Possible Involvement of µ Opioid Receptor in the Antidepressant-Like Effect of Shuyu Formula in Restraint Stress-Induced Depression-Like Rats

Abstract

Recently μ opioid receptor (MOR) has been shown to be closely associated with depression. Here we investigated the action of Shuyu, a Chinese herbal prescription, on repeated restraint stress induced depression-like rats, with specific attention to the role of MOR and the related signal cascade. Our results showed that repeated restraint stress caused significant depressive-like behaviors, as evidenced by reduced body weight gain, prolonged duration of immobility in forced swimming test, and decreased number of square-crossings and rearings in open field test. The stress-induced depression-like behaviors were relieved by Shuyu, which was accompanied by decreased expression of MOR in hippocampus. Furthermore, Shuyu upregulated BDNF protein expression, restored the activity of CREB, and stimulated MEK and ERK phosphorylation in hippocampus of stressed rats. More importantly, MOR is involved in the effects of Shuyu on these depression-related signals, as they can be strengthened by MOR antagonist CTAP. Collectively, these data indicated that the antidepressant-like properties of Shuyu are associated with MOR and the corresponding CREB, BDNF, MEK, and ERK signal pathway. Our study supports clinical use of Shuyu as an effective treatment of depression and also suggests that MOR might be a target for treatment of depression and developing novel antidepressants.

Figure 1

HPLC chromatogram of Shuyu preparation. Six major constituents were detected. The concentration, linearity, limit of detection (LOD), limit of quantification (LOQ), and interbatch variation (CV) are listed in Table 1.

Figure 2

Behavior changes of stressed rats treated with/without Shuyu and/or CTAP. Body weight was monitored before stress (0 day) and just before sacrifice (5 day) (a). Forced swimming test and open field test were performed just before sacrifice. Duration of immobility (b), number of crossings (c), reflecting range of motion, and the number of rearings (d), reflecting exploratory behaviors were recorded as explained in the Materials and Methods. Values are shown as mean ± SEM (each n = 14). ^## P < 0.01 compared with the normal control group. ^** P < 0.01 compared with RRS group. ^&& P < 0.01 compared with Shuyu treated RRS group.

Figure 3

Expression of MOR in the CA1 and CA3 region of hippocampus detected by immunofluorescence staining (a) (×400) and by Western blot analysis (c). The numbers of MOR-immunoreactive neurons in the hippocampus were calculated (b). The intensity of immunoblot bands were quantified by Alphalmager TM 2000 and presented as mean ± SEM (each n = 14). ^## P < 0.01 compared with the normal control group. ^** P < 0.01 compared with RRS group. ^&& P < 0.01 compared with Shuyu treated RRS group.

Figure 4

BDNF protein expression in the rat hippocampus examined by Western blot analysis. The intensity of immunoblot bands was quantified by Alphalmager TM 2000 and presented as mean ± SEM (each n = 14). ^## P < 0.01 compared with the normal control group. ^** P < 0.01 compared with RRS group. ^&& P < 0.01 compared with Shuyu treated RRS group.

Figure 5

Phosphorylation of CREB in the rat hippocampus presented by the ratio of pCREB/CREB protein expression as examined using Western blot analysis. The intensity of immunoblot bands was quantified by Alphalmager TM 2000 and expressed as mean ± SEM (each n = 14). ^## P < 0.01 compared with the normal control group. ^** P < 0.01 compared with RRS group. ^&& P < 0.01 compared with Shuyu treated RRS group.

Figure 6

Phosphorylation of MEK in the rat hippocampus presented by the ratio of pMEK/MEK protein expression as examined using Western blot analysis. The intensity of immunoblot bands was quantified by Alphalmager TM 2000 and expressed as mean ± SEM (each n = 14). ^## P < 0.01 compared with the normal control group. ^* P < 0.05, ^** P < 0.01 compared with RRS group. ^&& P < 0.01 compared with Shuyu treated RRS group.

Figure 7

Phosphorylation of ERK1/2 in the rat hippocampus presented by the ratio of pERK1/2 and ERK1/2 protein expression as examined using Western blot analysis. The intensity of immunoblot bands was quantified by Alphalmager TM 2000 and expressed as mean ± SEM (each n = 14). ^## P < 0.01 compared with the normal control group. ^** P < 0.01 compared with RRS group. ^&& P < 0.01 compared with Shuyu treated RRS group.