Paeoniflorin, the Main Active Ingredient of Shuyu Capsule, Inhibits Cav1.2 and Regulates Calmodulin/Calmodulin-Dependent Protein Kinase II Signalling

Abstract

The aim of this study was to explore the mechanism underlying the antidepression activity of paeoniflorin, the main active ingredient of paeony extract and Shuyu capsules, and determine its effect on the calmodulin/calmodulin-dependent protein kinase II (CaM/CaMKII) signalling pathway and on the possible target, the voltage-gated calcium channel (Ca_v). Rats at the nonacceptance stage were selected for premenstrual syndrome (PMS) depression modelling. Behavioural assays were used for model testing. Rats were given Shuyu capsules, paeony extract, and bupleurum. Western blot analysis was used to assess the expression levels of calcium voltage-gated channel subunit alpha 1 C (CACNA1C), brain-derived neurotrophic factor, and CaM/CaMKII signalling pathway proteins. Intracellular Ca²⁺ concentration in CHO cell line was measured using Fluo-4-AM and whole-cell patch clamps. The PMS depression model was successfully established and demonstrated that Shuyu can mitigate depressive behaviour in a rat PMS model. Paeony extract did not affect CACNA1C protein expression in rat hippocampi but did affect Ca_v1.2-mediated CaM/CaMKII signalling pathways. Paeoniflorin significantly inhibited KCl-induced increases in intracellular Ca²⁺ concentration and Ca_v1.2 current density. Further, it may function via the CaM/CaMKII pathway and its downstream signalling molecules by regulating Ca_v1.2, thus playing an important role in the treatment and alleviation of affective disorders.

Figure 1

Behavioural assays. (a) Body weight test. (b) Open-field test. (c) Sucrose preference test. For all assays, testing was performed both before and after modelling. Moreover, the following groups were analysed: the control/normal group, model group, Shuyu capsule group, bupleurum group, and paeony group. The statistical analysis for the behaviour assays was performed by one-way ANOVA (n = 10, ^∗∗∗P < 0.001).

Figure 2

Western blot and analysis of hippocampus tissue samples from the control/normal group, model group, Shuyu capsule group, bupleurum group, and paeony group. (a) CACNA1C protein level analysis (n = 6). (b) Analysis of calmodulin (CaM) protein level (n = 6, ^∗P < 0.05). (c) Analysis of phosphorylated calmodulin-dependent protein kinase II (p-CaMKII) level (n = 6, ^∗P < 0.05 and ^∗∗P < 0.01). (d) Analysis of brain-derived neurotrophic factor (BDNF) protein expression (n = 3, ^∗∗P < 0.01).

Figure 3

Intracellular calcium ion concentrations of primary culture of hippocampal neurons from groups including blank control, 50 μM paeoniflorin treatment, 100 μM paeoniflorin treatment, and 200 μM paeoniflorin treatment. (a) Morphology and fluorescence intensity of hippocampal neurons in each group before and after KCl and paeoniflorin treatment. Pf: paeoniflorin treatment. (b) Fluorescence intensity of each group over time (0–191 s). (c) Statistical comparison of fluorescence intensity at 25 s and 150 s in each group (n = 5, ^∗P < 0.05 and ^∗∗P < 0.01). Scale bar: 20 μm.

Figure 4

Whole-cell patch clamp analysis of CHO cells with LTCC constitutive expression. (a) Ca_v1.2 current density was recorded and analysed for the following groups: blank control, positive control (1 μM nifedipine treatment), 50 μM paeoniflorin treatment, 100 μM paeoniflorin treatment, and 200 μM paeoniflorin treatment. (b) Inhibition ratios for 1 μM nifedipine treatment, 50 μM paeoniflorin treatment, 100 μM paeoniflorin treatment, and 200 μM paeoniflorin treatment.