Experimental Study Of Electroacupuncture On The Reuptake Of MiRNA-16 And 5-HT In Hippocampus Of CUMS-induced Depression Model Rats

1 ObjectiveDepression is classified as a common mental illness.Clinical symptoms include a high suicide rate,a mutilation rate and a recurrence rate,which cause serious economic burden and mental stress to patients and families.Studies have suggested that the etiology and pathological mechanisms of depression might be caused by environment and gene factors,and the imbalance of monoamine neurotransmitters,Selective serotonin reuptake inhibitors(SSRIs)such as fluoxetine are commonly used to treat patients with depression,but approximately 50%of patients fail to respond to therapy.A great number of studies have shown that under the guidance of traditional Chinese medicine theory,electroacupuncture therapy has shown certain advantages in improving the clinical symptoms of depression patients and depression model animals in experimental research.Chronic unpredictable mild stress(CUMS)was used to establish a rat model of depression and the current study aimed to investigate the effects and possible mechanisms of fluoxetine and electroacupuncture therapy,alternative treatment options may provide novel antidepressant strategies.2 MethodsA total of 72 Sprague-Dawley(SD)rats with similar baseline characteristics were randomly divided into four groups:were randomly divided into four groups(18 rats per group):no-stress stimulation as the control(C)group,a CUMS model(M)group,a fluoxetine(F)group,and an electroacupuncture(E)group,which were adaptable for a week before the experiment.The CUMS model was established as previously described with minor modifications.All rats were provided with free access to food and water except during the periods of stress stimuli.Stimulation was performed at 10a.m.each day.The M,F,and E groups received randomized stress stimulation once a day from day 8 to day 36 for 4 weeks(28 days).In brief,stress stimuli included forced swimming in 4? water for 5 min,deprivation of either water and/or food for 24 h,inversion for 24 h,tail fastening and clipping for 3 min,cage tilting for 24 h,humid environment for 24 h,and immobilization for 3 h.Intragastric administration of fluoxetine(2mg/kg)diluted insaline was performed for the F group.The acupuncture needles(one-time use)were pricked into the skin at GV20 and GV29 to a depth of 5 mm.After twitching slightly,the needle handle was connected to Han's Electroacupuncture Instrument(Beijing Huawei Industry Co.,Ltd.).Continuous wave electroacupuncture was provided at a frequency of 2 Hz and intensity of 1 mA.The stimulation level was maintained at a level where head muscles vibrated slightly and vocalizations were minimized.All rats in the E group were subjected to electroacupuncture therapy once a day for 20 minutes.Treatments were conducted during the same period after 1h of stress exposure once daily.3 Research results3.1 Sucrose Preference TestThere are no significant differences in rats with similar baseline characteristics of sucrose preference index between groups prestimulation(P>0.05).However,after stress stimulation for 4 weeks,compared with rats in the C group,rats in the M group had lower sucrose preference index(P<0.01).Compared with the M group,the effects of electroacupuncture and fluoxetine treatment on regulating the sucrose preference index,the F and E groups had significantly increased the sucrose preference index in the depression rat model(P<0.05;P<0.05).3.2 Open-Field TestThere are no significant differences in rats with similar baseline characteristics of horizontal and vertical scores between groups prestimulation(P>0.05;P>0.05).However,after stress stimulation for 4 weeks,compared with rats in the C group,rats in the M group had lower horizontal and vertical scores(P<0.01;P<0.01).Compared with the M group,the fluoxetine treatment reversed the upregulation of the lower horizontal and vertical scores.E groups had significantly increased the horizontal and vertical scores in the depression rat model(P<0.05;P<0.05).3.3 The morphology of hippocampal neurons and the number of Nissl bodies in hippocampus CA1 of each group.The results of HE staining and Nissl body test showed that the hippocampal cells in the M group had reduced the number of neurons,unclear structure,the neuron cell body form is irregular,and the number of glial cells were increased significantly after CUMS procedures.Compared with the M group,the effects of electroacupuncture and fluoxetine treatment on regulating the loss of hippocampal neurons by CUMS procedures,reversed the phenomenon of neuron lysis and deletion,maintaining cell morphology,and the treatment could improve nissl body to protect the neurons in hippocampus CA1 area.3.4 Expression of miRNA-16 in hippocampus and serum of each group.Compared with the C group,the M group had higher levels of miRNA-16(P<0.01),the miRNA-16 levels in the serum also showed an increasing trend,but there was no statistical difference compared with C group(P>0.05).miRNA-16 levels in the hippocampus are significantly reduced in the F and E groups compared to those in the M group.It is worth noting that the levels of miRNA-16 in the hippocampus were significantly reduced in the E group compared with the F group(P<0.01).3.5 Effect of electroacupuncture treatment on 5-HT and reuptake 5-HT based on the hippocampal miRNA-16 abnormal expression.The results of RT-PCR and WB showed that the relative expressions of SERT mRNA and SERT protein in the hippocampus of group M were significantly higher than those in group C(P<0.01;P<0.01).Compared with the M group,the fluoxetine and electroacupuncture treatment could reverse the upregulation of the SERT mRNA and SERT protein expressions in the hippocampus,and the levels of SERT mRNA and SERT protein in the hippocampus are significantly reduced in the E group compared with the M group(P<0.05;P<0.05).Immunofluorescence analyses revealed that the levels of SERT positive cells in the hippocampus CA1 of group M were significantly higher than those in group C(P<0.01).Compared with the M group,the fluoxetine and electroacupuncture treatment could reverse the upregulation of the SERT positive cells in the hippocampus CA1 by CUMS procedures(P<0.01;P<0.01).The results of High-Performance Liquid Chromatography(HPLC)showed that the hippocampal 5-HT levels of group M were significantly lower than those in group C(P<0.05).Hippocampal 5-HT levels were significantly increased in F and E groups versus M group(P<0.05;P<0.05),resulting in near-normal levels of 5-HT in the hippocampus.4 Conclusion(1)Our results showed that the rats in the M group have showed depression-like behaviors,and depression model has been successfully set up at postintervention.However,utilizing fluoxetine and electroacupuncture treatment during CUMS can prevent depression-like behavioral changes,fluoxetine and electroacupuncture both have antidepressant effects.(2)CUMS stimulation procedures induced neuronal damage in hippocampus of rats.Fluoxetine and electroacupuncture treatment for the protective effect of chronic stress depression hippocampal CA1 neurons,reversing the phenomenon of neuron lysis and deletion,maintaining cell morphology,and the treatment could improve nissl body to protect the neurons in hippocampus CA1 area.(3)Epigenetics mainly involve the influence of environmental factors pathological gene factors on depression.Our results showed that long-term chronic stress could induce the M group had higher levels of miRNA-16 of hippocampus,but there was no statistical difference of miRNA-16 levels in the serum compared with C group.Future studies would be to further explore which miRNA related to depression,which is very important for the prevention and treatment of depression.(4)Electroacupuncture/fluoxetine treatment might inhibit the expression of SERT protein via miRNA-16,which further inhibited the reuptake of 5-HT resulting in near-normal levels of miRNA-16 and 5-HT in the hippocampus.