| BackgroundDepression is a mental illness with high morbidity, recurrence rate and suicide rate, which harms humans’ physical and psychological health seriously. The pathogenesis of depression is associated with multiple factors as heredity, physiology, psychology and social effects, etc. In clinic, it’s characterized by constant down in spirits, slow thought and hebetude, always accompany with some physical symptoms such as sleep disorders, taciturnity, anorexia and weight reduction.At present, pharmacotherapy is common treatment in clinical in the word, but pharmacotherapy has toxic and side effect and be with poor patients’ compliance. Acupuncture is a holistic therapy that has multiple targets, so it gets more and more attention of people in the clinical therapy in last ten years. According to the statistics of clinical study, Dv-20 (Baihui) and Dv-29 (Yin Tang) acupoints are the most usually used to treat depression.In recent years, with the thorough research of pathogenesis of depression and mechanism of drugs acting on depression, cell signal transduction pathways have attracted attention of researchers. ERK signal pathway plays an important role in development, differentiation and regeneration of nerve cells, as well as regulating the plasticity of nerve cells, which can influence learning and memorizing. The pioneer study found that the depression-like behaviors of chronic stress rats were connected with the reduction of key proteins related to ERK signal pathway in hippocampus.On this study, we established chronic unpredictable stress and separation rat model, which pretreated with PD98059, an inhibitor of ERK, to discuss the relationship between the antidepressant-like effects of acupuncture and ERK signal pathway in hippocampus on model rats.Methods1. Animals and Groups.Sixty-eight male Sprague-Dawley rats (180±10 g) were randomly divided into eight groups (eight rats in each group):the Normal group, the Model group, the Acupuncture (Acu) group, the Fluoxetine (Flu) group, the Model+DMSO group, the Model+PD98059 group, the Acu+PD98059 group, the Flu+PD98059 group.2. Chronic Unpredictable Mild Stress Procedure. Rats in stressed groups were exposed to CUMS after 1 week for acclimatization period under the housing conditions. Seven different stressors were used to induce a depressive state, including food deprivation (24h), water deprivation (24 h), reversal of the light/dark cycle, wet bedding (24 h), cage shaken on a rocking bed (30 min), tail clip (3 min), behavior restraint on a restraining device (2 h). The CUMS procedure was carried out in stressed animals once per day for 3 weeks.3. Treatments. The Normal group was given no stress for 3 weeks; the Model group was exposed to CUMS for 3 weeks; the Acu group received acupuncture treatment (Dv-20 (Baihui) and Dv-29 (Yin Tang) acupoints) once every day during the 3week stress period; the Flu group was given once daily oral gavage administration of Fluoxetine (1.8mg/kg). The Model+DMSO group and the Model+PD98059 group were conditioned with same as the Model group except for the intracerebroventricular injection of DMSO (10μl) or PD98059 (dissolved in DMSO,10 μM,10μl) 1h before CUMS exposure; the Acu+PD98059 group and the Flu+PD98059 group were conditioned with same as the Acu group or the Flu group except for the intracerebroventricular injection of PD98059 30min before Acu/Flu delivery.4. Behavior Tests and index detection.We use open-field test, sucrose intake test and body weight to evaluate the model’behaviors. Western-blot was used to detect the protein levels of Raf, p-Raf, ERK, p-ERK, Rsk, CREB, p-CREB and BDNF in hippocampus. RT-PCR was used to detect the level of Bc1-2 mRNA and Bad mRNA.Results1. Behavior Tests. Before the experiment, there was no significant difference among groups. After 21-day stress procedure, the Model group showed a significant decrease of the number of crossing and rearing, sucrose intake and the body weight compared to the Normal group (P<0.01); the Acu group and the Flu group showed a significant increase of the number of crossing, sucrose intake and the body weight compared to the Model group (p< 0.05); the Model+PD98059 group showed a significant decrease of the number of crossing and body weight compared to Model group (p<0.05); both the Acu+PD98059 group and the Flu+PD98059 group showed a significant decrease of the number of crossing, sucrose intake and the body weight compared to the Acu group and the Flu group (p<0.01).2. The protein levels of ERK signal pathway. Western blot analysis revealed that there were significant difference in protein level of Raf and p-Raf between the Model group and the Normal group (P<0.01); Acupuncture and Fluoxetine all can increase the levels of Raf and p-Raf compared to the Model group (p< 0.01).The Model group showed a significant decrease in protein levels of ERK1/2 and p-ERK1/2 compared to the Normal group (P<0.01); the Model+PD98059 group showed a significant decrease in protein levelof p-ERK1/2 compared to the Model group (p<0.05); the Acu group and the Flu group showed a significant increase in protein levels of ERK1/2 and p-ERK1/2 compared to the Model group (p<0.05); the Acu+PD98059 group showed a significant decrease in protein levels of p-ERK1/2 compared to the Acu group (p<0.05). The Model group showed a significant decrease in protein levels of Rsk compared to the Normal group (P<0.01); the Acu group and the Flu group showed a significant increase in protein levels of Rsk compared to the Model group (p<0.05); the Acu+PD98059 group showed a significant decrease in protein levels of Rsk compared to the Acu group (p<0.05).3. The protein levels of CREB and p-CREB in hippocampus. The Model group showed a significant decrease in protein levels of CREB and p-CREB in hippocampus compared to the Normal group (P<0.01); the treatment of acupuncture and fluoxetine increased both CREB and p-CREB compared to the Model group (P<0.05); the Acu+PD98059 group showed no significant difference compared to the Acu group (p>0.05), but the Acu+PD98059 group showed a downtrend compared to the Acu group in hippocampus.4. The protein levels of BDNF in hippocampus. The Model group showed a significant decrease in protein levels of BDNF in hippocampus compared to the Normal group (P<0.01); the treatment of acupuncture and fluoxetine could increase the protein level of BDNF of rats exposed to CUMS (p<0.05); the Acu+PD98059 group showed no significant difference compared to the Acu group (p>0.05), but the Acu +PD98059 group showed a downtrend compared to the Acu group in hippocampus.5. The levels of Bcl-2 mRNA and Bad mRNA. RT-PCR analysis revealed that the Model group showed a significant decrease in Bcl-2 mRNA and increase in Bad mRNA compared to the Normal group in hippocampus (p<0.01); the treatment of acupuncture and fluoxetine could increase Bcl-2 mRNA in hippocampus (p <0.01) and decrease Bad mRNA in hippocampus(P<0.05); the Acu+PD98059 group showed a downtrend compared to the Acu group (P>0.05).Conclusions1. Chronic Unpredictable Mild Stress has a marked impact on rats behaviors. In this study, the Model group showed a low exploration to external environment and insensitivity to reward. Both acupuncture and fluoxetine could alleviated the depressive-like behaviors, and acupuncture had a better effect on alleviating the sensitivity to reward and maintaining the normal increase in body weight than fluoxetine.2. Acupuncture could raise the upstream of ERK signal pathway protein levels, such as Raf, p-Raf, ERK, p-ERK, and downstream protein levels,such as Rsk. The results suggested that the antidepressive-like effects of acupuncture was associated with activating the proteins in ERK signal pathway.3. Acupuncture could raise the protein levels of CREB, p-CREB and BDNF in hippocampus. The combine of BDNF and TrkB can activate the CREB through ERK signal pathway, which can phosphorylate CREB, and further promote genetic transcription and protein synthesis of BDNF. The results suggested that the antidepressive-like effects of acupuncture was associated with promoting synaptic remolding and nerve cell regeneration.4. Acupuncture could regulate the levels of Bcl-2 mRNA and Bad mRNA. The results suggested that the antidepressive-like effects of acupuncture was associated with reducing hippocampal neuron apoptosis.5. Pretreatment with PD98059 would exacerbate the model rats’ depressive-like behaviors. PD98059 is the inhibitor of MEK1 which is an upstream kinase of ERK. In this study, PD98059 could partly up regulate ERK phosphorylation and downstream proteins, such as Rsk, CREB, p-CREB and BDNF, as well as Bcl-2 mRNA.6. PD98059 blocked the upregulating roles of acupuncture on p-ERK, Rsk, CREB, p-CREB, BDNF and Bcl-2 mRNA. The results suggested that acupuncture had an effect on ERK signal pathway, which roles could be blocked by PD98059.7. According to this study, it’s suggested acupuncture had various roles, including promoting nerve cells to develop, differentiate and regenerate, regulating the plasticity of nerve cells, as well as preventing nerve cells apoptosis through activating the ERK signal pathway (BDNF-TrkB, Raf, ERK, Rsk, CREB, Bcl-2).
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