Mechanisms Underlying the Antidepressant Effect of Acupuncture via the CaMK Signaling Pathway

Abstract

The CaMK pathway has been proven to play an important role in regulating cognitive function and emotional response. Acupuncture through the CaMK pathway improves depression-like behavior and the molecular mechanism related to its antidepressant remains to be explored. In this study, we aimed to determine whether the ability of acupuncture at Baihui (GV20) and Shenting (GV24) points to treat depression is related to the regulation of key proteins in the CaMK pathway. A rat model of depression was induced by chronic unpredicted mild stress (CUMS). Model rats in the electroacupuncture group were subjected to acupuncture at the Baihui (GV20) and Shenting (GV24) acupoints once a day for 20 min. Model rats in the fluoxetine group were gavaged with fluoxetine (1.8 mg/kg). Immunohistochemistry and Western blotting assays were used to evaluate immunoreactivity for and the protein expression levels of CaMKII, CaMKIV, and CaM. The results showed that electroacupuncture had a significant effect in rats with depression. Electroacupuncture and fluoxetine regulated the expression of key proteins in the CaMK signaling pathway, which is related to depression, in the hippocampi of rats. This indicates that acupuncture at Baihui (GV20) and Shenting (GV24) may alleviate depressive symptoms and reduce work- and life-related burdens and stress by regulating the CaMK signaling pathway.

Keywords: Baihui; CAMK signaling pathway; Shenting; depression; electroacupuncture.

Figure 1

(A) Experimental procedures and (B) experimental animal intervention method. All the experimental rats were fed for 1 week. A CUMS protocol was used to induce depression in the rats in the CUMS group, electroacupuncture group and fluoxetine group, and all of the rats were raised in isolation except those in the control group. The CUMS rats were exposed to different stimuli during the experimental period. The stimuli mainly included fasting (for 24 h), water deprivation (for 24 h), tail clipping (for 3 min), swimming in cold water (10°C, for 5 min), horizontal turbulence (for 2 min), day and night reversal (for 24 h) and heat exposure (40°C, for 5 min), and the stimuli were presented in an unpredictable manner. The model rats were subjected to the OFT and SPT. The BWs of the rats and other behavioral indexes data were measured at the appropriate time points. The animals in the CUMS group were exposure to the CUMS protocol, during which they were subjected to different stimuli. The animals in the electroacupuncture group were subjected to electroacupuncture for 20 min once a day before stress. The Baihui (GV20) and Shenting (GV24) acupoints, which correspond to human anatomical regions, were located according to “Acupuncture and Moxibustion.” The intensity of acupuncture was sufficient to induce microvibration of the rat head. The animals in the fluoxetine group were administered fluoxetine (1.8 mg/kg, 0.18 mg/ml distilled water) once a day 1 h before the stress exposure. BW, body weight; SPT, sucrose preference test; OFT, open-field test, CUMS, chronic unpredicted mild stress.

Figure 2

A total of 120 healthy SD rats were selected; 30 were used as control rats, and the remaining 90 were divided into the CUMS group (n = 30), electroacupuncture group (n = 30), and fluoxetine group (n = 30). These 90 rats were used for the construction of a depression model (CUMS) and subjected to the corresponding interventions. Compared with those of the rats in the control group, the OFT scores, sucrose water consumption, and BW of the rats in the CUMS group were significantly lower, indicating that significant depression-like behavior. In the CUMS group, the electroacupuncture group and the fluoxetine group, OFT scores, sucrose water consumption and BW were higher than those in the control group, and there was no significant difference between the groups. The data are expressed as the mean ± standard error of the mean (SD). (A) Changes in Body Weight. (B) Changes in Sucrose Preference. (C) Changes in OFT Scores. **P < 0.01 vs. the control group; ^#P < 0.05, ^##P < 0.01 vs. the CUMS group.

Figure 3

The tissues of interese were removed, and hippocampus tissues from both cerebral hemispheres were collected. Western blotting was used to compare the expression of CaMKII and CaMKIV at each time point on the basis of integrated optical density (IOD) values. On day 7, the expression of CaMKII in the hippocampus in the CUMS group was downregulated compared with that in the control group. However, there was no significant statistical difference in the expression of CaMKIV between the four groups on day 7. On the 14th and 21st days, the expression of CaMKII and CaMKIV in the hippocampus of in the CUMS group was significantly downregulated compared with that in the control group. Compared with that in the CUMS group, the expression of CaMKII in the electroacupuncture group and the fluoxetine group was significantly upregulated, whereas the expression of CaMKIV in the fluoxetine group was also significantly upregulated on day 14. Compared with that in the CUMS group, the expression of CaMKII and CaMKIV in the electroacupuncture group and the fluoxetine group was significantly upregulated on day 21. There were no significant differences in CaMKII and CaMKIV expression between the electroacupuncture group and the fluoxetine group. The data are expressed as the mean ± SD. CaMKII, calcium/calmodulin-dependent protein kinase II; CaMKIV, calcium/calmodulin-dependent protein kinase IV. (A) Expression of CaMKII on the hippocampus was shown by western blot. (B) Expression of CaMKIV on the hippocampus was shown by western blot. (C) CaMKII protein expression level. (D) CaMKIV protein expression level. *P < 0.05 vs. the control group; **P < 0.01 vs. the control group; ^#P < 0.05, ^##P < 0.01 vs. the CUMS group.

Figure 4

On the 7th, 14th and 21st days after the intervention, the expression in the hippocampus of rats was analyzed by immunohistochemistry. The expression of CaM1 in the CUMS group was significantly lower than that in the control group on day 7, day 14 and day 21. On day 14 and day 21, the CaM1 content in the hippocampus in the electroacupuncture group and the fluoxetine group was significantly higher than that in the CUMS group, and the difference between the electroacupuncture and fluoxetine groups was not statistically significant. The data are expressed as the mean ± SD. (A) Expression of CaM1 on the hippocampus was shown by immunohistochemistry. (B) CaM1 protein expression level . **P < 0.01 vs. the control group; ^##P < 0.01 vs. the CUMS group.