| BackgroundDepression was regraded as a major mental disorder in the world.It has become the most serious social problem in contemporary society.Challenging the depression is a hot topic in the research of human medicine due to its high prevalence,high recurrence rates,high incident rats.The mechanism of depression and the mechanism of therapeutic effect mainly involve neurotransmitter,neuroendocrine system,neuron nutrition and apoptosis,cytokine theory,and cell signal transduction pathway.JNK(c-jun N terminal kinase,JNK)signal pathway,belonging to the MAPK family,can be activated by external stress stimulation.The activation of JNK pathway can lead to apoptosis of Prefrontal Cortex neurons.In recent years,the Encephalic Region is mainly concentrated in hippocampus,prefrontal cortex,cingulate gyrus,amygdala,lateral habenular nucleus and so on.Basing on the previous confirmation of the effectiveness of acupuncture in treating depression,the aim of the study is to observe the effect of JNK signaling pathway on Prefrontal Cortex in rats with chronic unpredicted mind stress CUMS,so as to reveal its underlying mechanism in relieving depression.ObjectiveThis study specifically activated the JNK signaling pathway,and observed the changes of the protein expression levels of JNK,c-jun,p-c-jun of JNK signal pathway in the frontal lobe of chronic stress depression model rats.To verify the validation of the research,and continue to explore and improve the anti-depression mechanism of acupuncture,which involved the activate of JNK signaling pathway.Methods1.Modeling methodsCombine solitary with chronic unpredictability mild stress(CUMS)method to establish depression model.CUMS method including:food deprivation(24 h),water deprivation(24 h),bound(2 h),the oscillation(30 min);tail clip(3 min),wet bedding(24 h),the night light(12 h).In the process of experiment,each type of stress stimulation is conducted 3 times in alternate way in 21 days.A week before the building,model + DMSO group,model + SP group,the flu+ SP group and acupuncture + SP group receive lateral ventricle casing embedment surgery.2.Intervention methodsAcupuncture treatment of selected "Nei guan" and "San yin jiao",use twirling stimulation methods at the depth of 0.5?1 cm,retaining needle 20 min once a day;Fluoxetine is mixed with distilled water at 1.8 mg/kg of suspension liquid,with 10 ml/kg lavage.Reduce additional stimulus when the above process is operated.3.Experimental groups48 male Sprague-Dawley rats(180±10g)were randomly divided into eight groups(six rats in each group):the control group,the model group,the model + acupuncture(Acu)group,the model + fluoxetine(Flu)group,the model + DMSO group,the model + SP group,the Model+Flu +SP group,the Model + Acu + SP group.4.Process methodsDo not give any stimulus to control group,which are group housed.Other groups are feed separately receiving 21 days CUMS stimulation.At the same time,the acupuncture group receive acupuncture treatment 30 min before modeling on each day.Flu group receive fluoxetine lavage 30 min before stimulated on each day.Model + DMSO group receive 10ul/Id DMSO lateral ventricle injection 1h before stimulated.Model + SP group receive 10ul/1d block inhibitor SP600125 diluent lateral ventricle injection 1 h before stimulated.Acupuncture + SP group,flu + SP group receive 10ul/Id block inhibitor SP diluent lateral ventricle injection 1h before stimulated,and then are separately given acupuncture treatment and fluoxetine lavage 30 min before stimulated.5.Detection index and methodsUsing open-field test,body weight to evaluate the behavior change of depression model rats.Using Western blot method to detect protein expression levels of JNK,c-jun,p-c-jun in the prefrontal cortex of rats.Results1.Behavioral change1.1 Body qualityDay 0:there is no significant difference in body quality between all groups of rats(all P>0.05).Day 21:Compared with the blank group,the body quality of model group and model +DMSO group declined significantly(P<0.01),and model + flu + SP group and model + Alu +SP group had no significant difference(P>0.05).Compared with the model group,model + SP group and model + DMSO group rats had no significant difference(P>0.05),model + flu group and model + Alu group increased significantly(P<0.01).Compared with the model +DMSO group,model + SP group,model + flu + SP group and model + Alu + SP group had no significant difference(P>0.05).Compared with the model + flu group,model + flu + SP group declined significantly(P<0.01);compared with the model + Alu group,model + Alu + SP group declined significantly(P<0.01).1.2 Open-field test(horizontal direction)Day 0:there is no significant difference in body quality between all groups of rats(all P>0.05).Day 21:Compared with the blank group,all group declined significantly(P<0.01).Compared with the model group,model + SP group and model + flu group group rats had no significant difference(P>0.05),and model + Alu group increased significantly(P<0.01).Compared with the model +DMSO group,model + SP group and model + flu + SP group had no significant difference(P>0.05),and model + Alu + SP group increased significantly(P<0.01).Compared with the model +SP group,model + flu group and model + Alu + SP group had no significant difference(P>0.05),model + flu + SP group model + Alu + SP group increased significantly(P<0.01).Compared with the model + flu group,model + flu + SP group increased significantly(P<0.05);compared with the model + Alu group,model + Alu + SP group declined significantly(P<0.05).1.3 Open-field test(vertical direction)Day 0:there is no significant difference in body quality between all groups of rats(all P>0.05).Day 21:Compared with the blank group,the body quality of all group declined significantly(P<0.01).Compared with the model group,model + SP group and model + flu group group rats had no significant difference(P>0.05),and model + Alu group increased significantly(P<0.01).Compared with the model +DMSO group,model + SP group,model + flu + SP group and model + Alu + SP group increased had no significant difference(P>0.05).Compared with the model + flu group,model + flu + SP group had no significant difference(P>0.05);compared with the model + Alu group,model + Alu + SP group declined significantly(P<0.05).2.Change of JNK signaling pathway in frontal cortex2.1 The expression of JNKCompared with the blank group,the expression of JNK were up-regulated in model group.Compared with the model group,model + SP group and model + flu group group rats had no significant difference(P>0.05),and model + Alu group down-regulated significantly(P<0.01).Compared with the model +DMSO group,model + SP group and model + flu + SP group increased had no significant difference(P>0.05),model + Alu + SP group down-regulated significantly(P<0.01).2.2 The expression of c-junCompared with the blank group,the expression of c-jun were up-regulated in model group.Compared with the model group,model + SP group and model + flu group group rats down-regulated significantly(P<0.05),and model + Alu group down-regulated significantly(P<0.01).Compared with the model +DMSO group,model + SP group and model + flu + SP group increased had no significant difference(P>0.05),model + Alu + SP group down-regulated significantly(P<0.05).2.3 The expression of p-c-junCompared with the blank group,the expression of p-c-jun were up-regulated in model group(P<0.001).Compared with the model group,model + flu group group and model + Alu group down-regulated significantly(P<0.01;P<0.001).Compared with the model +DMSO group,model + SP group increased had no significant difference(P>0.05),model + flu + SP group and model + Alu + SP group down-regulated significantly(P<0.01;P<0.001).Conclusion1.CUMS model is a reliable chronic model.This study reflects the two behavioral indicators of depression core symptoms:the body mass and the open field behavior are significantly decreased,suggesting that the model is successful.2.Combining solitary keep with chronic stress can significantly activate activation of JNK signaling pathways by up-regulating the expression of JNK.c-jun?p-c-jun.3.Acupuncture and fluoxetine intervention can significantly improve the depressive behavior of CUMS rats.4.The result of WB:it suggest that JNK signaling pathways activated by CUMS can be regulated by acupuncture and fluoxetine.5.The blocking action of JNK signaling pathway blocker SP600125 on JNK signaling pathway occurred after JNK phosphorylation,and the JNK in the upstream of phosphorylation was not affected by SP600125;fluoxetine also did not interfere with the JNK protein but interfered with the expression of c-jun and p-c-jun downstream.6.SP600125,fluoxetine and acupuncture played a synergistic role in inhibiting the phosphorylation level of JNK signaling pathway.Innovation1.On the basis of previous work of our team,it is confirmed that the abnormal expression of JNK,c-jun and p-c-jun in JNK signaling pathway of CUMS rats can be regulated by acupuncture.2.The results of this study confirm that the differential expression of acupuncture and fluoxetine on the JNK signaling pathway in the depression model,after JNK phosphorylation,interfered with the JNK signaling pathway in the upstream of fluoxetine intervention.3.The results of this study confirm that SP600125,fluoxetine and acupuncture have synergistic effects on the inhibition of the phosphorylation level of JNK signaling pathway,which is reflected in behavioral and signaling pathways. |
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