The Role Of Synapse Pruning Mediated By Complement C3aR In Depression-like Behavior In Mice And The Intervention Of Gypenoside ⅩⅦ

Depression is a common mental disorder that is the result of a combination of social,psychological and biological factors.Its biological characteristics are not only manifested by the deficiency of monoamine neurotransmitters,but also accompanied by persistent neuroinflammation.The complement system is extensively involved in the immune mechanism of the body,producing different activating complements such as C1 q and C3 during activation to mediate the inflammatory response.Gypenosides are the active part of Gynostemma pentaphyllum and exert a potential antidepressantlike effects.Gypenoside ⅩⅦ is one of the active components of Gypenosides.Previous studies have shown that Gypenoside ⅩⅦ can significantly reduce the inflammatory response of the body.However,whether Gypenoside ⅩⅦ can reduce neuroinflammation by regulating the complement C3 system to improve depressive symptoms remains unknown.In the present study,how complement components C3 and C3a R affect astrocyte-microglia-neuron interactions in the pathogenesis of depression was investigated.Then,the C3/C3 a R signaling pathway mediated synaptic pruning in the antidepressant-like effects of Gypenoside ⅩⅦ was also elucidated.Firstly,a chronic unpredictable mild stress(CUMS)depression-like model was established and the C3 a R antagonist SB290157(1 mg/kg)was injected intraperitoneally for intervention.Sucrose preference test was used to assess depressive-like symptoms,and proteomic analysis and functional prediction were used to analysis protein differences between depressed and normal mice.The results revealed that the expression of complement C3 and C1 q proteins increased in the prefrontal cortex of depressed mice,and the synaptic pruning signal mediated by the complement system was involved in the pathogenesis of depression.Subsequently,lipopolysaccharide(LPS)inflammatory depression model and CUMS model were constructed to assess the dynamic changes of complement C3/C3 a R throughout the pathogenesis of depression,and the depression-like symptoms of mice were evaluated by behavioral experiments.The expression of C3/C3 a R in the prefrontal cortex of depressed mice was analyzed by Western Blot and immunofluorescence.The results showed that C3/C3 a R was activated rapidly in the early stages of depression.Microglia are resident macrophages of the central nervous system,which mainly exist in two states: resting and activated.In the resting state,microglia project the impulse to sense changes in their environment and secrete brain-derived neurotrophic factors that contribute to the balance of the central nervous system and neuronal plasticity.Once the changes such as neuron damage are sensed,microglia will enter an activated state,secrete pro-inflammatory cytokines and reactive oxidation substances,and enhance phagocytosis.If microglia remain activated for a long time,they can cause damage to nerve cells.In the brain,the complement system is involved in the regulation of synaptic pruning of microglia: complement labels the synapse and induces phagocytosis of labelled cellular components by microglia through binding to complement receptors on microglia.Therefore,in order to investigate whether blocking C3/C3 a R pathway could improve depressive symptoms and microglial activity,the C3 a R antagonist SB290157(1mg/kg)was treated in CUMS mice,and the depressionlike behaviors were evaluated.The expression of C3/C3 a R was detected by Western blot and immunofluorescence.Meanwhile,to investigate the influence of C3/C3 a R pathway on APT2/DHHC7 palmitoylation and activation of STAT3 in microglia,Western blot was performed to detect the expression of related signaling proteins,triple labelling immunofluorescent technique to detect protein co-expression,quantitative real time polymerase chain reaction(q RT-PCR)and enzyme-linked immunosorbent assay(ELISA)to detect the expression of pro-inflammatory cytokines.To investigate the effect of C3/C3 a R on synaptic pruning in microglia,triple labelling immunofluorescent was used to detect the labelling of synaptic proteins by C3 and microglia,and Golgi staining was used to detect changes in dendritic spine density.The results showed that blockade of C3 a R signaling reduced C3/C3 a R expression in the prefrontal cortex and attenuated depressive-like behaviors.C3 a R signaling blockade inhibits APT2/DHHC7 palmitoylation mediated STAT3 phosphorylation and proinflammatory cytokine expression in microglia and reduced numbers of microglia and C3 content in the prefrontal cortex.The triple labelling immunofluorescent of microglia showed that complement C3 chemotaxis and activates microglia into the amoebic form,phagocytic synaptic proteins,and inhibit the growth of synaptic spines.Blocking the C3 a R pathway can efficiently reduce the synaptic labeling and synaptic pruning by complement C3.The expression of complement C3 is regulated by cytokines and other inflammatory molecules.In the brain,complement C3 is expressed primarily by astrocytes.The receptor for cytokine IL-1β,IL-1R,is located primarily on the cell membrane of astrocytes.The study then further explored whether IL-1R could regulate the expression of complement C3 in the depression-like mice.Anakinra(10mg/kg),an IL-1R antagonist,was treated in CUMS mice,and the depression-like behaviors were evaluated by experimental ethology.The effects of blocking IL-1β/IL-1R signaling pathway on the expression of IL-1R,NF-κB and C3 in astrocytes were detected by Western blot and immunofluorescence assay.We found that blocking IL-1R reversed depression-like behavior in mice by inhibiting the NF-κB/C3 signaling pathway and reducing the release of complement C3 by astrocytes.Finally,the present study continues to investigate the antidepressant-like effects of Gypenoside ⅩⅦ and to elucidate whether Gypenoside ⅩⅦ exerts the effects through the inhibition of complement C3/C3 a R.The results showed that acute administration of Gypenoside ⅩⅦ exerted the antidepressant-like effects and reduced serum corticosterone levels.Long-term administration with Gypenoside ⅩⅦ reversed the activation of C3/C3 a R/STAT3 signaling pathway and release of pro-inflammatory cytokines,inhibited prefrontal cortex microglia activation and C3 expression,attenuated synaptic pruning,and alleviated depression-like symptoms in CUMS mice.In summary,this study demonstrates that stress activates the astrocyte IL-1R/NF-κB/C3 signaling pathway,releasing large amounts of complement C3,which induces synaptic pruning and further causes the expression of inflammatory factors through chemotaxis and activation of the microglia C3 a R/APT2/DHHC7/STAT3 signaling pathway,and this abnormal astrocyte-microglia-neuron interaction is involved in the pathogenesis of depression.Meanwhile,Gypenoside ⅩⅦ exerts the antidepressantlike effects,which were associated with the blocking of C3/C3 a R/STAT3 signaling pathways and inhibition of synaptic pruning.