| Purpose:To explore the pathogenesis of brain center of chronic restraint stress premenstrual dysphoria(PMDD)rats with liver qi depression syndrome,and to improve the effective components of Radix Bupleuri combined with Radix Paeoniae Alba,and then to explain the brain center mechanism of the treatment of liver qi depression in the basic theory of traditional Chinese medicine.Materials and methods:① based on the study of TCM depression and Shugan Jieyu treatment,combined with the study of modern science on PMDD liver qi depression syndrome,the hypothesis that "Shugan Jieyu Decoction and Chinese herbal Chaihu combined with white peony can treat PMDD liver qi depression syndrome" was put forward.② PMDD rat model of liver qi depression was established by chronic restraint stress.The behavioral indexes of rats were tested by open field experiment,high price cross maze experiment and forced swimming experiment.③ The mRNA expression of a 2,α4,α5,β 2,γ 2L,δ and other subunits of GABAA receptor was detected in hippocampus and hypothalamus of different groups of rats.④The available components of Radix Bupleuri and Radix Paeoniae Alba were screened by network pharmacology and literature search.⑤ The binding of GABAA receptor to[H3]labeled target drugs and the primary screening components of Radix Bupleuri and Radix Paeoniae Alba was studied.⑥ The primary neurons of fetal rat cortex were infused with different components selected from Radix Bupleuri and Radix Paeoniae Alba to detect the changes of cell membrane potential.Results:① rat model evaluation:rat body mass:there was no significant difference in the measurement of rat body mass before modeling.There was no significant difference in body weight between the model group and the blank control group(P=0.9859).After administration,compared with the model group,there was no significant difference in body weight between the bupleurum combined with white peony group and fluoxetine group.Open field experiment:compared with the blank control group,there was no significant difference in the total distance of each group(P=0.7344).After modeling,compared with the blank control group,the distance between the model group and the open field central area was significantly different(P<0.0001),and the number of central area entry was significantly different(P=0.0001).Compared with the model group,there was no significant difference in the distance between Bupleurum combined with white peony group and fluoxetine group,but there was a rising trend(P=0.0965)and a rising trend in the central area stay time(P=0.1933).Elevated cross maze test:compared with the blank control group,there was a significant difference in the percentage of open arm access times in the model group(P<0.0001),There was a significant difference in the percentage of open arm stay time(P=0.0042).After treatment,compared with the model group,there was a significant difference in the percentage of open arm stay time in fluoxetine group and Chaishao group(P<0.0001),and there was a significant difference in the percentage of open arm entry times(P<0.0001).Forced swimming test:suspension time ratio:compared with the blank control group,the suspension time ratio in the model group was significantly different in the non acceptance period(P=0.0028),and there was no significant difference in the suspension time ratio in the acceptance period(P=0.9910);after the treatment,compared with the model group,the suspension time in the non acceptance period in the bupleurum combined with white peony group and fluoxetine group decreased significantly Low(P<0.0001).② Real time fluorescence quantitative PCR experiment:through the detection of different subunits gene expression of GABAA receptor in the hippocampus and hypothalamus of PMDD rats with liver qi depression syndrome,it was found that there was no significant difference in the expression of α 2(P=0.2169),α 5(P=0.0788),β2(P=0.0796)between different groups in the hippocampus;α 4(P=0.0188),γ 2(P=0.0207),δ(P=0.0071)subunits gene expression trend or obvious The difference of authorship.There was no significant difference in the expression of α 2(P=0.9864),α 5(P=0.7445),γ 2(P=0.4360),α 4(P=0.0325),β 2(P=0.0306)and other subunits.③ The possible effective components of Radix Bupleuri are:stigmasterol,α-spinagosterol,troxerutin,quercetin,baicalin and rutin;the possible effective components of Radix Paeoniae Alba are β-sitosterol,betulinic acid,paeoniflorin and paeoniflorinone.④ Radioreceptor ligand binding assay:Quercetin(inhibition rate=146%),paeoniflorin(inhibition rate=63%),rutin(inhibition rate=100%)in Radix Bupleuri combined with Paeoniae Alba were found to be able to compete with[H3]labeled flunitrazepam for the binding site of GABAA receptor in the brain cell membrane of PMDD model rats with liver qi depression.⑤ Cell patch clamp test:30 um paeoniflorin,rutin and quercetin can induce obvious inward current,while 30 um GABA and compound paeoniflorin,rutin and quercetin(100/300/1000 uM)can significantly reduce the induced current,and show significant dose-response glycoside(inhibition rate=47.31%),rutin(inhibition rate = 34.01%),quercetin(inhibition rate=58.71%).Conclusion:the whole animal behavior experiment confirmed that the combination of Radix Bupleuri and Radix Paeoniae Alba can effectively improve the Depression Behavior of PMDD rats with liver qi depression and the abnormal expression of GABA-AR related subunits in the brain.Therefore,we infer that the abnormal expression of GABA-AR related subunits in the brain is one of the pathogenesis of liver qi depression caused by insufficiency of liver catharsis.Through screening,we can find out the ingredients that can interact with GABA-AR in the mixture of Radix Bupleuri with classical Shugan Jieyu recipe:paeoniflorin,quercetin and rutin.We infer that the action of GABAAR in the brain is one of the brain central mechanisms of Liver Soothing and depression relieving therapy,and the compatibility of Radix Bupleuri with Radix Paeoniae Alba can achieve the effect of Liver Soothing and depression relieving through the action of paeoniflorin,quercetin and rutin on GABA-AR in the brain. |
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