Study On The Underlying Mechanism Of Dsorders Cause By The Liver Failing To Conver And Disperse-Allopregnanolone Mediated GABAARα4 Sensitivity In The Pathogenesis Of Specific Brain Regions Of PMDD

Purpose:The main content of TCM visceral image theory is "liver dominates catharsis,regulating emotion",revealing its deep micro mechanism is the only way to promote visceral image theory from phenomenon description to essence explanation.Therefore,focusing on the key problem of brain central mechanism of "liver dominating and relieving,regulating emotion",this paper takes PMDD rat model of Liver Qi reverse syndrome as the carrier,and on this basis,observes the pathogenesis of PMDD Liver Qi Reverse Syndrome(PMDD-LIS)and the relationship between allo mediated GABAAR function in specific brain areas.In order to explain part of the central mechanism of li ver catharsis,to prove the necessity of classification treatment,and to provide experimental evidence for clinical effective treatment of PMDD.Methods:①Through combing the literatures of traditional Chinese medicine and modern medicine of PMDD-LIS,present a hypothesis that "ALLO-mediated GABAAR dysfunction is one of the mechanisms of PMDD-LIS pathogenesis";②Using the Rat model of PMDD-LIS that induced by social isolation and residential invasion stress,observed rats’s aggression behavior,EPM and OFT,hormone and neurotransmitter contents in serum and specific brain areas,and the expression of GABAARa4 subunit in specific brain areas.③Administration of ALLO and its antagonists for PMDD-LIS rats,the hypothesis was further proved by the observation of aggression behavior,EPM and OFT,hormone and neurotransmitter content in serum and specific brain area,and the expression of GABAARα4 subunit in specific brain area.④Using the serum containing medicine in the above experiment to intervene the neurons of hippocampus of rats,observe the change of Cl-channel,and further clarify that allo mediates the change of GABAAR function,which is one of the important pathogenesis of PMDD-LIS.Results:①Behavioral study:PMDD-LIS Rats showed anxiety and irritability in the aggressive behavior test,and the aggressive behavior score was much higher than the Normal Group(P<0.01),after give exogenous ALLO,the rats attacked score was significantly lower than the Model Group(P<0.01),but the aggressive behavior score of the Finasteride Group was higher than Model Group(P<0.05).In the EPM,the number of times the Model Group entered the open arm percentage(OE%)and the percentage of Open arm residence time(OT%)were significantly reduced(P<0.01,P<0.05),and the OE%and OT%of the ALLO Group were increase(P<0.01).In the OFT,the total distance and the number of rats entering the central zone of the PMDD-LIS rats were significantly lower than the Normal Group(P<0.01,P<0.05).After give exogenous allo,the total distance and the times of entering the central area were increased(P<0.05).The results showed that the behavior of PMDD-LIS model rats showed aggressive and anxiety-like behavior,exogenous ALLO could significantly improve their abnormal behavior,which indicated that the occurrence of abnormal emotions in PMDD-LIS model rats was closely related to ALLO.②Research results of hormone and neurotransmitter contents in serum and specific brain areas:Compared with the Normal Group,the PROG in serum of the Model Group increased significantly(P<0.05),and the ALLO and CORT concentration decreased(P<0.05);In prefrontal and hippocampus,PROG content decreased significantly(P<0.01,P<0.05);in hypothalamus,PROG content increased significantly(P<0.01),ALLO content decreased significantly(P<0.01);in amygdala,allo content decreased significantly(P<0.01).After administration of exogenous ALLO,compared with the Normal Group,the content of PROG and ALLO in the prefrontal lobe of the ALLO Group increased significantly(P<0.05),and only the content of PROG increased in the hippocampus(P<0.05);the content of PROG in the hypothalamus significantly decreased(P<0.05),ALLO content increased(P<0.01);ALLO concentration in the amygdala increased(P<0.01)In the UHPLC-MS/MS,compared with the Normal Group,Glu,GABA and Glu/GABA in the serum of the Model Group decreased significantly(P<0.01,P<0.05),5-HT content increased(P<0.05).After ALLO was given,GABA in the serum increased(P<0.05),5-HT content decreased(P<0.01).It is suggested that ALLO may play a role by increasing the concentration of GAB A in serum of rats.The above results indicate that the pathogenesis of PMDD is closely related to the decrease of allo content in peripheral and central amygdala,hippocampus,prefrontal cortex and hypothalamus,and the significant decrease of GABA and Glu/GABA,while exogenous ALLO can significantly improve the above abnormal expression,and further clarify that the regulation of ALLO on central and peripheral GABA and Glu/GABA is closely related to the pathogenesis of PMDD-LIS.③In the Immunofluorescence Technic and RT-PCR,the change trend of α4 subunit was the same.Compared with the Normal Group,the expression of GAB AARα4 in the hypothalamus and amygdala of the Model Group rats decreased(P<0.01),while the expression of GAB AAR α4 in the hippocampus increased significantly(P<0.01).After give exogenous ALLO,the expression of GABAARα4 in amygdala increased(P<0.05),and the expression of GABAARα4 in hippocampus decreased(P<0.05).This is suggested that the pathogenesis of PMDD-LIS Model Rats is closely related to the expression of GABAARα4 in amygdala and hippocampus.There was no significant difference in the expression of GABAAR α 4 in prefrontal lobe and hypothalamus.④Whole-Cell Patch-Clamp study:Through the preparation of hippocampal neurons,we observed the effect of serum of PMDD-LIS model rats on chloride channel of hippocampal neurons.compared with the Normal Group,the Cl-current density of neurons in the Model Group decreased significantly(P<0.01,P<0.05)after the intervention of serum.After the intervention of exogenous ALLO,the Cl-current density of neurons increased significantly(P<0.05),there was no significant difference in Cl-current density between Finasteride Group and Model Group.Furthermore,the abnormal changes of chloride channel current related to GABAAR function mediated by allo are closely related to the pathogenesis of PMDD-LISConclusion:Through literature review and experimental research,we can draw the following conclusions:①PMDD-LIS model rats show aggressive and anxiety-like behavior,and exogenous ALLO can improve their aggressive and anxiety-like behaviors;②PMDD-LIS model rats show significant decrease in ALLO,GABA,Glu/GABA in peripheral and specific brain areas,The expression of GABAARα4 was abnormal in amygdala and hippocampus.The serum of PMDD-LIS interfered with hippocampal neurons,and Cl-current density was significantly reduced,while exogenous ALLO could significantly improve the above abnormal expression.In conclusion,ALLO mediated changes in GABAAR function is one of the pathogenesis of PMDD,mainly through changes in the expression of GABAARα4 in the amygdala and hippocampus.