| Purpose:1.The rat model of PMDD liver-qi inversion syndrome was established by natural screening-residential invasion method and hormone induction-residential invasion method respectively.The scientific nature and applicability of PMDD rat model were confirmed by OFT,EPM,LDB and other behavioral evaluation,and a perfect rat model in line with the standard of combination of PMDD liver-qi inversion syndrome was established to provide a carrier for the study of animal model with TCM characteristics and the pathogenesis of PMDD and drug intervention.2.The rat model of PMDD liver-qi inversion syndrome was established by hormone induction-residential invasion method.By detecting the levels of behavior,neurosteroids and neurotransmitters in each group,the expression and distribution of key factors of progesterone and allopregnanolone anabolism transduction pathway in hippocampus and other brain regions were detected to clarify the role of progesterone and allopregnanolone anabolism transduction pathway in the occurrence and development of PMDD liver-qi inversion syndrome and the intervention mechanism of paeonol.Thus,it partly reveals the microcosmic mechanism of liver catharsis,which adds new understanding to enrich and innovate the theory of liver governing catharsis,and enriches the connotation of the basic theory of modern traditional Chinese medicine.3.The rat model of PMDD liver-qi inversion syndrome was established.The rats were intervened with paeonol and treated with flumazenil.by detecting the levels of behavior,neurosteroids and neurotransmitters in each group,and preliminarily exploring whether paeonol could play a role in treating PMDD by regulating GABAA receptor(benzodiazepine receptor site),and explaining the intervention mechanism of paeonol.The purpose of this study is to provide reference and basis for clinical diagnosis,treatment,drug research and pathogenesis of PMDD with liver-qi inversion syndrome.Methods:1.The rat model of PMDD liver-qi inversion syndrome was established by natural screening-residential invasion method:the rats with regular estrous cycle were naturally screened by vaginal resistance method,and the screening cycle was divided into three estrous cycles,about 12 days.According to the score of attack and fight in the non-receptive period,the rats were randomly divided into normal control group(normal saline group and CMC-Na group),model group,fluoxetine group,paeonol group,paeonol-flumazenil group and flumazenil group.2.The rat model of PMDD liver-qi inversion syndrome was established by hormone induction method-residential invasion method:the estrous cycle was induced by hormone induction method.According to the score of attack and struggle in non-receptive period,the rats were randomly divided into normal control group,model group,fluoxetine group and paeonol group.3.Behavioral evaluation and drug intervention:the drug was given by intragastric administration while making the model,before and after treatment,and the behaviors of rats were evaluated by OFT,EPM,LDB,RIT and so on.in the non-receptive period after two complete cycles(8 days),the blood of abdominal aorta of rats was collected and decapitated to separate the hippocampal brain area of rats,and stored in the refrigerator at-80 ℃ for examination.4.The levels of hormones(progesterone,allopregnanolone,alloprenolone,dehydroepiandrosterone)in peripheral serum of rats were detected by ELISA.5.The contents of neurotransmitters(glutamic acid,γ-aminobutyric acid,serotonin,norepinephrine)in serum of rats were detected by UHPLC-MS/MS.6.The protein expressions of GABAAR α 4,β 2,δ subunits,BDNF,CYP11A1(P450 enzyme),5 α reductase(SRD5A2)and TSPO(PBR)in hippocampal region of rats were detected by Western blotting method.7.The localization and expression of GABAAR α 4 and δ subunits,TSPO and CYP11A1(P450 enzyme)proteins in the hippocampus of rats in each group were detected by double immunofluorescence localization.Results:1.The rat model of PMDD liver-qi inversion syndrome was established by natural screening-residential invasion method and hormone-induced residential invasion method,respectively,and the open field,EPM,LDB and aggressive behavior of rats were evaluated during the non-receptive period.The results were as follows:(1)Behavioral evaluation of the rat model of PMDD liver-qi inversion syndrome made by natural screening-residential invasion:Compared with the control group,the stay time in the central area of OFT in the model group was significantly lower than that in the control group(P<0.05).The percentage of entry number(OE%)and the percentage of time(OT%)entering the open arm of EPM decreased significantly,the time and distance of movement in the bright area of LDB decreased significantly(P<0.0001),and the score of aggressive behavior increased significantly(P<0.05).②Behavioral evaluation of the rat model of PMDD liver-qi inversion syndrome induced by hormone-residential invasion:compared with the control group,the total distance of exercise in OFT,the distance of exercise in the central area and the stay time in the central area of the model group were significantly lower than those in the control group(P<0.05).In the EPM test,OE%and OT%were significantly decreased,and the total distance of exercise in LDB was significantly lower than that in the control group(P<0.05).The exercise distance and stay time in the bright area decreased significantly(P<0.01),and the score of aggressive behavior in the model group was significantly higher than that in the control group(P<0.0001).(2)The effect of paeonol on the behavior of rats with PMDD liver-qi inversion inversion syndrome induced by hormone-intrusive method:compared with the control group,the total distance of exercise and the distance of exercise in the bright area in the model group were significantly lower than those in the control group(P<0.001),and the number of times entering the bright area was significantly decreased(P<0.0001).There was no significant difference between fluoxetine group and paeonol group(P>0.0001).Compared with the model group,the total distance of exercise.in OFT and the distance of exercise in the central area were significantly increased in paeonol group(P<0.001,P<0.05).OE%and OT%in EPM test were significantly increased(P<0.05,P<0.001),and the total distance of exercise in LDB and stay time in bright area were significantly increased(P<0.05),and the score of attack was significantly decreased(P<0.01).In fluoxetine group,the total exercise distance in OFT and LDB increased significantly(P<0.01),the exercise distance and stay time in LDB bright area increased significantly(P<0.0001),and the score of aggressive behavior decreased significantly(P<0.05).2.Effect of paeonol on serum neurotransmitters and neurosteroids in rats with PMDD liver-qi inversion syndrome induced by hormone-intrusive method:compared with the control group,the contents of Glu and DHEA in serum of the model group were significantly decreased and DHEA increased significantly(P<0.05).Compared with the model group,the content of Glu in serum of paeonol group increased significantly,while the content of 5-HT and NE decreased significantly(P<0.05).The content of ALLO and AP increased significantly(P<0.05,P<0.0001),The content of DHEA decreased significantly(P<0.0001).Compared with the model group,the contents of Glu and ALLO in serum of fluoxetine group increased significantly,while the content of 5-HT、NE and DHEA decreased significantly(P<0.05,P<0.001,P<0.0001).3.The effect of paeonol on the protein expression of GAB AAR-α 4,GAB AAR-β 2,GABAAR-δ,CYP11A1,BDNF,SRD5A2 and TSPO in the hippocampus of rats with PMDD liver qi inversion syndrome induced by hormone induced:compared with the control group,the content of BDNF in the model group was significantly lower than that in the control group(P<0.05).Compared with the control group,the content of TSPO in the hippocampus of the model group was significantly higher(P<0.05),while the content of TSPO in the paeonol group and fluoxetine group was significantly lower than that of the model group.There was no significant difference in the protein expression of GAB AAR-α 4,GAB AAR-β 2,GAB AAR-δ,CYP11A1 and SRD5A2 in the hippocampus of all groups.3.The expression of GABAAR-α 4/GABAAR-δ and PBR/CYP11A1 protein in the hippocampus of rats in each group was detected by double immunofluorescence method:compared with the control group,the cumulative fluorescence average intensity of GABAAR-α 4 in the model group was significantly higher than that in the model group,and the cumulative fluorescence intensity of GABAAR-α 4 protein in the paeonol group was significantly lower than that in the model group(P<0.05).Compared with the control group,the average cumulative fluorescence intensity of TSPO in the model group was significantly higher than that in the control group(P<0.01),but there was no significant difference in GABAAR-δ and CYP11A1 in each group(P>0.05).The results of double fluorescence labeling showed that the co-expression of GABAAR-α 4/GABAAR-δ and TSPO/CYP11A1 protein was not enhanced or weakened in the hippocampus of the model group.4.The effect of paeonol on the behavior of PMDD rats with liver-qi inversion syndrome treated with flumazenil:the results of open field test showed that the distance of movement and the time of staying in the central area in the model group were significantly less than those in the control group(P<0.001),and the times of entering the central area in the paeonol group were significantly lower than those in the model group(P<0.01).Compared with flumazenil group,paeonol group had no significant effect on open field behavior of model rats treated with flumazenil group(P>0.05).The scores of aggressive behavior in the model group were significantly higher than those in the control group(P<0.01).Compared with the model group,the aggressive behavior of rats in paeonol group and fluoxetine groupConclusion:1.The rat model of PMDD liver-qi inversion syndrome was successfully established by natural screening-residential invasion method and hormone-induced residential invasion method.The movement distance,residence time and times of entering the central area in the OFT,and the OE%and OT%of EPM,aggressive behavior score were cyclically dependent of menstrual cycle.Among them,the rat model of PMDD liver-qi inversion syndrome made by hormone induction-residential invasion method is stable and reliable,a high success rate,good credibility and practicability,and can better simulate the clinical characteristics of PMDD liver-qi inversion syndrome,so it can be used as a good carrier to study the pathogenesis of PMDD and the mechanism of drug intervention.2.The movement distance,stay time and times of entering the central area in the OFT,the movement distance,stay time of the bright area in the LDB,OE%and OT%in the EPM test and aggressive behavior score are the key behavioral indexes to evaluate the rat model of PMDD liver-qi inversion syndrome.The occurrence of PMDD liver-qi inversion syndrome may be related to the decrease of the content of GABA and the increase of the contents of DHEA and NE in the serum of rats.Paeonol could significantly decrease the contents of NE and DHEA,increase the contents of Glu、ALLO and AP,improve the physical and activity ability of model rats,and relieve anxiety and aggressive behavior of rats.3.By detecting the protein expression and distribution of GAB AAR-α 4,β 2,δ,CYP11A1,BDNF and other key molecules of progesterone and allopregnanolone synthetic metabolic transduction pathway in the hippocampus of rats with PMDD liver qi inversion syndrome,it was found that the protein expression and distribution of key proteins TSPO,BDNF and GABAAR-α 4 in the hippocampus of PMDD liver qi inversion syndrome rats were abnormal.The interventionof paeonol can reduced the expression of TSPO,GABAAR-α 4 and other proteins in the hippocampus of rats.The results of flumazenil intervention experiment showed that the intervention pathway of paeonol may not be related to the benzodiazepine site of GABAA receptor.To sum up,the abnormality of GABA energy system mediated by progesterone and allopregnanolone synthetic metabolic transduction pathway is closely related to the occurrence of PMDD liver-qi inversion syndrome.Paeonol can regulate GABA energy system mediated by this pathway.The above studies can provide reference and direction for the pathogenesis,clinical diagnosis and treatment of PMDD liver-qi inversion syndrome. |
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