| Depression is a global mental complaint which is characterized with mood disorder.It can lead to cognitive impairment,serious influence of human’s life and work and enhancement of the financial burden of society with high prevalence,high rate of relapse suicide.Based on the data reported by the World Health Organization,up to 2020,depression will be ranked to number 2 in which diseases affect human health and enhance burden of society.However,the pathogenesis of depression is still unclear till now.In the theory of traditional Chinese medicine,depression belongs to the constraint syndrome category.The weakness of organs’ Qi is the internal cause and usually induced by affect-mind dissatisfaction.The mainly pathogenesis is stagnation of liver and spleen deficiency and lack of spirit preservation.In the theory of modem medicine,monoamine hypothesis and neurotransmitter receptor hypothesis are the most important and classic currently.Dysfunction of neuroendocrine,especially HPA axis,neuroplasticity,brain-derived neurotrophic factor,neuroimmunology and signal pathway which can enrich the recognition of the mechanism of depression.For the treatment of depression,antidepressant drugs are still the main method in clinical practice,including tricyclic antipsychotics,monoamine oxidase inhibitor,selective serotonin reuptake inhibitor,which has the disadvantages of work slowly(about 2-3 hours),gastrointestinal reaction,headache,drowsiness or insomnia,sexual dysfunction and et al,but the most serious is that antidepressant drugs can not prevent the suicide risk of the early patients with severe depression in time and about 1/3 to 2/3 patients has no response to the initial antidepressant.None pharmacotherapy includes psychotherapy and physiatrics.Cognitive behavior therapy is considered as one of the most effective psychotherapy with the disadvantages of long time cost which is only an adjuvant therapy of drugs.Vagus nerve stimulation(VNS)is a common physiatrics on depression which has been approved by FDA to treat with refractory depression.However,this surgery costs a lot and leads to postoperative risk of infection and the effective rate after 10 weeks’treatment is only 40%.Based on the results of neuro-anatomy,rich auricular vagus nerve distributes on the region of concha.Our team has been studying transcutaneous auricular vagus nerve stimulation(taVNS)on the treatment of depression and showed that taVNS can significant decrease the score of Hamilton’s Depression Scale which can not only produce a similar effect of VNS but also overcome the above deficiencies of the invasive VNS.Though,taVNS is still in the early stage,it plays an important role in the treatment of depression.Better yet,the auricular points widely used in the depression are just the region where auricvagus nerve distributed.Therefore,we think that auricular acupuncture and taVNS are the same or similar diagnosis and treatment procedures guided by the two different theories.However,neither the special frequency of taVNS on depression nor the mechanism of the anti-depressive effect has been evaluated and interpreted.We would like to compare the anti-depressive effect of three different levels of taVNS frequencies,including 5Hz,20Hz,100Hz,detected by behavioristics,like sucrose preference,forced swimming and open-field,and by the evaluation on the function of HPA axis,like plasma CORT,ACTH and CRF.Additionally,the size of hippocampus reduction is the main pathogenesis of depression and ERK(Extracellular Signal-Regulated Kinase)is closly related to neuro growth,differentiation,apoptosis and proliferation.In this study,we would like to explore the correlation between anti depressive effect of taVNS and the expression of upstream and downstream proteins in ERK signal pathway in hippocampus on the CUMS depression model rats.Methods1.Effect of different frequencies of taVNS on behavioristics of CUMS ratsFifty male Sprague-Dawley rats weighting 180-230g were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences(Certificate no.SCXK(jing)2014-0013).The rats were randomly divided into five groups,i.e.the control,model,5Hz-taVNS,20Hz-taVNS and 100Hz-taVNS groups(n=10 per group).Except the control group,all animals were exposed to CUMS combined with isolation for 21 days to make depression model.The stressors included 5 minutes’ ice water swimming,12 hours’ day and night inversion,24 hours’ wet pecking,1 minute’s tail clipping,5 minutes’ hot plate,24 hours’ food deprivation and 24 hours’ water deprivation.After 21 days of CUMS procedure,the three experimental groups received taVNS at the frequency of 5Hz,20 Hz and 100Hz respectively by an electrical stimulator(Huatuo SDZ-IIB,Suzhou,China)each day for consecutive 28 days.Under 2%isoflurane inhalation anesthesia(Matrx VIP 3000,Midmark corporation,USA),the positive and negative electrodes were placed over the skin inside and outside of the rats’auricular concha region to ensure the electric current pass through the ear.Other taVNS parameters used for the three experimental groups were the same(2mA intensity,30 min per day)and were proved effective by our previous studies.taVNS procedure was administrated daily at the same time between 9 and 11am to weaken the influence of biological rhythm.Sucrose preference,forced swimming test and open-field test were measured once a week during the experimental procedure,which were extensively used in the evaluation of behavioristics of depression.2.Effect of different frequencies of taVNS on function of HPA axis of CUMS ratsAll rats were sacrificed and the neck venous blood was collected at the same time by the end of the whole experiment(day 28).The administration of groups,model and intervention methods are the same as the experiment 1.All the sample were tested by enzyme linked immunosorbent assay(ELISA)test.Plasma CORT,ACTH and CRF levels were measured to evaluate the function of HPA axis.3.Effect of taVNS on ERK signaling pathway in hippocampus of CUMS ratsThirty SD rats were divided into control,model,M/taVNS,M/PD98059,M/DMSO,M/PD98059/taVNS groups(each n=5).CUMS for 21 days were used to make depression model(the same as experiment 1),intracerebroventricular injection PD98059(an inhibitor of ERK pathway)or DMSO(a common organic solvent)and taVNS procedure were administrated daily for the consecutive 28 days.The expression of Raf,p-Raf,ERK,p-ERK,RSK,CREB,p-CREB in hippocampus were measured by Western blot to explore the mechanism of ERK signaling pathway in hippocampus on CUMS depression model rats and the target of antidepressant effect of taVNS.Results1.Effect of different frequencies of taVNS on behavioristics of CUMS rats1.1 Effect of different frequencies of taVNS on sucrose preference of CUMS rats There was no statistical difference in sucrose preference among the groups at the baseline(-21 day)of the study.During the 21 days of CUMS procedure,compared with the control group,a descending trend of sucrose water intake was observed in the model,5Hz-taVNS,lOHz-taVNS and 20Hz-taVNS groups and significant differences were shown in day 0 when taVNS administration started,indicating that the depression model was made successfully.After 21 days of taVNS intervention,rats in the 20Hz group showed significantly more sucrose consumption than that of the other groups(P<0.05)except for the control group.Result on Day 28 was the same(P<0.01),and no statistical difference was found among the model,5Hz-taVNS,and 100Hz-taVNS groups(P>0.05 for all).Compared with the control group,the sucrose consumption in all the CUMS groups,including the 20Hz-taVNS group,was still at a significantly low level(P<0.05).1.2 Effect of different frequencies of taVNS on immobility time of CUMS ratsOn day-21.the immobility time of different groups had no statistical difference(P>0.05).However,rats exposed to 21 days of CUMS procedure performed poorly in FST with the immobility time much less than that of the control group(P<0.01),but the FST performance could be improved by 20Hz taVNS intervention for 28 days(P<0.01).Rats in the 5Hz-taVNS and 100Hz-taVNS groups had no obvious improvement in FST compared with the model group(P>0.05).1.3 Effect of different frequencies of taVNS on open-field test score of CUMS ratsThe horizontal and vertical movement scores of rats in the open-field test significantly decreased on day 0 compared to the baseline(day-21)in all groups except the control group(P<0.01).After taVNS intervention,both the horizontal and vertical movement scores of the 20Hz-taVNS group significantly increased compared with those of the model group(P<0.01).Especially.the increase of horizontal movement score could be observed on day 21(P<0.05),earlier than the increase of vertical movement score(P>0.05 on day 21,P<0.05 on day 28).However,no significant difference was found in the OFT scores of the 5Hz-taVNS and 100Hz-taVNS groups.compared with the scores of the model group.2.Effect of different frequencies of taVNS on plasma CORT and ACTH of CUMS ratsAs detected from samples taken at the end of the last intervention,CUMS exposure significantly increased the concentration level of CORT(P<0.01 for all comparisons)compared with the control group.A decreasing trend of the mean concentration of plasma CORT was observed in all groups exposed to CUMS,but significant difference was only found between the 20Hz group and the model group(P<0.01)Plasma concentration of ACTH in the four CUMS groups showed significant increases compared with that of the control group(P<0.05 for the 20Hz-taVNS group and P<0.01 for the others).A significant decline of high level concentration of ACTH was shown in the 20Hz-taVNS group in contrast to that of the CUMS model group(P<0.01).3.The effect of taVNS on the ERK signal pathway in the hippocampus on CUMS model rats3.1 Comparison of sucrose preference in different time pointsThere was no statistical difference in sucrose preference among the groups at the baseline(-21 day)of the study.During the 21 days of CUMS procedure,compared with the control group,a descending trend of sucrose water intake was observed in the model,M/taVNS,M/PD,M/DMSO and M/PD/taVNS groups and significant differences were shown in day 0 when taVNS administration started,indicating that the depression model was made successfully.After 28 days of taVNS intervention,rats in the model,M/PD,M/DMSO and M/PD/taVNS groups showed significantly more sucrose consumption than that of the other groups(P<0.05)except for the M/taVNS group.Only M/taVNS group showed significant increase compared with model group(P<0.01)and M/PD and M/PD/taVNS showed significant decrease compared with M/taVNS group(P<0.01),but others groups showed any statistic differences(P>0.05).3.2 Comparison of Raf and p-Raf in hippocampusAll the rats are sacrificed at day 28,the expression of Raf and p-Raf in hippocampus were measured by Western blot.Compared with the control group,the level of Raf decreased in model,M/PD,M/DMSO and M/PD/taVNS groups(P<0.01,P<0.01,P<0.01,P<0.05).It increased significantly in M/taVNS and M/PD/taVNS groups compared with model group(P<0.05,P<0.05).Compared with the M/taVNS group,the level of Raf decreased obviously in M/PD and M/DMSO groups(P<0.05,P<0.05).There were no statistic difference in other groups(For all P>0.05).Compared with the control group,the level of p-Raf decreased in model,M/PD and M/DMSO groups(P<0.01,P<0.01,P<0.01)decreased obviously.The results of p-Raf expression in M/taVNS and M/PD/taVNS groups increased significantly compared with the model group(P<0.01,P<0.01).And the level of p-Raf in M/PD and M/DMSO groups decreased significantly than M/taVNS group(P<0.01,P<0.01).There were no statistic difference in other groups(P>0.05).3.3 Comparison of ERK and p-ERK in hippocampusThe expression of ERK and p-ERK in hippocampus decreased significantly in model,M/PD and M/DMSO groups compared with control group(P<0.01,P<0.01,P<0.01).Compared with the model group,ERK expression in M/taVNS and M/PD/taVNS groups increased obviously(P<0.05,P<0.05)and the result in M/DMSO group only decreased compared with M/taVNS group.For the expression of p-ERK in hippocampus,model,M/PD,M/DMSO and M/PD/taVNS groups showed significantly decreased(P<0.01,P<0.01,P<0.01,P<0.01)compared with control group.The results in M/taVNS group were higher than that in model group(P<0.05).Compared with M/taVNS group,the expression of p-ERK in hippocampus decreased significantly in M/PD,M/DMSO and M/PD/taVNS groups(P<0.01,P<0.01,P<0.05).There were no statistic difference in other groups(For all P>0.05).3.4 Comparison of Rsk in hippocampusCompared with the control group,the expression of Rsk in hippocampus decreased significantly in model,M/PD,M/DMSO and M/PD/taVNS groups(P<0.01,P<0.01,P<0.01,P<0.01)and rats in M/taVNS group increased obviously compared with model group(P<0.01).The results in M/PD,M/DMSO and M/PD/taVNS group showed significant decrease compared with M/taVNS group(P<0.05、P<0.05、P<0.01).There were no statistic difference in other groups(For all P>0.05).3.5 Comparison of CREB and p-CREB in hippocampus The expression of CREB and p-CREB in hippocampus decreased significantly in model,M/PD,M/DMSO and M/PD/taVNS groups compared with control group(P<0.01,P<0.01,P<0.01,P<0.01).Compared with the model group,CREB expression in M/taVNS and M/PD/taVNS groups increased obviously(P<0.05,P<0.05)and the result in M/PD and M/DMSO groups decreased compared with M/taVNS group(P<0.05,P<0.05).For the expression of p-CREB in hippocampus,model,M/taVNS,M/PD,M/DMSO and M/PD/taVNS groups showed significantly decreased(P<0.01,P<0.01,P<0.01,P<0.01,P<0.01)compared with control group.The results in M/taVNS group were higher than that in model group(P<0.01).Compared with M/taVNS group,the expression of p-CREB in hippocampus decreased significantly in M/PD,M/DMSO and M/PD/taVNS groups(P<0.01,P<0.01,P<0.05).There were no statistic difference in other groups(For all P>0.05).Conclusions1.Rats exposed CUMS for 21 days show obvious depression-like behaviors like decrease of sucrose preference,long immobility time and increase of horizontal and vertical scores of open-field test.taVNS at 20Hz frequency has an anti-depressive effect which provides an optimized frequency of taVNS in treatment of depression.2.taVNS can significantly improve the hyperactivity of HPA axis in CUMS model rats and 20Hz frequency shows a better curative anti-depressive effect than 5 Hz or 100Hz.3.taVNS can up-regulate the upstream proteins,like Raf,p-Raf,ERK,p-ERK,and downstream protein,like RSK,of the ERK signal pathway in hippocampus of CUMS model rats which indicates that taVNS may develop the anti-depressive effect and promote the protection of neurons through activating upstream and downstream proteins in the ERK signal pathway. |
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