Pharmacological Mechanism Of Baixiangdan Capsule For Treatment Of PMS Model Rats With Liver-qi Invasion—Effect Of Medicated Serum On The Expression Of5-HTR2C And The Signaling Pathways In Neonate Rat Hippocampal Neurons Cultured In Vitro

Objective:Our aims are to explore the pharmacological mechanisms of Baixiangdan capsule by observing the expression level of5-hydroxytryptamine2C receptor(5-HTR2C) and the changes of its signaling pathways in neonate rat hippocampal neurons cultured in vitro. At the same time,we want to seek for parts of the pathogenesises of Premenstrual Syndrome liver-qi invasion,and study the effects of Baixiangdan capsule acting on the liver-qi invasion.Methods:1.Premenstrual syndrome(PMS) model rats with liver-qi invasion were induced by multiple emotional stimulations and Baixiangdan capsule was administered after PMS induction.The medicated serum were used to intervene in neonate rat hippocampal neurons.The expression level of5-HTR2C in the neurons was evaluated by western blot and the concentrations of IP3and DAG were measured with Elisa method.Changes of [Ca2+]i in hippocampal neurons were detected with confocal laser scanning microscopy by using Ca2+fluorescent dyes Fluo-3/AM.2.First,paeoniflorin level of the normal rat serum was detected by HPLC in different time after the treatment of Baixiangdan capsule.According to the peak time of paeoniflorin concentration-time change curve,we got the rat blood for drug serum. Second,we used the medicated serum to intervene in the hippocampal neurons and activated the signaling pathways of5-HTR2C.Third,we used Elisa method to detect the concentrations of IP3and DAG in hippocampal neurons and we determined the changes of [Ca2+]i with Fluo-3/AM calcium fluorescence probe.Results:1.The behavioral test results:Compared with the normal group, the scores of the PMS liver-qi invasion model group rats display significant difference (P<0.001). Meanwhile, when treating with drugs, the significant difference disappears(P<0.01;P<0.05;P<0.01). Western blot findings:Compared with the normal group,the protein expression of5-HTR2C in PMS liver-qi invation group increased obviously(P<0.001).Compared with model group,all three drug groups decreased significantly (P<0.001;P<0.05;P<0.001).We also find the concentrations of IP3/DAG/Ca2+in PMS liver-qi invation group were significantly higher in neurons than the normal group (P<0.001;P<0.001;P<0.01).After treated with drugs, the contents of IP3/DAG/Ca2+in neonate rat hippocampal neurons were significantly improved(P<0.05;P<0.001;P<0.05) and all of them had no significant difference compared with the normal group.2.By RP-HPLC chromatography,we find that when the normal rats had been given Baixiangdan and Paeonia lactiflora P.E. by gavage for2hours,the paeoniflorin level of the medicated serum was maximum.After activating the signaling pathways of5-HTR2C in hippocampal neurons,compared with the normal group, the concentrations of IP3and DAG in both Baixiangdan group and Baishao group were significantly reduced(P<0.001;P<0.001),but Baishao group was not as good as Baixiangdan group.Also,[Ca2+]i both in Baixiangdan group and Baishao group were lower than the normal group(P<0.001;P<0.05).Conclusion:The Premenstrual syndrome(PMS) model rats with liver-qi invasion can be prepared successfully by multiple emotional stimulations.5-HTR2C in hippocampal neurons is closely related to the pathogenesises of PMS liver-qi invasion.Baixiangdan capsule medicated serum can change the expression of5-HTR2C and influence on the concentrations of IP3/DAG/Ca2+in cellular signaling pathways.Baixiangdan capsule might partially plays its role in ameliorating PMS by regulating the expression of5-HTR2C and change the contents of IP3/DAG/Ca2+.5-HTR2C may be one of the drug targets of Baixiangdan capsule.