| Objective:In the practice of forensic medicine,it is often encountered that the victims suffer different degrees of mental injury after being subjected to restriction of personal freedom such as continuous binding and bondage.However,because the mechanism of injury is not clear,there is still a lack of systematic and reasonable scientific explanation for the identification of such facts,so it is difficult to issue an objective and fair appraisal opinion.The common feature of this kind of cases is that the victims often experience strong mental and psychological stress reaction after being bound.An in-depth study of the mechanism of excessive stress induced mental injury is of great significance for revealing the mechanism of mental injury and even death caused by such indirect violent injury.The previous work in our laboratory confirmed that stress can cause damage to many tissues and organs,such as the heart,kidney,amygdala and hippocampus of rats,suggesting that the damage effect of stress on the body should not be ignored.The main neuroendocrine reaction of stress is the strong excitement of hypothalamic-pituitary-adrenal cortex axis,and the increase of the synthesis and secretion of Glucocorticoid（GC）.Exposure to moderate stress,GC plays a regulatory role in maintaining homeostasis in the body,but exposure to excessive stress,the abnormally elevated GC level will have adverse effects on the body.Recent studies have confirmed that neuroinflammatory response caused by stress plays an important role in the pathogenesis of depression,post-traumatic stress disorder and other diseases.Microglia（MG）are the main immune cells of the central nervous system,and play a key role in mediating neuroinflammatory response.GC mainly includes cortisol and corticosterone,and corticosterone is the dominant one in rodents.It has been confirmed that murine-derived MG may secreted proinflammatory cytokine after Corticosterone（CORT）treatment,suggesting that GC-activated MG may play an important role in stress-induced neuroinflammatory response,but its molecular mechanism remains unclear.Damage associated molecular patterns are endogenous intracellular molecules and extracellular matrix molecules released by activated or necrotic cells,which are considered to play a key role in the neuroinflammatory response.High mobility group box 1 protein（HMGB1）,its representative material,is an important initiator of the neuroinflammatory response.Under physiological conditions,HMGB1 is mainly involved in various biological processes in the nucleus.Once stimulated by harmful factors,HMGB1expression is increased,transferred from the nucleus to the cytoplasm,and then secreted into the extracellular,where it recognizes cell surface receptors and initiates inflammation-related signal transduction pathways.Our previous study found HMGB1 translocation in amygdala microglia of rats under bondage stress,suggesting that HMGB1 may be involved in the neuroinflammatory response induced by stress.What is the regulatory effect of CORT on the production of HMGB1?What is the role of HMGB1 in the activation of MG by CORT?None has been reported in the literature.Based on the above research background,this study intends to use CORT to activate microglial BV2 cells and explore the role of HMGB1 in CORT activation of MG,so as to provide a scientific basis for clarifying the mechanism of neuroinflammatory response caused by stress and an experimental basis for establishing the identification system of stress induced central damage.Methods:1.Mouse BV2 cells were cultured in vitro,and the expression of IBA1（MG marker）in BV2 cells was detected by immunofluorescence staining.This cell line basically has the morphology,phenotype and various functional characteristics of primary culture microglia cells,and is used as the most commonly used cell model to study the inflammatory response in the brain.2.Different concentrations of 10-8M,10-7M,10-6M and 10-5M CORT were used to treat BV2 cells,and IL-1βexpression was detected by ELISA to determine the concentration of CORT in subsequent experiments.3.The experiment was divided into 6 groups:Control group,CORT group,CORT+RU486（glucocorticoid receptor inhibitor）group,RU486 group,CORT+GLY（HMGB1 inhibitor）group,and GLY group.3.The expression of inflammatory cytokines IL-1β,TNFαand damage associated molecular pattern HMGB1 in BV2 cells were detected by Western blot.4.The expression of inflammatory cytokines IL-1β,TNFαand damage associated molecular pattern HMGB1 in BV2 cells were detected by cellular immunofluorescence assay.5.SPSS25.0 software was used for statistical analysis,and all data were expressed as Mean±SEM.Oneway analysis of variance（ANOVA）was used to compare the Mean of each group.The Least Significant Difference（LSD）method was used to make pair comparison.The statistical results showed that P<0.05 was considered statistically significant.Results:1.The results of cellular immunofluorescence showed that all BV2 cells expressed IBA1 and were identified as microglia cell lines,which could be used for subsequent experiments.2.ELISA results showed that compared with Control group,BV2 cells were treated with 10-7M,10-6M,10-5M CORT for 24h,BV2 cells IL-1βrelease was significantly increased（P<0.05）and was concentration dependent.There was no significant difference in IL-1βrelease between 10-8M groups compared with Control group.Therefore,10-5M CORT was selected for subsequent experiments.3.Changes in IL-1βexpression levelImmunofluorescence staining and Western blot results showed that the expression of IL-1βin CORT group was significantly increased compared with the Control group,while the expression of IL-1βin CORT+RU486 group was significantly decreased compared with CORT group,suggesting that CORT can promote the secretion of IL-1βin microglia cells through receptors.Compared with the Control group,the expression of IL-1βdid not change significantly in RU486 group.Compared with CORT group,the expression level of IL-1βwas significantly decreased in CORT+GLY group,suggesting that HMGB1 plays an important role in CORT promoting the secretion of IL-1βby microglia.Compared with the Control group,the expression of IL-1βdid not change significantly in GLY group.4.Changes in TNF-αexpression levelImmunofluorescence staining and Western blot results showed that the expression level of TNF-αin CORT group was significantly increased compared with the Control group,while the expression level of TNF-αin CORT+RU486 group was significantly decreased compared with CORT group,suggesting that CORT can promote the secretion of TNF-αin microglia cells through receptors.There was no significant change in TNF-αexpression between RU486 group and Control group.Compared with CORT group,the expression level of TNF-αin CORT+GLY group was significantly decreased,suggesting that HMGB1plays an important role in CORT promoting the secretion of TNF-αby microglia.There was no significant change in TNF-αexpression between GLY group and Control group.5.Changes in HMGB1 expression levelsWestern blot results showed that the expression of HMGB1 in CORT group was significantly increased compared with the Control group,while the expression of HMGB1 in CORT+RU486 group was significantly decreased compared with CORT group,suggesting that CORT can promote the secretion of HMGB1 by microglia cells through receptors.There was no significant change in HMGB1 expression between RU486 group and Control group.Compared with CORT group,the expression level of HMGB1 was significantly decreased in CORT+GLY group,indicating that administration of HMGB1 inhibitor could significantly inhibit CORT-induced HMGB1expression.There was no significant change in HMGB1 expression between GLY group and Control group.6.Changes in expression position of HMGB1Immunofluorescence results showed that HMGB1 was only expressed in the nucleus of the Control group,while HMGB1 was expressed in the nucleus and cytoplasm of the CORT group and its expression level was increased.Combined with Western blot results,CORT could induce the increase of HMGB1 expression and transposition,and some HMGB1 in the nucleus was translocated into the cytoplasm to play a role.Compared with the CORT group,HMGB1 was only expressed in the nucleus of CORT+RU486 group,which further suggested that CORT could promote HMGB1 secretion and translocation in microglia cells.There was no significant change in HMGB1expression between RU486 group and Control group.Compared with CORT group,HMGB1 was only expressed in the nucleus of CORT+GLY group,and the expression level also showed a downward trend.There was no significant change in HMGB1 expression between GLY group and Control group.Conclusions:As a damage associated molecular pattern,HMGB1 plays an important role in the secretion of inflammatory cytokines by microglia induced by corticosterone. |
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