Suanzaoren decoction exerts its antidepressant effect via the CaMK signaling pathway

Abstract

Calmodulin-dependent protein kinases (CaMKs) are widely regarded as "memory molecules" due to their role in controlling numerous neuronal functions in the brain, and the CaMK signaling pathway plays a crucial role in controlling synaptic plasticity. Suanzaoren decoction (SZRD) can improve depression-like behavior and thus has potential benefits in the clinical treatment of depression; however, its mechanism of action is not fully understood. In this study, we found that key proteins in the CaMK signaling pathway were regulated by the decoction used to treat depression. The purpose of this research was to ascertain if the SZRD's therapeutic efficacy in the treatment of depression is associated with the modulation of key proteins in the CaMK signaling pathway. A rat model of depression was created by exposing the animals to chronic, unexpected, mild stress. Model rats were given intragastric administration of SZRD or fluoxetine every morning once a day. Protein and mRNA relative expression levels of CaM, CaMK I, and CaMK IV in the hippocampus were measured by Western blot, quantitative polymerase chain reaction, and immunohistochemistry in the hippocampus. Our findings demonstrated that SZRD significantly improved the mood of depressed rats. This indicates that SZRD, by modulating the CaMK signaling system, may alleviate depressive symptoms and lessen work and life-related pressures.

Keywords: CaMK signaling pathway; Suanzaoren decoction; depression; hippocampus.

Figure 1

The CaM, CaMK I, and CaMK IV activation cascades and function in the central nervous system. The concentration of Ca²⁺ in the cytoplasm increases when voltage-sensitive Ca²⁺ channels, ligand-gated ionotropic channels, or G-protein-coupled receptors are activated in the plasma membrane. It may also increase when Ca²⁺ is released from the endoplasmic reticulum by IP3R or RyR. Subsequently, CaM forms a complex with Ca²⁺ and triggers the activation of CaMKK, which in turn exerts its influence on CaMK I, and CaMK IV. CREB, cAMP response element-binding protein; IP3, inositol trisphosphate; IP3R, IP3 receptor; RyR, ryanodine receptors.

Figure 2

(a) Experimental procedures and (b) the experimental animal intervention method. Before the start of the trial, all rats were fed ad libitum for a full week. All rats were raised in isolation with the exception of the control group, and all rats in the CUMS, SZRD + CUMS, and fluoxetine + CUMS groups were subjected to the CUMS protocol to induce depression. Over the course of the experiment, the CUMS rats were exposed to a variety of stimuli from 8:00 to 9:00 a.m. every day for 28 days. The SZRD doses were 2.5, 5, and 10 g/kg, which were equivalent to 1/2, 1, and 2 times the equivalent clinical dose, respectively. Rats in the fluoxetine + CUMS group were given the drug (1.8 mg/kg, dissolved in a concentration of 90% normal saline) once daily 1 h before being exposed to the stress stimulus. BW, body weight; SPT, sucrose preference test; OFT, open-field test; and CUMS, chronic unpredicted mild stress.

Figure 3

The effect of SZRD treatment on behavior in CUMS model rats. (a) BW. (b) Total liquid consumption (ml). (c) Sugar water consumption (ml). (d) Pure water consumption (ml). (e) Sugar water preference (%). (f) Horizontal score. (g) Vertical score. ^* p < 0.05, ^** p < 0.01 vs control; ^△ p < 0.05, ^△△ p < 0.01 vs CUMS. n = 20 per group.

Figure 4

Ca²⁺ titer assay and CaM activity assay. The data were normalized to the control group. ^* p < 0.05, ^** p < 0.01 vs control; ^△ p < 0.05, ^△△ p < 0.01 vs CUMS; n = 4 per group.

Figure 5

The effects of SZRD treatment on CaMK protein and mRNA expression in the hippocampus at 28 days after CUMS. (a) Western blot analysis of representative bands and (b) quantification of CaM, CaMK I, and CaMK IV expression in the hippocampus. (c) Quantitative analysis of relative CaM, CaMK I, and CaMK IV mRNA expression in the ipsilateral basal ganglia by qPCR. The data were normalized to the control group. The results were consistent with the Western blot trend. ^* p < 0.05, ^** p < 0.01 vs control; ^△ p < 0.05, ^△△ p < 0.01 vs CUMS; n = 4 per group.

Figure 6

The effect of SZRD treatment on the relative expression levels of various proteins in hippocampus at 28 days after CUMS. (a) CaM protein expression level (IOD) after 28 days. (b) CaMK I protein expression level (IOD) after 28 days. (c) CaMK IV protein expression level (IOD) after 28 days. (d) Histogram of CaM, CaMK I, and CaMK IV IOD values. The data were normalized to the control group. The results were consistent with the Western blot trend. ^* p < 0.05, ^** p < 0.01 vs control; ^△ p < 0.05, ^△△ p < 0.01 vs CUMS; n = 4 per group.