt administration group significantly increased,and the effect decreased with increasing dose(0.65 g·kg-1dose group,P<0.001;1.3 g·kg-11 dose group,P<0.01;2.6 g·kg-11 dose group,P<0.05),the different dosage groups of At showed significant difference compared with the model group（P<0.001）.In the medicine pair group,when Pt and At was administered in a ratio of 1:1,the effect was the strongest,with a significant difference（P<0.001）,which was higher than the same dose of Pt′s and At′s effect when they were used alone.3.Changes of Glu and GABA contents in mouse hippocampus:ethanol has no effect on Glu content in mice hippocampus（P?0.05）;GABA neurotransmitter in the hippocampus of model group mice decreased significantly,from 9.73±2.79 nmol/L to 0.97±0.45 nmol/L（P<0.001）,compared with the model group.The GABA content of the mice in the Pt administration group was significantly increased（P<0.001 or P<0.01）,and with the increase of the dose,there was a dose dependence.All of them had significant differences（P<0.001）.At this time,the single medicine of Pt and At were more effective than the combination of the two drugs（P<0.001 or P<0.01 or P<0.05）.The effect of At was better.4.Western blot results:compared with normal control mice,the expression of GAT1,VGAT on vesicles,GAD67 was increased（P<0.05 or P<0.01 or P<0.001）,and the expression of GABAAR protein in the hippocampus of the model group was decreased（P<0.05）.Compared to the model group,the administration group can significantly lower the elevated level of VGAT expression（P<0.001 or P<0.01）,two drug compatibility group has the most significant effect;in contrasted to the model group,GAT1 was reduced significantly in the hippocampus of mice in the At administration and two-drug-compatible group（P<0.01 or P<0.001）,while the Pt group had a reduction effect,but there was no significant difference（P?0.05）.In contrasted to the model group mice,all the groups lower the level of GAD67expression（P<0.001）,and has statistically significant.Compared with the model group,GABAAR in the hippocampus of the Pt administration group was significantly increased（P<0.01）.4.Cell experiment research results show:compared with the control group,the survival rate of cells was significantly reduced when the concentration of ethanol was400mM.Polygala saponin,Polygalanone,and 3,6-disaperoyl sucrose,have no protective effect on the cell damage model caused by 400 mM ethanol;when the concentration ofα-asarone,β-asarone is 5μg·mL-1,there is obviously protective effect（P<0.05,P<0.001）;the serum of the mouse containing At has a strong protective effect on the model（P<0.001）.Conclusions:1.When the concentration of ethanol was 50%and gavage dose is 2 g·kg-1,the learning ability of mice was reduced.Pt and At could improve the memory reappearence disorder induced by ethanol in mice,the effcet was most significant when the propotion was 1:1.2.Ethanol can disturb the Glu/GABA balance in the hippocampus of mice.Pt and At can regulate the GABA content by regulating the GABAergic neuron system,thereby regulating the nervous system’s excitation/inhibition balance,thereby improving the mice’s learning ability.3.The active substance in Polygala tenuifolia Willd hadn’t effect on the ethanol model cells.Acorus tatarinowii had palpable protective effect on the nerve cells damaged by ethanol,which can be considered for further exploration. |
