Effects Of Acupuncture On BDNF Protein And Gene Expression Based On Epigenetics And PKA Signaling Pathway

ObjectiveTo study the effects of acupuncture in regulating the depression-like behaviors and the expression of brain-derived neurophic factor through epigenetics (DNA methylation and histone acetylation) and PKA signaling pathway in rats exposed to chronic unpredicted mild stress and to provide the evidence for the antidepressant mechanism of acupuncture.Methods1.40 adult male SD rats, body weight (220-240) g, were randomly divided into 4 groups: control group, model group, model+flueoxetine group (FLX group) and model+ acupuncture group (Acu group). Except the control group, other rats exposed to chronic unpredictable mild stress (CUMS) for 28 days. The FLX group rats were treated with 1.8 mg kg-1 d-1 fluoxetine i.g 30 minutes before CUMS. Acupuncture stimulation was carried out at acupoints of Baihui and Yintang in the Acu group 30 minutes before CUMS. Both acupuncture and fluoxetine carried out each day during the experimental procedure. The sucrose intake and the open-field tests and the body weight were used to determine the degree of depression and were performed at the 0 day,7th day,14th day,21th day and 28th day. The serum brain-derived neurophic factor (BDNF) was examined using ELISA. The level of BNDF mRNA in the hippocampus was examined using RT-PCR. The proteins of BDNF, acetylation of histone 3 lysine 9 (acH3K9) and histone deacetylase 2 (HDAC2) were examined using western blot. The methylation status of BDNF DNA promoter I was examined by MS-PCR.2.60 adult male SD rats, body weight (220-240) g, were randomly divided into 6 groups: control group, model group, model+flueoxetine group (FLX group), model+acupuncture group (Acu group), model+flueoxetine+H89 group (FLX+H89 group), model+ acupuncture+H89 group (Acu+H89 group). Except the control group, other rats exposed to CUMS for 21 days. The performance of Acu group and FLX group were the same as above. The groups in which H89 was administered were exposed to intracerebroventricular injection of H89 (10μM,5μL) 60 minutes before the CUMS once every other day, and received acupuncture stimulation or fluoxetine treatment 30 minutes before the CUMS each day. The sucrose intake and the open-field tests and the body weight were performed at the 0 day and 21th day. The proteins of PKA, cAMP response element binding protein (CREB) and phosphor-CREB (p-CREB) were examined using western blot.Results1. The effects of acupuncture and fluoxetine on the model rat behaviorsBefore the experiment, there was no significant difference among groups (P> 0.05). After 28-day stress procedure, the model group rats showed significant decreases in the locomotory numbers, the sucrose preference and the body weight (P<0.01, for all). The FLX group showed increases in the locomotory numbers, the sucrose preference and the body weight (P< 0.05, P< 0.01, P< 0.01, respectively) compared to the model group. The Acu group also showed increases in the locomotory numbers, the sucrose preference and the body weight (P< 0.05, P< 0.01, P< 0.05, respectively) compared to the model group. However, the trend of acupuncture on the behaviors was different with that of fluoxetine.2. The effects of acupuncture and fluoxetine on the expression of BDNF protein in the hippocampus and serum and the level of BDNF mRNA in the hippocampusCompared with the control group, the model group rats showed significant decreases of BDNF protein in the serum and hippocampus (P< 0.01, for both) and BDNF mRNA in the hippocampus (P< 0.01). Fluoxetine treatment significantly increased the BDNF protein in the serum (P< 0.01) and BDNF mRNA in the hippocampus (P< 0.01) compared with the model group. Acupuncture stimulation significantly increased the BDNF protein in the serum and hippocampus (P< 0.01, for both) and BDNF mRNA in the hippocampus (P< 0.01) compared with the model group. However, the increases of serum BDNF protein and hippocampal BDNF mRNA in the Acu group were lower than that in the FLX group (P<0.05, P< 0.01, respectively).3. Correlation analysis between serous BDNF protein, hippocampal BDNF protein and the ethologyThere was a positive correlation between serous BDNF protein and hippocampal BDNF protein (P< 0.05). The locomotory numbers had a positive correlation with serous BDNF protein (P< 0.05) or hippocampal BDNF protein (P< 0.05). The sucrose preference had no correlation with serous BDNF protein (P> 0.05), but a significant positive correlation with hippocampal BDNF protein (P< 0.01).4. Correlation analysis between BDNF protein and BDNF mRNA in the hippocampus and the ethologyThere was no correlation between BDNF protein and BDNF mRNA in the hippocampus (P> 0.05). However, BDNF mRNA in the hippocampus had a significant positive correlation with the locomotory numbers (P< 0.000) or the sucrose preference (P< 0.005).5. The effects of acupuncture and fluoxetine on the BDNF DNA promoter Ⅰ methylation statusThere was no significant difference in the status of BDNF DNA promoter Ⅰ methylation among groups (P> 0.05).6. The effects of acupuncture and fluoxetine on the proteins expression of acH3K9 and HDAC2 in the hippocampusCompared with the control group, the model group rats showed a significant decrease of acH3K9 protein in the hippocampus (P< 0.05). Both fluoxetine and acupuncture had positive effects on the expression of acH3K9 protein, but the differences were not significant (P> 0.05, for both). Besides, there was no significant difference between acupuncture and fluoxetine (P> 0.05).Compared with the control group, the model group rats showed a significant increase of HDAC2 protein in the hippocampus (P< 0.05). Both fluoxetine and acupuncture decreased the expression of HDAC2 protein (P< 0.01, for both). There was no significant difference between acupuncture and fluoxetine (P> 0.05).7. Correlation analysis between acH3K9 protein, HDAC2 protein and BDNF mRNA in the hippocampusThere was a significant negative correlation between acH3K9 and HDAC2 proteins in the hippocampus (P< 0.005). The BDNF mRNA had a positive correlation with acH3K9 protein in the hippocampus (P< 0.05) and a significant negative correlation with HDAC2 protein in the hippocampus (P< 0.000).8. Correlation analysis of acH3K9 and HDAC2 proteins in the hippocampus with the ethologyThe locomotory numbers had a positive correlation with the expression of acH3K9 in the hippocampus (P< 0.05) and a significant negative correlation with the expression of HDAC2 in the hippocampus (P< 0.000).The sucrose preference had no significant correlation with the expression of acH3K9 protein in the hippocampus (P> 0.05), but had a significant negative correlation with the expression of HDAC2 in the hippocampus (P< 0.000).9. The effects of acupuncture and fluoxetine on the PKA signaling pathwayPretreatment with H89, an inhibitor of PKA, inhibited the improvements of fluoxetine or acupuncture on the ethology (the numbers of crossing and rearing and the sucrose intake) of rats exposed to CUMS. The effect of H89 pretreatment on fluoxetine was more effective than that on acupuncture.There was no significant difference of the expression of CREB protein in the hippocampus among groups (P> 0.05). However, CUMS decreased the proteins expression of PKA-a and p-CREB (P<0.01, for both) in the hippocampus. Fluoxetine increased the expression of PKA-a protein (P<0.05), but not p-CREB (P> 0.05). Acupuncture significantly increased the proteins expression of PKA-a and p-CREB (P<0.01, P<0.05, respectively). There was no significant difference between acupuncture and fluoxetine on the proteins expression of PKA-a and p-CREB (P> 0.05, for both). Pretreatment with H89 inhibited the improvements on p-CREB induced by acupuncture or fluoxetine (P<0.01, for both), but not PKA-a (P> 0.05, for both).Conclusions1. CUMS decreased the autonomous exploration and body weight and inhibited the reward system. Acupuncture ameliorated the depression-like behaviors induced by CUMS.2. The reductions of the locomotory numbers and the sucrose preference induced by CUMS were related with the decreases of the expressions of BDNF protein and BDNF mRNA in the hippocampus. Acupuncture could increase the levels of BDNF protein and BDNF mRNA to ameliorate the depression-like behaviors.3. The depression-like behaviors and the decrease of BDNF mRNA in the hippocampus induced by CUMS were related with the increase of HDAC2 protein in the hippocampus and the decrease of acH3K9 protein. Acupuncture could decrease the HDAC2 protein and increase the acH3K9 protein to ameliorate the depression-like behaviors.4. The depression-like behaviors induced by CUMS were related with the inhibition of the PKA signaling pathway. Acupuncture could activate PKA signaling pathway.5. The BDNF, acH3K9, HDAC2 and PKA signaling pathway in the hippocampus play an important role in the pathophysiology of depression. The present study showed that acupuncture could ameliorate the depression-like behaviors through the above indexes.