补体系统C1q/C3介导的胶质细胞激活在小鼠抑郁样行为中的作用

The Role of Glial Cell Activation Mediated by Complement System C1q/C3 in Depression-like Behavior in Mice

【目的】探讨小鼠的杏仁核中C1q/C3补体系统在诱导抑郁样行为过程中的作用途径。【方法】本实验分3部分:①采用慢性束缚压力方法建立小鼠抑郁模型,将12只8周龄雄性C57BL/6小鼠随机分为对照(WT)组和抑郁模型(CRS)组,各6只,抑郁模型组造模2周。行为学测试系统悬尾实验(TST)、强迫游泳实验(FST)测试小鼠抑郁状态,采用免疫荧光染色法检测小鼠杏仁核中突触素(Syn)和突触后致密物(PSD95)变化情况,小胶质细胞(Iba-1)和星形胶质细胞(GFAP)细胞荧光强度,C1q、C3含量。②6只8周CX3CR1-GFP雄性鼠,随机分为GFP组以及GFP模型(GFP+CRS)组,各3只,采用免疫荧光染色法观察小胶质细胞及星形胶质细胞相互作用情况。③12只8周龄雄性C57BL/6和12只C1q^(-/-)小鼠随机分为对照(WT)组,抑郁模型(CRS)组,C1q敲除(C1q^(-/-))组,C1q敲除模型(C1q^(-/-)+CRS)组,各6只,模型组组造模2周,行为学TST、FST检测4组小鼠抑郁状态,采用免疫荧光染色法检测四组小鼠杏仁核部位突触Syn和PSD95变化。【结果】行为学分析结果显示:与WT组比较CRS组在TST和FST中不动时间明显升高(P<0.0001);免疫荧光法测定,与WT组比较CRS组杏仁核内突触Syn、PSD95含量明显降低(P<0.0001;P=0.0038);与WT组比较CRS组杏仁核内Iba-1和GFAP明显激活(P=0.0004,P=0.003)且与WT组比较CRS组杏仁核内小胶质细胞与星形胶质细胞相互作用明显;与WT组比较CRS组杏仁核内C1q与C3产生明显增多(P=0.0002,P=0.0119);行为学分析,WT、CRS、C1q^(-/-)、C1q^(-/-)+CRS 4组综合比较,在TST中,与CRS组比较,WT组不动时间明显升高,C1q^(-/-)+CRS组不动时间也明显增高(均P<0.001);在FST中,与CRS组比较,WT组不动时间明显升高,C1q^(-/-)+CRS组不动时间也明显增高(均P<0.001);与CRS组比较WT组与C1q^(-/-)+CRS组杏仁核内突触Syn明显增多(P<0.001;P<0.05),PSD95明显增多(P<0.05)。【结论】抑郁模型小鼠杏仁核内发生神经炎症,胶质细胞激活,表达补体C1q、C3,进一步激活小胶质细胞和星形胶质细胞修剪突触,导致突触含量降低,促使小鼠产生抑郁样行为;C1q基因缺陷阻断CRS诱导的小鼠突触丢失和抑郁样行为。

【Objective】To investigate the pathway of C1q/C3 complement system in the amygdala of mice in the induction of depressive-like behavior.【Methods】Three parts were included.Firstly,the depression model of mice was established by chronic restraint stress.Twelve 8-week-old male C57BL/6 mice were randomly divided into control(WT)group(n=6)and depression model(CRS)group(n=6).The depression model group was established for 2 weeks.The depressive state of mice was tested by tail suspension test(the tail suspension test,TST)and forced swimming test(the forced swimming test,FST).The fluorescence intensity of the changes of synaptic Synaptophysin(Syn)and post synaptic density protein 95(PSD95),microglia(Iba-1)and astrocytes(GFAP)and the contents of complement component(C1q)and C3 in amygdala were detected by immunofluorescence staining.Secondly,six 8-week-old CX3CR1-GFP male mice were randomly divided into GFP group(n=3)and GFP model group(GFP+CRS)with 3 mice in each group.The interaction between microglia and astrocytes was observed by immunofluorescence staining.Thirdly,twelve 8-week-old male C57BL/6 and C1q^(-/-)mice were randomly divided into control(WT)group,depression model(CRS)group,C1q knockout(C1q^(-/-))group and C1q knockout model(C1q^(-/-)+CRS)group,with 6 mice in each group.depression model was established for 2 weeks.Behavioral test TST and FST were used to detect the state of depression in four groups.Immunofluorescence staining was used to detect the changes of synaptic Syn and PSD95 in the amygdala of the four groups.【Result】The results of behavioral measurement showed that the immobility time in TST and FST in CRS group was significantly higher than that in WT group(P<0.0001);the result immunofluorescence assay shows that the content of Syn and PSD95 in amygdala in CRS group was significantly lower than that in WT group(P<0.0001;P=0.0038);the activation of Iba-1 and GFAP in amygdala in CRS group was significantly higher than that in WT group(P=0.0004,P=0.003);and the interaction between microglia and astrocytes in amygdala in CRS group was more obvious than that in WT group;compared with WT group,the production of C1q and C3 in amygdala was significantly increased in CRS group(P=0.0002,P=0.0119);Comprehensive comparison of WT,CRS,C1q^(-/-),C1q^(-/-)+CRS,the immobility time in TST in WT group was significantly lower than that in CRS group and in C1q^(-/-)+CRS group was also significantly lower than that in CRS group(both P<0.001),the immobility time in FST in WT group was significantly lower than that in CRS group and in C1q^(-/-)+CRS group was significantly lower than that in CRS group(both P<0.001).The Syn in amygdala in WT group and C1q^(-/-)+CRS group were significantly higher than those in CRS group(P<0.001).The PSD95 in amygdala in WT group and C1q^(-/-)+CRS group were significantly higher than those in CRS group(P<0.05).【Conclusion】In the depressive model mice,neuroinflammation occurred in the amygdala,where glial cells were activated to express C1q and C3,which further activated microglia and astrocytes to prune synapses,resulting in the decrease of synaptic content and the depression-like behavior in mice.C1q^(-/-)deficiency prevented CRS-induced synaptic loss and depressive-like behavior.