| Objective:Premenstrual Syndrome(PMS)is a series of symptoms manifested by women in physiology,emotion,and behavior.It is characterized by obvious periodicity and often appears during the premenstrual phase(luteal phase),disappearing after menstruation.Premenstrual Dysphoric Disorder(PMDD)is one of the common types of PMS and severely affects the quality of life and physical and mental health of affected women.It manifests as severe mental and emotional disorders such as irritability,depression,anxiety,and sensitivity.The pathogenesis of PMDD is not yet clear,and research suggests that it is related to sex hormones and neurotransmitters.Clinical treatments mainly consist of selective serotonin reuptake inhibitors(SSRIs)or hormone therapy,but they have limited efficacy and certain side effects.Based on the theory of "the liver as the regulator of circulation,"this study explores the relationship between the pathogenesis of PMDD and changes in the progesterone metabolite allopregnanolone(ALL0).The study clarifies the main components of Shuyu Capsules and verifies their effects on PMDD liver-qi stagnation syndrome rats and central brain region response.Based on this,the study discovered the disease and drug-targeting mechanisms around the y-aminobutyric acid(GABA)A receptor-mediated diseases and Shuyu Capsule intervention mechanisms in the periaqueductal gray(PAG)around the midbrain aqueduct.Methods:(1)Blood was taken from the mandibular venous plexus of non-treated rats while awake to determine the levels of progesterone and ALLO in the serum of normal and stressed rats during different estrous cycles.Behavioral indicators were tested through forced swimming experiments,and Shuyu Capsules and the positive drug fluoxetine were used for treatment to determine the effect of Shuyu Capsules in improving PMDD depressive behavior.(2)Using a slow-release pump to simulate the rapid withdrawal of ALLO,the open field test and forced swimming experiment were selected as behavioral paradigms to detect the phenotypic behavior and ALLO content in plasma to explore the relationship between the pathogenesis of PMDD liver-qi stagnation syndrome and rapid ALLO withdrawal.(3)Real-time fluorescence quantitative PCR(RT-PCR)and protein immunoblotting experiments were used to detect the expression of GABAA receptor alpha 4,betal,delta subunit proteins,and gene transcription in the PAG brain region of rats after drug treatment to reveal the possible targeting mechanisms of the drug.(4)Using mass spectrometry analysis system to detect the main components of Shuyu Capsules,the PMDD liver-qi stagnation syndrome rat model was obtained using forced swimming as a modeling method.Results:(1)In the non-treatment experiment,there were significant differences in the levels of progesterone and ALLO in the serum of rats in the normal group during each estrous cycle[F3,8=195.6,P<0.0001;F3,8=29.26,P=0.0001].The progesterone content was significantly higher in the late stage of estrus than in the early stage of estrus,pre estrus,and estrus(P<0.0001,p=0.0583,p<0.0001),and the ALLO content was also significantly higher(p=0.0002,p=0.0007).There were significant differences in the levels of progesterone and ALLO in the serum of rats in the stress group during each estrous cycle[F3,8=142.8,p<0.0001;F3,9=10.01,p=0.0032].The progesterone content was significantly higher in the late estrous period than in the early estrous period,pre estrous period,and estrous period(p<0.0001,p=0.0004,p<0.0001),and the ALLO content was also significantly higher(p=0.0134,p=0.0589,p=0.0017).In the forced swimming modeling group experiment,compared with the control group,the stress group showed a significant increase in immobility time(p<0.0001)and immobility times(p=0.0001)during the late diestrus phase,and there was no significant difference between the two groups during the estrus phase.In the forced swimming evaluation experiment after drug treatment,compared with the model group,the Shuyu group and fluoxetine group showed a significant decrease in immobility time(p<0.0001,P=0.0036,P=0.0018)and immobility times(p<0.0001,p<0.0001,p<0.0001)during the late diestrus phase,similarly,there was no significant difference between groups during the estrus phase.(2)In the ALLO rapid withdrawal simulation experiment using a microdialysis pump,microscopic examination showed that the plasma ALLO levels of the PMDD liver-qi stagnation model rats were decreased compared with the control group(p=0.0037),and both the Shuyu group and fluoxetine group showed a significant increase in ALLO levels during the late diestrus stage(p=0.0423,p=0.0479).Behavioral experiments showed that in the open field test,there was no difference in total distance traveled during the late diestrus stage among the groups.Compared with the model group,the Shuyu group showed a significant increase in the central area distance and duration during the late diestrus stage(p=0.0019,p=0.0012),while the fluoxetine group showed a trend of significant increase in the central area distance and duration during the late diestrus stage.In the forced swimming experiment,compared with the model group,the Shuyu group showed a significant decrease in immobility time and immobility counts during the late diestrus stage(p=0.0261,p=0.0198),while the fluoxetine group showed a significant decrease in immobility time(p=0.0332)and a trend of decrease in immobility counts during the late diestrus stage.(3)In the PAG neuronal GABAA receptor subunit distribution and protein expression detection experiment in PMDD liver-qi stagnation model rats,RT-PCR detection showed that compared with the control group,the model group showed an increase in GABAAR β 1 and δ subunit levels(p=0.0120,p=0.0587),while both the Shuyu group and fluoxetine group showed a significant decrease in GABAAR β1 and δ subunit levels during the late diestrus stage(p=0.0131,p=0.0631 for Shuyu).There was no significant difference in the level of GABAAR α 4 among the groups.WB detection showed that compared with the control group,the model group showed a trend of increase in GABAAR β 1 andδ subunit levels,while both the Shuyu group and fluoxetine group showed a trend of decrease in GABAAR β1 and δ subunit levels during the late diestrus stage.(4)Chemical component identification revealed that the main compounds of Shuyu capsules were rutin,glycyrrhizic acid,saikosaponin c,and ferulic acid.Conclusion:(1)By detecting the concentrations of progesterone and ALLO in the serum of normal and stressed rats during different estrous cycles,it was determined that the peak values of progesterone and ALLO appeared in PMDD rats during the late estrous period,and rapidly decreased during the pre-estrous period,indicating that there was a significant abnormal fluctuation in the ALLO of PMDD rats.Through forced swimming tests,the PMDD liver-qi stagnation syndrome rat model was successfully established and evaluated for drug efficacy.Results showed that Shuyu Capsules could improve typical depressive-like behavior in PMDD rats during specific phases of the estrous cycle.(2)Furthermore,simulation of rapid withdrawal of allopregnanolone(ALLO)using a slow-release pump induced the PMDD liver-qi stagnation syndrome behavior phenotype.By testing behavior and plasma ALLO levels,our hypothesis was confirmed that "the mechanism of action of Shuyu Capsules in treating PMDD liver-qi stagnation syndrome may counteract the rapid physiological decline of ALLO during the late luteal phase,exerting a steroid-like effect and thereby affecting the reactivity of the brain region to ALLO withdrawal and subsequent changes in behavior responses."(3)The expression of GABAA receptor subunits α 4,β 1,and δ in the periaqueductal gray(PAG)region of PMDD model rats fluctuated with changes in ALLO levels.Shuyu Capsules may improve depressive-like behavior by correcting the changes in GABAA receptor subunit expression caused by PMDD. 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