| Depression is a severe illness and the prevalence increased gradually in the past decades according to large epidemiological studies. It is estimated by the WHO that, by2020, depression will become a dangerous life-threatening illness second only to cardiovascular disease. In that case, it is crucial to prevent the onset of the disease and approach effective treatment. Medication is the most common method in the treatment of depression, unfortunately, it alleviates the blues in about70%of patients who complete the regimen. On the other hand, during the treatment, the inevitable side effect-high incidence of relapse makes it reasonable to look for a more effective treatment without side effect. Acupuncture has long been used as a treatment for somatic and mental disorders in Traditional Chinese Medicine (TCM). This approach generates few side effects, and is considered as a new, alternative method of medicine in Western countries. To date, pathogenesis of depression is complicated and unclear.Depression is a devastating illness which may be caused by genetic and environmental factors such as stress. However, the biological basis of depression and the precise mechanism of antidepressant efficacy remain unclear. The stress may be the key cause of the depression. The Biogenic Amine Hypothesis states that depression is caused by a deficiency of monoamines, particularly noradrenaline and serotonin.To date, available chemical antidepressants including monoamine oxidase inhibitors(MAOIs), tricyclic antidepressants (TCAs), and selective serotonin or norepinephrine reuptake inhibitors (SSRIs, SNRIs, respectively) have been developed out of the monoaminergic deficit hypothesis of depression. Laboratory tests indicate that antidepressants such as TCAs and MAOIs increase available neurotransmitters quite rapidly-within a matter of hours. Yet, typically, clinical relief takes much longer. A person suffering from depression may not experience significant relief for as long as6to8weeks. Since this, elucidating new mechanisms for depression is critical to the development of more effective antidepressant drug. Our early results show that the proliferation of neural progenitors in hippocampus of CUS rats was decreased significantly; further the clomipramine and EA can upregulated the decreased proliferation of the neural progenitor and glial cells. The late studies give some clues that the major depression may be mediated by the genetic materials and the epigenetic regulation. The research about the depression has been related to the neuroendocrine, neuro-development and epigenetic regulation.Epigenetic changes are long-lasting modifications in gene function that do not involve changes in gene sequences. Recent evidence suggests that these changes may occur in both dividing and nondividing cells and may be transmitted intergenerationally. The earlier work shows protracted L-methionine treatment exacerbates schizophrenia symptoms; L-methionine supplies a methyl residue in a DNA methylation reaction. Recently, growing evidences showed that some gene expressions were up-regulated or down-regulated in depression. However, no assured gene mutation was found in depression. This hinted that the other causes such as epigenetic modification may result the abnormality of the gene expression. As mentioned above, the etiology of depression may be nearly related with the epigenetics. As such, the interplay between a relatively fixed genome and an often variable environment stress involves epigenetic modification and the onset of depression.The role of epigenetic modification in the mechanism of acupuncture and antidepressant were warranted further investigation. The further research focuses on the gene expression changes and the epigenetic modification and the interaction between them. In this study, we observed the antidepressant efficacy of EA and some antidepressants, and whether EA and some antidepressants perform the function through the epigenetic modification or not.The DNA methyltransferase (DNA MTase) catalyzing the transfer of a methyl group to DNA, is an important enzyme in epigenetic modification. DNMT3L contains DNA methyltransferase motifs and is required for establishing maternal genomic imprints, despite being catalytically inactive. DNMT3L is expressed during gametogenesis when genomic imprinting takes place. The loss of DNMT3L leads to bi-allelic expression of genes normally not expressed by the maternal allele. DNMT3L interacts with DNMT3a and DNMT3b and co-localized in the nucleus. Though DNMT3L appears incapable of methylation, it may participate in transcriptional repression. The DNMT3L is the observed target in this study. We observed the protein and mRNA changes of the DNMT3L after the EA or antidepressant treatment of the depression model rats. Myelin basic protein (MBP) is a protein improved to be important in the process of myelination of nerves in the central nervous system (CNS). MBP are commonly one of the assessed biomarkers of brain injury. The late studies showed that MBP levels in serum and in the cerebrospinal fluid (CSF) may be related to the medullary sheath changes in the onset of epilepsy. In addition, the earliest study in1986showed that the MBP level in serum was increased in schizophrenia. But, the MBP changes in depression were not reported yet. Therefore, we observed the MBP changes in depression model rats and after the antidepressant treatment.Our research includes two parts.The first one:Antidepressant-like effect and epigenetic mechanisms of EA, Escitalopram and Clomipramine treatment with helpless rats.(1) The inescapable foot-shocks induced learned helpless rats.(2) The mechanical threshold and thermal threshold were detected with Paw Stimulator Analgesia Meter and von Frey hair.(3) Observing the change of behavior after treatment of EA, Escitalopram and Clomipramine with shuttle box test, forced swimming test, open field test and elevated plus-maze test.(4) Using the differential methylation hybridization (DMH), we analyzed the whole genome of CpG island methylation between the hippocamus of the normal rats and the helplessness rats. Then, we detected the Dnmt3L and MBP genes which are possible genes associate with the cause of depression.(5) We detect the mRNA levels of Dnmt3L and MBP in the hippocamus by RT-PCR and Real-Time PCR test between the normal group, the helpless group, the saline group, the Escitalopram group and the Clomipramine group.(6) We detect the protein levels of Dnmt3L and MBP in the hippocamus by Western blot test between the normal group, the helpless group, the saline group, the Escitalopram group and the Clomipramine group.The second:Antidepressant-like effect and epigenetic mechanisms of EA treatment with CUS induced depression model rats.(1) Copy the CUS induced depression rats model(2) Observing Behavioral changes of the EA treatment by forced swimming test, open field test and elevated plus-maze test. (3) Observing the change of Dnmt3L and MBP mRNA level in the hippocampus after EA treatmentResults:1. Antidepressant-like effect and epigenetic mechanisms of EA, Escitalopram and Clomipramine treatment with helpless rats.1.1Making helpless ratsWe picked20Wistar rats to model the learned helplessness depression rat. In the1st,2nd, and3rd day after inescapable foot-shocks (IS), their failures to escape to escapable foot-shocks increased. The numbers were stable in the4th and5th day, there is no significant statistic difference between them, but they are both significantly higer than the numbers in the1st and2nd day. That indicated these rats expressed stable helpless action after IS.We tested10helpless rats with the active avoidance reaction in the3th to7th day after IS. The result is that all rats kept helpless in the3rd to5th day;9rats were helpless till the6th day; about70%rats could keep this state for one week.1.2The stimulus response threshold in the helpless ratsThe mechanical threshold, and thermal threshold of normal, helpless and non-helpless rats were tested in pre-IS and the1st,3rd,5th,7th day after IS. The mechanical and thermal threshold in the helpless group is increased compraed with the normal group and the non-helpless group. After the treatment with Clomipramine, the mechanical threshold is decreased, but not the thermal threshold.1.3Behavioral changes of the EA, Escitalopram and Clomipramine treatment1.3.1Behavioral changes of the EA treatmentConsidering the genic homozygous and phenotypic consistency, EA treatment was praticed on depression models of inbred lines Wistar rats and closed population SD rats respectively. They were divided into five groups:normal, model, depression+sham EA, depression+EA, and depression+Clomipramine.1.3.1.2Shuttle box test For both of Wistar and SD rats treated with EA, in shuttle box test of escapable foot-shocks, the number of failures to escape was not different from the models’, but the Clomipramine treated rats were significantly lower than models’. That indicated EA treatment can’t help the helpless rat’s active avoidance reaction.1.3.1.2Forced swimming test, FSTAfter EA or Clomipramine treated, the struggling times of treated SD rats were significantly higer than the model rats. That indicated EA treatment can improve the helpless rat’s despair reaction.1.3.1.3Open field test, OFTIn this test, EA or Clomipramine treated Wistar rats’horizontal Total distance of travel and number of rearing are significant higer than model rats’. That indicated EA treatment can significantly improve the helpless rats’spontaneous motor activity and exploration.1.3.1.4Elevated plus-maze test, EPMIn this test, for both of the Wistar rats and SD rats treated with EA or Clomipramine, percent of time in open arm were significant higer than model rats’. That indicated EA treatment can improve significantly helpless rats’anxiety.1.3.2Behavioral changes of the Escitalopram and Clomipramine treatmentConsidering the genic homozygous and phenotypic consistency, Escitalopram Intraperitoneal injection treatments were praticed on depression model of inbred lines Wistar rat and closed population SD rat, respectively. They were grouped into five groups:normal, model, depression+Saline, depression+Escitalopram, and depression+Clomipramine.1.3.2.1Shuttle box testFor both of Wistar and SD rats treated with Escitalopram, in shuttle box test of escapable foot-shocks, the number of failures to escape was significant lower than the model’s, and the result was totally similar to Clomipramine treated rats’. That indicated Escitalopram and Clomipramine treatment can improve the helpless rat’s active avoidance reaction significantly. 1.3.2.2Forced swimming test, FSTAfter Escitalopram or Clomipramine treated, the struggling times of treated SD rats were significantly higer than the model rats.That indicated EA treatment can improve the helpless rat’s despair reaction.1.3.2.3Open field test, OFTIn OFT test, Escitalopram or Clomipramine treated Wistar or SD rats’horizontal total distance of travel and number of rearing were significant higer than model rats’. That indicated Escitalopram or Clomipramine treatment can improve significantly the spontaneous motor activity and exploration of helpless rats.1.3.2.4Elevated plus-maze test, EPMIn this test, for both of the Wistar rats and SD rats treated with Escitalopram or Clomipramine, percent of time in open arm were significantly higer than model rats’. That indicated helpless rats’anxiety can be improved significantly by Escitalopram or Clomipramine treatment.1.4Screening the differentially methylated gene DNA through the whole genome of CpG island methylation chipUsing the differential methylation hybridization (DMH), we analyzed the whole genome of CpG island methylation between the hippocamus of the normal rats and the helplessness rats. Then, we detected the Dnmt3L and MBP genes which are possible genes associate with the cause of depression through the analysis of gene classification and function and the number of log-Ratio is more than five as the significant difference. Here, we could not supply the detailed location or degree of the two methylated genes, because the sequencing is still in progress by methylation specific polymerase (MSP) chain reaction。1.5RT-PCR and Real-Time PCR test resultsWe detected the mRNA levels of Dnmt3L and MBP in the hippocamus by RT-PCR and Real-Time PCR test between the normal group, the helpless group, the saline group, the Escitalopram group and the Clomipramine group.The mRNA level of MBP in the hippocamus in the helpless group was significantly increased compared with the normal group. However, the mRNA level of MBP in the hippocamus after Escitalopram or Clomipramine treatment was significantly decreased compared with the helpless group, but still decreased compared with the normal group.The mRNA level of Dnmt3L in the hippocamus in the helpless group was significantly decreased compared with the normal group. However, the mRNA level of MBP in the hippocamus after Escitalopram or Clomipramine treatment was significantly increased compared with the helpless group.1.6Western blot test resultsWe detected the protein levels of Dnmt3L and MBP in the hippocamus by Western blot test among the normal group, the helpless group, the saline group, the Escitalopram group and the Clomipramine group.The protein levels of MBP in the hippocamus in the helpless group and the saline group were significantly increased compared with the normal group, the Escitalopram group and the Clomipramine group.The protein levels of Dnmt3L in the hippocamus in the helpless group and the saline group were significantly decreased compared with the normal group, the Escitalopram group and the Clomipramine group. In addition, the the protein level of Dnmt3L in the hippocamus in the Escitalopram group was significantly increased compared with the normal group.1.7SummaryThe results above demonstrated that the rats exerted the steady-going helpless behaviors after the3days of the inescapable foot-shocks (IS). About70%rats could keep this state for one week.1. The mechanical and thermal threshold in the helpless group is increased compraed with the normal group and the non-helpless group. After the treatment with Clomipramine, the mechanical threshold was decreased, but not the thermal threshold.2. EA treatment with the helpless rats could ameliorate the behaviors of despair, exploration, anxiety and spontaneous motor activity, but not the active avoidance reaction.3. Escitalopram treatment with the helpless rats could ameliorate the behaviors of despair, exploration, anxiety and spontaneous motor activity, and these effects are similar with the Clomipramine treatment.4. Helplessness rats exerted differential methylation in the hippocampus compared with the normal group. Dnmt3L and MBP genes are possible genes associate with the cause of depression through the analysis of gene classification and function.5. The mRNA level of MBP in the hippocamus in the helpless group was significantly increased compared with the normal group. Escitalopram or Clomipramine treatment could reverse the level. The similar change was detected about the Dnmt3L gene.6. The protein level of MBP in the hippocamus in the helpless group is significantly increased compared with the normal group, but decreased about the Dnmt3L portein. In addition, Escitalopram treatment could inctease the protein level of Dnmt3L compareed with the normal group.2. Antidepressant-like effect and epigenetic mechanisms of EA treatment with CUS induced depression model rats2.1Copy the CUS induced depression rats modelThe CUS induced depression rats model was made by six random stressors during nine weeks. The experiment rats were divided into five groups:the normal group, the model group, the sham EA group, the EA group and the Clomipramine group.2.2Behavioral changes of the EA treatment2.2.1Forced swimming test (FST)The struggling time in the FST is significantly increased after EA treatment compared with the Model. The Clomipramine treatment had the similar change and didn’t have significant change compared with the model. These results suggest the EA treatment could ameliorate the despair behavior.2.2.2Open field test (OFT)The total distance of travel in the OFT was significantly increased after EA treatment or Clomipramine treatment compared with the model. EA group had significant change compared with the Clomipramine group. The number of rearings in the OFT is significantly increased after EA treatment and Clomipramine treatment compared with the model. These results suggest the EA treatment could ameliorate the exploration and spontaneous motor activity behavior.2.2.3Elevated plus-maze test (EPM)The percent of time in open arm in the EPM was significantly increased after EA treatment or Clomipramine treatment compared with the model. These results suggest the EA treatment could ameliorate the anxiety behavior.2.3The change of Dnmt3L and MBP mRNA level in the hippocampus after EA treatmentAccording to the Real-Time PCR test, the mRNA level of MBP in the hippocampus in the sham EA group was significantly increased copmared with the normal group. After EA treatment or Clomipramine treatment, the mRNA level of MBP in the hippocampus was significantly increased copmared with the sham EA group. The mRNA level of Dnmt3L in the hippocampus in the sham EA group was significantly decreased copmared with the normal group. After EA treatment or Clomipramine treatment, the mRNA level of Dnmt3L in the hippocampus was significantly increased copmared with the sham EA group.2.4SummaryThe results above demonstrated that EA treatment could ameliorate the the behaviors of despair, exploration, anxiety and spontaneous motor activity. Compared with the Clomipramine group, EA treatment didn’t have significant changes in the struggling time in the FST, the number of rearings in the OFT and the percent of time in open arm in the EPM, but had significant change in the total distance of travel in the OFT. Furthermore, the mRNA level of Dnmt3L in the hippocampus in the sham EA group is significantly decreased copmared with the normal group. After EA treatment or Clomipramine treatment, the mRNA level of Dnmt3L in the hippocampus was significantly increased copmared with the sham EA group. The mRNA level of MBP in the hippocampus in the sham EA group wais significantly increased copmared with the normal group. After EA treatment or Clomipramine treatment, the mRNA level of MBP in the hippocampus was significantly decreased copmared with the sham EA group. The present study suggested:1. The mechanical and thermal threshold of helpless rats will rise and Clomipramine elicited a drop in mechanical threshold, but showed no effects on thermal threshold.2. EA, Escitalopram and Clomipramine all showed the effect of antidepression. Escitalopram and Clomipramine make the behaviors of despair, exploration, anxiety and spontaneous motor activity better, but EA can’t improve active avoidance reaction.3. DNA Methylation and Demethylation in Promoter Region of Dnmt3L and MBP make the Gene Expression changes of helpless rats.4. EA has potent antidepressant-like effect on CUS induced depression model rats.5. EA or Clomipramine treatment possibly causes changes of DNA Methylation in Dnmt3L and MBP. |
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