| Background Depression is a neuroimmune disease characterized by persistent black mood and retardation of thinking.Neuroinflammation is a major pathological change in depression.Studies have found that in inflammatory diseases of central nervous system(CNS),abnormal glycosylation of some key proteins can affect immune regulation of body and prognosis of diseases.Glycosylation is an important post-translational modification of proteins,which can maintain the structure and function of proteins,and mediate cell adhesion and signal transduction.Maladjustment of glycosylation can lead to disorder of glycosyl structure and quantity,resulting in changes of biological functions of glycoproteins.Fucosylation and sialylation are common types of glycosylation,which are ubiquitous in organisms.?1,6-fucosyltransferase(Fut8)is the only enzyme that catalyzes core fucosylation.?-galactoside ?2,6-sialyltranferase 1(ST6GAL1)catalyzes terminal 2,6-sialic acid to connect N-Glycans and participates in sialylation modification.Studies have shown that fucosylation and sialylation are related to the formation of tissue inflammatory microenvironment.In rheumatoid arthritis and osteoarthritis,the expression of Fut8 was increased in synovial tissues,and the fucosylation and sialylation were enhanced.Microglia are the innate immune cells in CNS,and activated microglia are the main source of inflammatory factors in the brain.However,with the increase of peripheral and central inflammatory responses in depression,whether the serum,brain tissue proteins and microglia cells undergo fucosylation and sialylation changes,and how the related glycosyltransferases change,have not been reported yet.Objective To explore the changes of protein glycosylation in serum and brain tissues of inflammatory depression model mice,identify the changes of protein glycosylation in peripheral and CNS undergoing immune activation in depression,and provide a new perspective and information for the study of pathogenesis of depression.Methods In this study,we established a mouse model of inflammatory depression by intraperitoneal injection of lipopolysaccharide(LPS).The behavioral changes were tested by open field test(OFT),elevated plus-maze(EPM),forced swimming test(FST)and tail suspension test(TST).Changes of glycosylation were analyzed by Mass Spectrometry(MS)in serum and the level of fucosylation and sialylation were tested by Lectin blotting.Primary culture of mouse microglial cells and BV2 microglial cell lines in vitro.The levels of tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?)and interleukin-6(IL-6)in serum,brain and cells were detected by ELISA and q RT-PCR,and the levels of Fut8 and ST6GAL1 were detected by ELISA.Results 1.Established a mouse model of depression by LPS intraperitoneal injection Compared with the control,24 h after LPS(1 mg/kg)injection,the mobility time of mice in the central zone in the OFT was significantly reduced(p<0.001),and the mobility time in open arm in the EPM was significantly reduced(p<0.001).In the TST and FST experiments,the immobility time was significantly up-regulated compared with the control(p<0.05).The above behavioral results showed that the intraperitoneal injection of LPS induced depression-like behaviors successfully.2.Characteristics of glycosylation in serumof depression model mice.(1)The depression model mice developed a peripheral inflammatory response.ELISA test results showed that the expression levels of TNF-?,IL-6 and IL-1? in serum were raised in LPS 24 h group(p<0.05).(2)The levels of fucosylation and sialylation in the serum of depression model mice were up-regulated.After injection of LPS,the results of MS analysis showed that the levels of fucosylation and sialylationi in N-Glycans of serum proteins were significantly up-regulated(p<0.0001).Lectin blotting showed that ?1,6 linked fucosylation,terminal fucosylation,and after the LPS injection ?2,6 linked sialylation in the serum were significantly up-regulated compared with the control(p<0.001).(3)The expression of fucose transferase and sialyltransferase were increased in serum of depression model mice.Fut8 and ST6GAL1 levels in serum and liver were detected by ELISA.The results showed that the levels of Fut8 and ST6GAL1 in the serum and liver of LPS 24 h group were significantly increased compared with the control group(p<0.05).3.Glycosylation changes in the brain of depression model mice.(1)The expression of pro-inflammatory cytokines in the brain of depression model mice was up-regulated.RT-q PCR and ELISA results showed that protein and m RNA expression levels of IL-1?,IL-6 and TNF-? were significantly upregulated in LPS 24 h group(p<0.01).(2)Up-regulation of protein fucosylation and sialylation were found in the brain of depression model mice.Lectin blotting results showed that the levels of fucosylation and sialylation in the brain of LPS-injected group were significantly enhanced(p<0.05).(3)The expression of glycosyltransferase was increased in the brain of depression model mice The results of ELISA showed that the expression of Fut8 in the brain of LPS-injection group was significantly enhanced compared with the control group(p<0.01).Similarly,ST6GAL1 levels were significantly up-regulated than those in the control group(p<0.0001).4.Characteristics of glycosylation changes in immune-activated microglia.(1)LPS-treated microglia expressed more inflammatory cytokines.LPS(100 ng/m L)was added to the culture medium of primary cultured microglia and BV2 microglial cell lines,respectively,and incubated for 24 h.The results of RT-q PCR showed that the levels of IL-1?,IL-6 and TNF-? m RNAs in primary microglia and BV2 cells were significantly increased after 24 h incubation with LPS (p<0.01).(2)LPS stimulated microglia to increase protein glycosylation.Lectin blotting showed that the levels of fucosylation and sialylation of microglia protein after LPS(100 ng/m L)stimulation for 24 h were significantly higher than those of the unstimulated group(p<0.05).(3)Addition of LPS led to upregulation of glycosyltransferase expression in microglia.ELISA showed that after LPS(100 ng/m L)stimulation for 24 h,expression levels of Fut8 and ST6GAL1 protein were significantly increased in primary microglia and BV2 cells compared with the control group(p<0.05).Conclusions 1.Peripheral and CNS inflammatory responses are enhanced in mice with inflammatory depression,accompanied by increased levels of glycosyltransferase and glycosylation.2.Inflammatory responses in the brain may be related to increased levels of glycosyltransferase and glycosylation in immune-activated microglia. |
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