| Objective: To explore the location and expression change of tryptophan hydroxylase 2 and serotonin transporter mediated by a estrogen receptor beta in vaious brain regions(dorsal raphe nucleus, hippocampus, hypothalamus) with premenstrual syndrome rat model during receptive and non-receptive period, and the intervention effect by Shu-Yu capsule(a chinese medicine formula). To reveal the role of ERβ regulating TPH2 and SERT in PMS with liver-qi depression syndrome, try to find the target point of Shu-Yu capsule and provide the experimental evidence for application Shu-Yu capsule in clinic.Methods: 1.We use the open field test to select healty female rats and then we confirmed the estrum of rats(including receptive and non-receptive period) by observing the vagina cell shape and their behavior. The rats of non-receptive period were introduced for model rats with PMS liver-qi depression syndrome by restraint stress method. The body quality and open field test were used to evaluated the model. Immunofluorescence technique(IF), Western blot(WB) and semiquantitative RT-PCR method were used to detect the location and expression of ERβ, TPH2 and SERT in dorsal raphe nucleus, hippocampus, hypothalamus of rats with different estrous cycle. 2. The same method were use to select rats, and then the experiment were divided into six groups: normal group, model group, fluoxetine group, Shu-Yu group,ERβ antagonist and ERβ agonist group. Fluoxetine, Shu-Yu and ERβ antagonist and ERβ agonist were used to intervention the model rats when we prepare the model except normal and model group.After modeling, we choose non-receptive period rats, using the method same as experiment one to detect the same genes, in order to elucidate the effects of Shu-Yu capsule on the location and expression of TPH2 and SERT mediated by ERβ of PMS model rats with liver-qi depression syndrome.Results: 1. Macroscopic behavior evaluation results ①Compared with the the normal rats, the body weight of model group were significantly decreased(P < 0.05); compared with the model group, fluoxetine and Shu-Yu group increased significantly(P < 0.01); ② The open field test: compared with the normal group, the score of the model group decreased significantly(P < 0.05), compared with the model group,the open field score of fluoxetin and Shu-Yu group increased significantly(P < 0.05). 2.The molecular biology experiment results: compared with the normal group, the distribution,mRNA and protein expression level of ERβ, TPH2, SERT in dorsal raphe nucleus, hippocampus and hypothalamus of model group rat has no significant difference in the acceptance period of estrous cycle; at non-receptive period of rats, compared with the normal group, genes of model group were distributed clutterly, the ERβ expression level increased significantly(P < 0.05, P < 0.01); the SERT and TPH2 expression level was significantly decreased(P<0.05, P < 0.01); compared with the model group,the distribution of the cell in hippocampus, hypothalamus and dorsal raphe nucleus of the drug intervention groups(otherwise agonist group)has no difference. The expression of ERβ were significantly decreased(P < 0.05, P < 0.01); SERT and TPH2 of mRNA and protein expression level increased significantly(P<0.05 or P < 0.01).Conclusion:(1) The expression of ERβ, TPH2, SERT in dorsal raphe nucleus, hippocampus, hypothalamus of PMS model rats with liver-qi depression syndrome in non-receptive period of estrous cycle changed significantly, but have no changed in receptive period of estrous cycle. We hypothesized that ERβ, TPH2, SERT may play important role in the pathogenesis of PMS with liver-qi depression syndrome; and this model has estrus dependence which in accordance with the clinical symptoms.(2) Shu-Yu capsule can effectively correct the excess expression of ERβ and TPH2, decreased expression of SERT in the dorsal raphe nucleus, hippocampus, hypothalamus of PMS with liver-qi depression syndrome rats in non-receptive period, these result give us a cue that ERβ and its mediated TPH2 and SERT are targets of Shu-Yu capsule which may has ERβ antagonism effect, and may regulate this signal pathway as a signal switch to intervention. |
