| [Background]Chronic Fatigue Syndrome(CFS)refers to a group of syndromes in which chronic fatigue that has persisted or recured for more than 6 months and this fatigue doesn't significantly relieve after rest.The long-term fatigue caused by CFS and its accompanying clinical symptoms seriously affects the patient's work efficiency,quality of life and mental health.With the acceleration of the pace of work in modern life and the increasingpressure,its incidence is increasing year by year.Therefore,CFS has become a major issue affecting human health in the 21st century.The pathogenesis of CFS is complex and hasn't yet been fully elucidated.In recent years,with the in-depth study of the pathogenesis of CFS,it has been shown that central nervous inflammation played an important role in the development of CFS.Microglia(MG),an important effector cell of central nervous system inflammation,release a large number of cytokines after excessive activation,especially pro-inflammatory cytokines such as interleukin-1?(IL-1?),interleukin-6(IL-6)and tumor necrosis factor-a(TNF-a)aggravate the inflammatory response and produce cytotoxic effects.Clinical practice has proved that moxibustion could rectify theinsufficiency,relieve fatigue,and it is an effective method of clinical treatment of CFS.Our previous studies showed that moxibustion can down-regulate the levels of serum IL-1?,IL-6,and TNF-? in chronic fatigue rats.The effect of moxibustion on the activity of pro-inflammatory cytokines and microglial cells in the brain is still unclear.[Objective]In order to explore the mechanism of moxibustion chronic fatigue syndrome,we observedthe behavior of chronic fatigue rats,as well as the effects of pro-inflammatory cytokines and hippocampal microglial activity.[Methods]1.Thirty-six male SD rats were randomly divided into normal group,model group,and moxibustion group,12 in each group.2.Chronic fatigue rat model:Theloaded rats were forced to swim until exhaustion for 21 days.The exhaustive swimming time was determined whenthe swimming action of the rat was obviously disordered,and ratscould not return to the surface for 10s.3.Moxibustion treatment:From the first day of the experiment,moxa cones were applied on Zusanli acupoints in the rats,each point with 3 moxa cones.Moxibustion treatment was used every other day after swim for 11 times.4.Behavioral observation:(1)General situation:we observed the state of the rat's mental state,activity,hair,etc.,that reflected the overall state of the animal.(2)Body weight:we observedthe body weight in the day before,7th,14th,and 21st days of the experiment,which reflected the overall situation of the animal.(3)Exhaustive swimming time:observations were made on the first day,7th,14th,and 21th days of the experiment,which reflected the degree of fatigue of the animals.(4)Tail suspension immobility test:we observed the immobile tail suspension time of rats within 5 min after the completion of theexperiment,reflecting the physical condition and despair.(5)Open-field test:After the completion of experiment,the rats moved horizontally and vertically in the open field within 5 min to reflect the animal's inquiry behavior.5.Index detection:(1)Enzyme-linked immunosorbent assay(ELISA)was used to detectIL-1?,IL-6 and TNF-a content in the hippocampus.(2)The expression of IL-1? mRNA,IL-6 mRNA and TNF-a mRNA in the hippocampus were detected by real-time reverse transcription-PCR(RT-PCR).(3)Immunohistochemistry method was used to detect the expression of microglia cell marker Ion calcium adapter protein 1(Iba-1)in the hippocampus.[Results]1.Effect of moxibustion on behavior of rats(1)Generalcondition:After 21 days of normal feeding,the rats in the normal group had good mental status,free movement,flexible movement,soft and shiny hair,and normal posture.After exhaustive swimming for 21 days,the rats in the model group had poor mental status,decreased activity,dark and dull hair.Compared with the model group,the rats in the moxibustion group had better mental status,more activity,and more shiny hair,suggesting that moxibustion improved the overall state of chronic exhaustive swimming CFS rats.(2)Body Weight:There was no significant difference in body weight between the groups before exhausted swim(P>0.05).Compared with the normal group,the body weight of the rats in the model group was significantly reduced on the 7th,14th,and 21st day(all P<0.05);Compared with the model group,the body weight of the moxibustion group increased,but there was no significant difference(all P>0.05).(3)Exhaustive swimming time:on the first and seventh days of modeling,there was no significant difference in the exhaustion time between the model group and the moxibustion group(P>0.05);on the 14th and 21st days of modeling,compared with the model group,the time of moxibustion group rats increased significantly(P<0.01,P<0.05).(4)Tail suspension test:After the completion of the model,compared with the normal group,the immobility time of the model group increased,but there was no significant difference(P>0.05);compared with the model group,moxibustion group suspension tail immobility time decreased significantly(P<0.01).(5)Open-field test:Compared with the normal group,the horizontal and vertical movement distances of the open-field test in the model group had a decreasing trend after the completion of the model,but there is no significant difference(P>0.05);compared with the model group,the horizontal and vertical movement distances of the field test in the moxibustion group showed an increasing trend,but there was no significant difference(P>0.05)2.The effect of moxibustion on the contents of IL-1?,IL-6 and TNF-? in rat hippocampus:compared with the normal group,the content of IL-1?,IL-6 and TNF-? in the hippocampus of the model group increased significantly(all P<0.01).Compared with the model group,the content of IL-6 in the moxibustion group was significantly decreased(P<0.01),and the content of IL-1? and TNF-a in the moxibustion group decreased,but there was no significant difference(all P>0.05).3.The effect of moxibustion on the expression of IL-1?,IL-6 and TNF-? mRNA in the hippocampus of rats:compared with the normal group,the expression of IL-1?,IL-6 and TNF-a mRNA in the model group increased significantly(all P<0.05).Compared with the model group,the expression of IL-6 mRNA decreasedsignificantly(P<0.05).However,the IL-1?and TNF-? mRNA in the moxibustion group had a decreasing trend,and there was no significant difference(all P>0.05).4.Effect of moxibustion on Iba-1,a marker of microglia in hippocampus of chronic fatigue rats:Immunohistochemistry results showed that Iba-1 immunopositive cells were distributed in various regions of the hippocampus.In the normal group,the microglia cell body was elongated,with branches and slender curved protrusions extending in all directions,showing a typical "branching-like" pattern,indicating that normal rat glial cells were inactive.In the model group,the microglia of the hippocampus in each group of rats increased in size,the protrusions were short and thick,and the branch retraction decreased.The activated morphology of microglial cells in the moxibustion group was significantly improved compared with the model group.The hypertrophy of the cell body was reduced,and the morphology of the microglia had shifted toward resting.The results of image analysis showed that compared with the normal group,the mean optical density of Iba-1 positive cells in the microglia cells in the CA1 and CA3 regions of the model group was increased significantly(all P<0.01).Compared with the model group,the average optical density of Iba-1 positive cells in the microglia cells in the CA1 and CA3 areas of the moxibustion group was significantly reduced(all P<0.01).[Conclusions](1)Exhaustive swimming time of chronic fatigue rats decreased significantly,and the time of immobility increased.The horizontal and vertical movement distance of the open field experiment decreased.This indicated that exhausted swimming could cause physical fatigue and mental fatigue in rats.Moxibustion treatment can significantly increase the time of exhausted swimming in chronic fatigue rats and reduce the immobility time of tail suspension,suggesting that moxibustion can significantly improve the fatigue state of rats.(2)Chronic fatigue rats hippocampus microglial cells were activated and produced a large number of pro-inflammatory cytokines IL-1?,IL-6 and TNF-?,confirming the presence of neuroinflammation in the brain of chronic fatigue rats,suggesting that activatedmicroglia mediating neuroinflammatory were participated in the pathogenesis of chronic fatigue syndrome.Moxibustion can significantly down-regulate the expression of pro-inflammatory cytokine IL-6 in the hippocampus and inhibit the activity of hippocampal microglial cells,suggesting that moxibustion can relieve the inflammatory response in the brain of chronic fatigue rats.(3)This study suggested that moxibustion,as an effective treatment to relieve chronic fatigue syndrome,may play a role in anti-fatigue by reducing the levels of pro-inflammatory cytokines and microglial cells in the central nervous system,thereby reducing the inflammation in the hippocampus. |
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