Effects of Paeonia lactiflora Extract on Estrogen Receptor β, TPH2, and SERT in Rats with PMS Anxiety

Abstract

This study is to investigate the effect of Paeonia lactiflora extract on PMS anxiety and on expression of estrogen receptor β (ERβ), tryptophan hydroxylase-2 (TPH2), and serotonin transporter (SERT) in the premenstrual syndrome (PMS) anxiety model rats. The vaginal smear and open field test were used to screen rats in nonreception phase of estrus cycle with similar macroscopic behaviors and regular estrus cycle. PMS anxiety model rats were prepared by electrical stimulation. RT-PCR and immunofluorescence were used to measure the expression of ERβ, TPH2, and SERT. Compared with normal rats, the total distance in the open field test of the model rats was significantly increased (P < 0.05). The model rats showed nervous alertness, irritability, and sensitivity to external stimuli. After treatment with the Paeonia lactiflora extract, the total distance of rats was significantly reduced (P < 0.05). In reception stage, there was no significant difference in the mRNA and protein expression of ERβ, TPH2, and SERT. In nonreception stage, the expression of ERβ and TPH2 in the model group was significantly decreased (P < 0.05) as compared with the control group, but not SERT. Abnormal changes of the above indicators were reversed after the administration of the Paeonia lactiflora extract. In conclusion, Paeonia lactiflora extract can increase the expression of ERβ and TPH2 and decrease SERT in PMS model rats, which may be one of the mechanisms underlying the effect of Paeonia lactiflora extract on PMS.

Figure 1

Experimental flowchart of PMS model establishment. Time points of the body weight measurements (circles), the open field test (hexagon), the vaginal smear test (triangles), and the sample collection (double triangles) are shown. Vaginal smear method was used to select the rats with regular estrous cycles (a cycle for 4 to 5 days). Then, these rats were weighed and subjected to open field test. After that, they were grouped. Each group contained both rats in the reception phase and nonreception phase. In the following estrus cycle, PMS modeling was carried out and corresponding drugs were given. After modeling, samples were collected.

Figure 2

Photomicrograph of cells in each phase of rat estrus cycle. (a) Preestrus. Vaginal smears included mostly nucleated epithelial cells, occasionally with a small amount of keratinization, and no leukocyte. (b) Midestrus. Vaginal smears included mostly nucleated keratinocytes, a small amount of epithelium, and no white blood cells. (c) Late estrus. Vaginal smears included keratinocytes, nucleated epithelial cells, and white blood cells. (d) Diestrus. Vaginal smears included a large number of white blood cells and a small amount of epithelial cells and mucous cells. Left panel, 10×; right panel, 40×.

Figure 3

Column chart of the total distance of rats in each group before and after modeling. ^∗∗P < 0.01 as compared with the normal group; ^#P < 0.05 as compared with the model group.

Figure 4

mRNA expression of TPH2, SERT, and ERβ in dorsal raphe nucleus in reception and nonreception stage. (a) TPH2 mRNA level, (b) ERβ mRNA level, and (c) SERT mRNA level. The mRNA expression of related genes in control, model, Paeonia lactiflora extract, fluoxetine, and ERβ agonist group were detected by RT-PCR. ^∗∗∗P < 0.001 as compared with the control group; ^# and ^##P < 0.05 and < 0.01 as compared with the model group, respectively.

Figure 5

Neuronal distribution pattern and protein expression of TPH2, SERT, and ERβ in dorsal raphe nucleus in reception and nonreception stage (IF 10x20). (a) TPH2 protein expression, (b) ERβ protein expression, and (c) SERT protein expression. The protein expression of related genes in control, model, Paeonia lactiflora extract, fluoxetine, and ERβ agonist group were detected by immunofluorescence. Left, neuronal distribution pattern; right, the fluorescence intensity. ^∗∗∗P < 0.001 as compared with the control group; ^# and ^##P < 0.05 and< 0.01 as compared with the model group, respectively.