| BACKGROUND&OBJECTIVEDepression,a common psychiatric disorder,is characterized by high prevalence,high disability rate,high recurrence rate and high social burden of disease.Presently,no established mechanism can exactly explain the etiology and pathological mechanisms of depression.And effective therapeutic strategies for depression are urgently needed.Data from clinical investigation and experimental study have suggested that the stress-induced activation of immune/inflammatory response is involved in the development and pathogenesis of depression.Nowadays,data from clinical trials and experimental funding has verified the efficacy and safety of acupuncture on treating depression.However,the mechanism of the antidepressant effect of acupuncture has not been fully elucidated.Accordingly,the rat model in the present study was established by exposure to chronic restraint stress(CRS)to simulate depressive disorder based on the preliminary research of our team.We hypothesize that the antidepressant effect of acupuncture is based on regulating the upper triggers of the stress-induced activation of immune/inflammatory response that is mediated by pattern recognition receptors toll-like receptor 4(TLR4)signaling pathway and nucleotide-binding oligomerization domain-like receptors,pyrin domain containing 3(NLRP3)inflammasome.This is the starting point and innovative point.METHODSForty-four male Sprague Dawley rats were randomly enrolled into normal group,model group,escitalopram group and acupuncture group.Except rats in the normal group,all the rats were exposed to chronic restraint stress(CRS)for 21 days continuously.Rats in the escitalopram group were subjected to escitalopram administration(30mg/kg)by gavage 1 hour before CRS procedures.Rats in acupuncture group were acupunctured at Baihui(GV20)and Yintang(GV 26)1 hour before CRS procedures.The experimental procedures were as follows.(1)The effects of acupuncture on ameliorating depression-like behaviors in the rat model of depression induced by CRS procedures.All the rats in each group were exposed to body weight assessment,sucrose preference test and open field test to evaluate the body weight,body weight gain,sucrose consumption,pure water consumption,total liquid consumption,sucrose preference index,horizontal movement scores and vertical movement scores at 0,7,14 and 21 days.Then we explored the effects of acupuncture on regulating the natural growth and development,overall states,anhedonia,spontaneous activity and exploratory behavior in the rats subjected to CRS to identify the antidepressant effect of acupuncture.(2)The effect of acupuncture on the immunological/inflammatory response and apoptosis mediated by microglia activation in the hippocampus of CRS rats.After the experimental procedures(21 days),the expression of microglia activation in the hippocampus was detected by immunohistochemistry technology(IHCT).And the effects of acupuncture on regulating the apoptosis of hippocampal neurons in the depression rat model were also investigated via detecting the changes of the state of hippocampal neurons by terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL)method.Meanwhile,the content of serum IL-1?,IL-10 and TNF-? was detected by enzyme-linked immunosorbent assay(ELISA)method.(3)The mechanism underlying the antidepressant effects of acupuncture on regulating the upper triggers of the stress-induced activation of immune/inflammatory response that is mediated by TLR4 signaling pathway and NLRP3 inflammasome.After experiment,the expression of the key hippocampal proteins on TLR4 signaling pathways(TLR4,MyD88,TRAF6,NF-?B p65,TNF-?)and the key trigger on the NLRP3 inflammasome(NLRP3,ASC,Caspase-1,IL-1?,IL-18)were detected by western blot(WB).The expression of the mRNA of TLR4,NLRP3,MyD88 and NF-?B p65 in hippocampus was detected by real-time quantitative polymerase chain reaction(Real-time Q-PCR).The objective was to elucidate the mechanisms of the antidepressant effect of acupuncture that was based on the stress-induced activation of immune/inflammatory response,mediated by TLR4 signaling pathway and NLRP3 inflammasome.RESULTS(1)The effects of acupuncture on ameliorating depression-like behaviors in the rat model of depression induced by CRS procedures.The results of behavioral assessment were as follows:? The changes of body weight and body weight gain at different time points indicated that there was no statistically significant difference among each group before experimental intervention.However,significant difference was found among each group following different intervention.Compared with normal group,the body weight and body weight gain of rats in model group began to decrease obviously after 7 days of CRS procedures.The body weight of rats in model group decreased significantly at 7,14,and 21 days when compared with that of normal group(P<0.01;P<0.01;P<0.01).The body weight gain in the model group was significantly lower than the normal group at 7 days and 14 days(P<0.01,P<0.01).Compared with model group,the body weight and body weight gain in acupuncture group were significantly higher than that of model group at 7 days,14 days and 21 days(P<0.01,P<0.01,P<0.01;P<0.01,P<0.01,P<0.05).Notably,The effects of acupuncture on reversing weight loss induced by CRS were earlier than that of escitaloplan at some certain extent.? The changes of sugar water consumption,water consumption,total liquid consumption and sugar preference index at different time points indicated that there was no statistically significant difference among each group before experimental intervention.With the change of time,the sugar water consumption,pure water consumption,total liquid consumption and sugar preference index of rats at different time points were statistically significant due to different intervention.The sugar water consumption,total liquid consumption and sugar preference index in model group decreased significantly when compared with that of normal group(P<0.01;P<0.01;P<0.01).The sensitivity of the reward of sugar water for rats exposed to CRS decreased obviously,and the pleasure deficiency was aggravated.However,acupuncture intervention effectively reversed the states.Acupuncture intervention exerted antidepressant effect.? The changes of horizontal motion score and vertical motion score at different time points indicated that there was no statistically significant difference among each group before experimental intervention.Compared with normal group,the horizontal motion score of rats in model group were significantly lower than normal group at 7 days,14 days and 21 days(P<0.01;P<0.01;P<0.01),and the vertical motion score of the model group was significantly lower than the normal group at 14 days and 21 days(P<0.01;P<0.01).The spontaneous activity and exploratory behavior of rats in model group were obviously inhibited.Compared with model group,the acupuncture intervention began to have a reverse effect on the 7th day.The horizontal motion score was significantly higher than the model group at 7 days,14 days and 21 days(P<0.01;P<0.01;P<0.01).The vertical motion score of rats in acupuncture group was significantly higher at 14 days and 21 days than model group(P<0.01;P<0.01).The horizontal motion score and vertical motion score of the escitalopram group were significantly higher than that of model group(P<0.01;P<0.01).Notably,the horizontal motion score of rats in acupuncture group was obviously higher than escitalopram group at 7 days(P<0.01),the vertical motion score at 14 days(P<0.01).(2)The effect of acupuncture on the immunological/inflammatory response and apoptosis mediated by microglia activation in the hippocampus of CRS rats.? Microglia(marked by IBA-1)of the hippocampal CA1,CA2,CA3 and DG area in normal group maintained as resident cells with small neuronal somata and less processes.The Microglia cells in the model group were activated.IBA-1 was highly expressed in the hippocampal CA1,CA2,CA3 and DG area in model group 21 days after CRS procedures.The amount of activated microglia significantly increased in the hippocampal CA1,CA2,CA3 and DG area(P<0.01;P<0.01;P<0.01;P<0.01).Compared with the model group,the amount of IBA-1 positive cells decreased obviously in acupuncture and escitalopram group(P<0.01;P<0.01;P<0.01;P<0.01),especially in CA1 and CA3 area,which indicating acupuncture and escitalopram intervention significantly reversed the activated microglia induced by CRS procedures.? In addition,the effects of acupuncture on regulating the apoptosis of hippocampal neurons in the depression rat model were also investigated via detecting the changes of the state of hippocampal neurons in CA1,CA2,CA3 and DG by TUNEL method.The results indicated that the cells of hippocampal CA1,CA2,CA3 and DG area in normal group distributed in the nuclei and with blue cell core.Little TUNEL positive cells were investigated in normal group.Compared with normal group,more TUNEL positive cells were investigated in hippocampal CA1,CA2,CA3 and DG area in model group,and the number of TUNEL positive cells increased obviously in the model group(P<0.01;P<0.01;P<0.01;P<0.01).Compared with model group,the number of TUNEL positive cells decreased obviously in acupuncture and escitalopram group(P<0.01;P<0.01;P<0.01;P<0.01),especially in CA1 and CA3 area.? The changes of the expression of serum IL-1?,IL-10,and TNF-? indicated that acupuncture could regulate the expression of serum IL-1?,IL-10,and TNF-?.(3)The mechanism underlying the antidepressant effects of acupuncture on regulating the upper triggers of the stress-induced activation of immune/inflammatory response that is mediated by TLR4 signaling pathway and NLRP3 inflammasome.The CRS procedures induced significant upregulation of the expression of the key hippocampal proteins on TLR4 signaling pathways(TLR4,MyD88,TRAF6,NF-?B p65,TNF-?)and the key trigger on the NLRP3 inflammasome(NLRP3,ASC,Caspase-1,IL-1?,IL-18).The same trend was also found in the expression of the mRNA of TLR4,NLRP3 and NF-?B p65 in hippocampus.Notably,acupuncture and escitalopram intervention can regulate the upper triggers of the stress-induced activation of immune/inflammatory response that is mediated by TLR4 signaling pathway and NLRP3 inflammasome.Compared with model group,acupuncture reversed the upregulation of the expression of the key hippocampal proteins of TLR4,MyD88,NF-?B p65,TNF-?,NLRP3 and Caspase-1 in model group(P<0.01;P<0.01;P<0.01;P<0.01;P<0.01,P<0.05).And acupuncture exerted therapeutic effects on reversing the upregulation of the expression of the key hippocampal mRNA of TLR4,MyD88,NF-?B p65 in model group(P<0.05;P<0.05;P<0.05).CONCLUSION(1)CRS procedures induced activation of immune/inflammatory response.The stress-induced activation of immune/inflammatory response that is mediated by TLR4 signaling pathway and NLRP3 inflammasome might be the key pathway linking stress and depression.(2)Acupuncture could regulate the stress-induced activation of immune/inflammatory response and reverse the microglia activation and the apoptosis of hippocampal neurons in the depression rat model.Accordingly,the antidepressant effect of acupuncture might be based on regulating the stress-induced activation of immune/inflammatory response that is mediated by TLR4 signaling pathway and NLRP3 inflammasome.(3)Notably,the antidepressant effect of acupuncture might be based on regulating the upper triggers of the stress-induced activation of immune/inflammatory response that is mediated by TLR4 signaling pathway and NLRP3 inflammasome. |
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