| [Background]Depression is a common mental disorder in modern society,which is often manifested as persistent depression,slow thinking,cognitive decline and other symptoms.Depression not only damages the health of patients,affects the quality of life,but also causes great burden to the family and society.Depression is expected to be the second highest disease burden in the whole world by 2022.The prevention and treatment of depression has been widely concerned by the society and the medical community.Although antidepressants are the main clinical treatment for depression,the have inevitable side effects.Acupuncture has benign regulative effects on the body,and can improve the clinical symptoms of patients with depression effectively.However the mechanism of its action is not clear,it is necessary to clarify in depth.Chronic stress is the main trigger of depression,it can cause the release of pro-inflammatory cytokines and a series of inflammatory reactions.Pro-inflammatory cytokines in the brain,such as interleukin-1β and interleukin-6 increased substantially,are the main factor that inducing depressive symptoms in the body.The role of central nervous system inflammatory response in the pathogenesis of depression has attracted increasing attention.Microglia is the main effector cell that mediates neuroinflarmmation.After overactivation,microglia will produce a large number of pro-inflammatory cytokines,thus aggravating the inflammatory response.Previous studies have found that acupuncture can reduce the serum levels of pro-inflammatory cytokines in rats with chronic stress depression.On the basis of previous work,this study investigates the effects of acupuncture on inflammatory cytokines,microglia activity and microglia-related TREM2 gene in prefrontal cortex of rats with chronic stress depression,in order to elucidate the antidepressant mechanism of acupuncture.[Objective]This study use chronic stress depression model rats,by observing the effects of acupuncture on behavior of chronic stress rats,inflammatory cytokines in the prefrontal cortex of brain,activity of Ionized calcium binding adapter molecule 1,and microglia-related The triggering receptor is expressed in myeloid cell 2 gene,to elucidate the mechanism of anti-depression by acupuncture,and provide scientific basis for the clinical application of acupuncture in the treatment of depression[Methods]1.Animals and groups:thirty-two SPF grade male SD rats with weight of 200±20g were randomly divided into normal group,model group,acupuncture group and fluoxetine group,with eight rats in each group.2.Preparation method of model rats with chronic stress depression:the model of depressed rats was prepared by six weeks of chronic unpredictable mild stress.Animals were randomly given one of the following stimuli every day:fasting for twenty-four hours,water prohibition for twenty-four hours,overnight lighting for twelve hours,strobing for two hours,binding for two hours,clip tail for two minute,and cold water swimming for five minutes at four degrees Celsius.Modeling continuously for six weeks.3.Treatment:Acupuncture treatment:During the six-week of modeling,starting from the first day,rats were acupuncture at Baihui(GV20)and Yintang(GV29)daily before modeling,the needle was kept for ten minutes.Acupuncture was performed once a day,and one day of rest was taken to ensure each six days of treatment.Medication:During six weeks of modeling,fluoxetine group was administered by gavage before daily modeling stress(10 mg/kg).4.Behavioral observation:(1)General condition:The reaction speed,mental state,softness and glossiness of hair and stool condition of the rats were mainly observed.(2)Open field test:Used to assess exploratory and autonomic activity in animals and was detected after the experiments.(3)Novelty-suppressed feeding:To observe the food consumption of animals in novel and strange environments,reflecting the anxiety and depression behaviors of the rats,and were tested after the experiment.5.Indicator detection:(1)Using ELISA to detect the contents of IL-1β,IL-6,IL-4 and IL-10 in the prefrontal cortex of rats.(2)Using real-time fluorescent quantitative PCR(RT-PCR)to detect the expression of TREM2 gene in the prefrontal cortex of rats(3)Using immunohistochemistry to detect the expression of Iba-1,a marker of microglia in the prefrontal cortex.6.Statistical method:The results were expressed as mean±standard deviation((?)±SD)form.One-way analysis of variance was used to statistically process the data,and P<0.05 was used as the standard for a statistically significant difference.[Results]1.The effects of acupuncture on the behavior of rats(1)General condition:Rats in the normal group were in good mental condition,with flexible movements and shiny fur.In the model group,the rats showed low spirits,less movement,loose and dim stool,easy hair loss and other performances.Rats in acupuncture group and fluoxetine group had better spirits and more active,with fluffy and shiny hair.(2)Open field test:Compared with the normal group,the number of horizontal crawling in the model group showed an decreasing trend(P<0.05).After treatment,compared with the model group,the number of horizontal crawling and vertical standing in the acupuncture group increased significantly,and the difference was statistically significant(P<0.05).Compared with the model group,the number of horizontal crawling in the fluoxetine group increased significantly,and the difference was statistically significant(P<0.05).The vertical standing times of fluoxetine group showed an increasing trend,but the difference was not significant(P>0.05).There was no significant difference between the acupuncture group and the fluoxetine group(P>0.05).(3)Novelty-suppressed feeding:Compared with the normal group,the feeding latency in the model group was significantly prolonged(P<0.05).Compared with the model group,the feeding incubation period in the acupuncture group and the fluoxetine group was significantly decreased(P<0.05,P<0.05).There was no significant difference between the acupuncture group and the fluoxetine group(P>0.05).2.The effects of acupuncture on the contents of pro-inflammatory cytokines IL-1β and IL-6 in the prefrontal cortex of rats:(1)Compared with the normal group,the contents of IL-1β in the prefrontal cortex of rats in model group was increased,and the difference was statistically significant(P<0.05).Compared with the model group,the contents of IL-1β in the prefrontal cortex of rats in acupuncture group decreased,and the difference was statistically significant(P<0.05).Compared with the model group,the contents of IL-1β in the prefrontal cortex of rats in fluoxetine group decreased,and the difference was statistically significant(P<0.05).There was no significant difference between the acupuncture group and the fluoxetine group(P>0.05).(2)Compared with the normal group,the contents of IL-6 in the prefrontal cortex of rats in model group increased significantly,and the difference was statistically significant(P<0.05).Compared with the model group,the contents of IL-6 in the prefrontal cortex of rats in acupuncture group decreased significantly,and the difference was statistically significant(P<0.05).Compared with the model group,the contents of IL-6 in the prefrontal cortex of rats in fluoxetine group decreased,and the difference was statistically significant(P<0.05).Compared with the fluoxetine group,the contents of IL-6 in the prefrontal cortex of rats in acupuncture group decreased,and the difference was statistically significant(P<0.05).3.The effects of acupuncture on the contents of anti-inflammatory cytokines IL-4 and IL-10 in prefrontal cortex of rats.(1)Compared with the normal group,the contents of IL-4 in the prefrontal cortex of rats in model group decreased significantly,and the difference was statistically significant(P<0.05).Compared with the model group,the contents of IL-4 in the prefrontal cortex of rats in acupuncture group increased,and the difference was statistically significant(P<0.05).There was no significant difference between the model group and the fluoxetine group(P>0.05).Compared with the fluoxetine group,the contents of IL-4 in the prefrontal cortex of the rats in acupuncture group increased significantly,and the difference was statistically significant(P<0.05).(2)Compared with the normal group,the contents of IL-10 in the prefrontal cortex of rats in model group decreased significantly,and the difference was statistically significant(P<0.05).Compared with the model group,the contents of IL-10 in the prefrontal cortex of rats in acupuncture group and fluoxetine group increased significantly,and the difference was statistically significant(P<0.05).There was no significant difference between the acupuncture group and the fluoxetine group(P>0.05).4.The effects of acupuncture on the expression of TREM2 gene in the prefrontal cortex of rats:Compared with normal group,the expression of TREM2 gene in the prefrontal cortex of rats in model group was significantly decreased,and the difference was statistically significant(P<0.05).Compared with the model group,the expression of TREM2 gene in the prefrontal cortex of rats in acupuncture group and fluoxetine group increased,and the difference was statistically significant(P<0.05).There was no significant difference between the acupuncture group and the fluoxetine group(P>0.05).5.The effects of acupuncture on microglia marker Iba-1 in prefrontal cortex of rats:Immunohistochemical results showed that Iba-1 immunopositive cells are widely distributed in prefrontal cortex of rats.In normal group,the microglia of rats showed spinous processes like multiple branchlets extending in all directions,the cell bodies are slender and long,showing a "branchlike" resting-state.In model group,the protuberance of the microglia of rats decreased,became short and thick,and even disappeared,the cell body became large and the morphology of the activated cells changed into a nearly circular shape,which was typical of"amoeba".Compared with model group,the cell bodies of microglia in acupuncture group and fluoxetine group get smaller and their form tended to resting-state.The results of immunohistochemical image analysis showed that compared with normal group,the expression of Iba-1 positive microglia in the prefrontal cortex of the rats was significantly increased in the model group(P<0.05).Compared with model group,the expression of Iba-1 positive microglia in the prefrontal cortex of the rats in the acupuncture group and fluoxetine group decreased significantly(P<0.05).There was no significant difference between the acupuncture group and the fluoxetine group(P>0.05).[Conclusion]1.The the number of horizontal crawling and vertical standing is decreased in open field test,and the incubation period of novel inhibition was significantly prolonged in chronic stress depression rats.Acupuncture can obviously vertical standing in open field test,improve the depressed state of rats,play an antidepressant role and shorten the incubation period and increase the number of horizontal crawling.2.The pro-inflammatory cytokines IL-1β and IL-6 in the prefrontal cortex of chronic stress depression rats were significantly increased,while the anti-inflammatory cytokines IL-4 and IL-10 were significantly decreased.The expression of TREM2 gene in the prefrontal cortex was down-regulated and the expression of microglia marker Iba-1 was increased,suggesting that microglia in the prefrontal cortex were overactivated,causing inflammation in the brain.Acupuncture can significantly decrease the contents of IL-1β and IL-6,and increase the contents of IL-4 and IL-10 in the prefrontal cortex,inhibit the overactivation of microglia,thus alleviating the inflammation in the brain and reducing brain tissue damage.3.In this study,we found that one of the mechanisms of acupuncture treatment for depression was to down-regulate the contents of pro-inflammatory cytokines in the prefrontal cortex and up-regulate the contents of anti-inflammatory cytokines.Inhibit the overactivation of microglia in the prefrontal cortex,thus alleviating the brain tissue damage caused by the inflammatory response in the brain.This experiment can provide important scientific basis for clinical acupuncture treatment of depression. |
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