| Depression is a mental illness which is characterized by significant and constant down in spirits, slow thought and hebetude as the main clinical features, with high morbidity, high recurrence rate and high 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.At present, pharmacotherapy is common treatment in clinical in the world, but pharmacotherapy has toxic and side effects, and also with poor patients’ compliance. Acupuncture is a holistic therapy that has multiple targets, so it draws 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. Many studies confirmed that acupuncture treated depression through multi-targets, multi-levels and multi-ways.In recent years, with the thorough research of pathogenesis of depression and mechanism of drugs acting on depression, cell signal transduction pathways attracted the attention of researchers. PKA 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 PKA signal pathway in prefrontal cortex.In this study, we established chronic unpredictable stress and separation rat model, which was pretreated with H89, an inhibitor of PKA, in order to discuss the relationship between the antidepressant-like effects of acupuncture and PKA signal pathway in prefrontal cortex 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 Acu+H89 group, the Flu+H89 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 (4 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 did not suffer 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 per day during the 3-week stress period; the Flu group was given once daily oral gavage administration of Fluoxetine (1.8mg/kg). the Acu+H89 group and the Flu+H89 group were conditioned with same as the Acu group or the Flu group except for the intracerebroventricular injection of H8930min 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. ELIS A was used to detect the level of IL-6 and TNF-a in serum. Western-blot was used to detect the protein levels of PKA, CREB, p-CREB of cAMP signal pathway in prefrontal cortex.Results1. Behavior Tests.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 sucrose intake and the body weight compared to the Model group (p<0.05); both the Acu+H89 group compared to the Acu group, the Flu+H89 compared to the Flu group showed a significant decrease of the number of sucrose intake and the body weight (p< 0.01).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 both acupuncture and flouxetine had a positive effect on alleviating the sensitivity to reward and maintaining the normal increase in body weight. 2. The protein levels of PKA signal pathway.Western blot analysis revealed that there were significant differences in protein level of PKA and CREB between the Model group and the Normal group (p<0.01); Both Acupuncture and Fluoxetine will increase the levels of PKA and CREB compared to the Model group (p<0.01).The Model group showed a significant decrease in protein levels of p-CREB compared to the Normal group (p<0.01); the Acu group and the Flu group showed a significant increase in protein levels of p-CREB compared to the Model group (p<0.01); the Acu+H89 group and the Flu+H89 group showed no significant differences in protein levels of p-CREB compared to the model group (p>0.05). Acu group showed a significant increase compared to the Acu+H89 group (p<0.05). Flu group showed a significant increase compared to and the Flu+H89 group (p<0.01).Acupuncture can raise PKA, CREB and p-CREB protein in PKA signal pathway in prefrontal cortex of CUMS rats.3. IL-6 and TNF-α levels in serumCompared with normal group, a remarkable increase of IL-6 and TNF-a in serum can be seen in model group (p<0.01); Compared with model group, there is a significant decline of IL-6 and TNF-a in Acu group, Flu group, Acu+H89 group and Flu+H89 group (p<0.01, p<0.05). The Acu+H89 group compared to the Acu group, the Flu+H89 compared to the Flu group showed no significant changing.Acupuncture and Fluoxetine can effectively reduce inflammatory cytokines IL-6, TNF-a in serum of rats with depression, and Fluoxetine has a better effect on improving IL-6 than acupuncture.Conclusions1. Both acupuncture and fluoxetine could alleviated the depressive-like behaviors. Fluoxetine regulates reward systems may through PKA signal pathway; Acupuncture regulating reward systems may through other mechanisms.2. Acupuncture treats depression through activating PKA signaling pathway, and this effect was blocked by H89, and fluoxetine has a closer relationship with PKA pathway in anti-depression mechanism. And acupuncture treat depression not only through PKA pathway but also through more targets.3. Fluoxetine has a better effect on improving IL-6 than acupuncture. This prompts that in the mechanism of treatmenting depression, acupuncture and fluoxetine may through different ways. |
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