Effect And Mechanism Of Complement Component 3 In Stress-induced Depressive-like Behaviors In Mice

Part ? The effect of C3 in stress-induced depressive-like behaviors in miceObjective: It has been reported that when the peripheral immune system continues activated by systemic infections,cancer or autoimmune diseases,the ensuing immune signaling to the brain would cause an exacerbation of sickness,even deteriorate the development of symptoms of depression in vulnerable individuals.Complement component 3(C3)and its fragment play an important role in immune,inflammation and neuropsychiatric disorders.It has been reported that activation of C3 induced the release of tumor necrosis factor,then wosen the inflammatory response and changed motivation behavior and emotion.It also has been reported that dificiency in C3 accelerated the deposition of ?-amyloid(A?)plaque.However,it still remains unknowns about the effect of C3 signal in depression.As to determine the role of C3 signal in depression,we established an animal model of depression by exposing to chronic social defeat stress(CSDS)or lipopolysaccharide(LPS)stress,and detected the mRNA and protein level of C3 in susceptible mice.Methods: Establishing animal models of depression by exposing to CSDS or LPS;Sucrose preference test(SPT),social interaction test(SIT),forced swim test(FST),tail suspension test(TST)and open field test(OFT)were utilized to detect the depressive-like behaviors and motivation;Quantitative real-time polymerase chain reaction(Q-PCR),western blotting and enzyme-linked immunosorbent assay(ELISA)were performed to detect the expression of C3 in peripheral blood and brain of susceptible mice;Intracerebroventricular microinjection of compstatin was utilized to detect the effect of C3 inhibitor on depressive behaviors induced by CSDS and susceptibility to CSDS.Results:(1)CSDS induced depressive-like behaviors in susceptible mice.Compared with the control,the sucrose preference of susceptible mice was remarkably decreased(P < 0.001).The time in the interaction zone with target(P < 0.001)and the interaction ratio(P < 0.001)of susceptible mice was significantly decreased when compared with control mice.(2)C3 gene expression was significantly increased(P < 0.01)in the blood of susceptible mice,and the C3 concentration in serum was increased(P < 0.05).(3)The mRNA level of C3 in mPFC was increased(P < 0.05)in susceptible mice,the C3 concentration of mPFC was also up-regulated in susceptible mice(P < 0.001).The protein expression of C3 in the mPFC of susceptible mice was increased significantly(P < 0.01).(4)Acute intraperitoneal injection of LPS induced depressive-like behaviors in the sucrose preference test and forced swim test.Compared with the control,the sucrose preference of susceptible mice was remarkably decreased(P < 0.001).The immobility time of LPS-treated mice(P < 0.01)in the FST was increased when compared with control.(5)The C3 concentration of LPS-injected mice was significantly up-regulated in the serum(P < 0.05)and mPFC(P < 0.001)by ELISA.(6)Pharmacological inhibition C3 in mPFC improved depressive-like behaviors.Acute bilateral infusions of C3 inhibitor in mPFC with a dose of 1 ?g/side,2 ?g/side,4 ?g/side,the dose of 4 ?g/side significantly decreased the immobility time in the TST(P < 0.05)and FST(P < 0.05),while there were no significant differences in the locomotor activity.(7)Acute bilateral infusions of compstatin significantly decreased the susceptibility to CSDS.As compared with the mice of PBS infusions,the proportion of susceptible mice in the group of C3 inhibitor infusions had a significant decrease,and the locomotor activity was not changed.Conclusion: CSDS and LPS stress induced depressive-like behaviors in susceptible mice.The mRNA and protein level of C3 in peripheral blood and mPFC of susceptible mice were increased significantly.Acute bilateral infusions of compstatin significantly decreased the susceptibility to CSDS.All results implied the depressive-like behaviors induced by CSDS or LPS were associated with C3 in peripheral blood and mPFC.Part ? The mechanism of C3 in stress-induced depressive-like behaviors in miceObjective: ? type complement receptor(CR3)was a member of adhesion molecules,belongs to the family of integrin,also a member of pattern recognition receptor which was microglia specific.Complement C3 was the ligand of CR3.It has been reported that CR3 bound to C3 could tag less active synapses and target them for elimination.It also has been reported that adult mice that knockdown CR3 in hypothalamus increased the density of synapses.However the underlying mechanism of depression remained unclear,little was known about the mechanism between the dysfunction of C3 and depression.In this part,we aimed to detect whether CR3 signal and microglia were implied in depression,and investigated the mechanism of C3 signal in chronic stress-induced depressive-like behaviors in mice.Methods: Establishing animal models of depression by exposing to CSDS or LPS stress;SPT,SIT and FST were utilized to detect the depressive-like behaviors after CSDS or LPS;Q-PCR,western blotting was performed to detect the gene and protein expression of CR3 in peripheral blood and brain of susceptible mice;CR3-sh RNA was used to specific knockdown the CR3 in mPFC,and detected the effect of CR3-sh RNA in the depressive behaviors induced by CSDS;Immunofluorescence was used to detect the expression of Iba1,gephyrin,psd95,synaptophysin in mPFC of mice after LPS and C3 inhibitor infusions;Antidepressants fluoxetine was utilized to treat the susceptible mice of CSDS,explored the effect of classic antidepressants fluoxetine in the depressive behaviors and the expression of C3 and CR3.Results:(1)CR3 mRNA level was significantly increased in the blood and mPFC of susceptible mice,while there were no significant difference in the CR3 gene expression of the hippocampus,amygdale and NAc.(2)The protein level of CR3 in the mPFC of susceptible mice was increased(P < 0.01).(3)The gene expression of CR3 was significantly increased(P < 0.05)in the blood of LPS-treated mice.Meanwhile,the protein level of CR3 in the mPFC of LPS-treated mice was up-regulated(P < 0.01).(4)Specific Knockdown CR3 in mPFC reversed depressive-like behaviors induced by CSDS.In the social interaction test,CR3 knockdown in the susceptible mice significantly increased the interaction ratio(P < 0.001).Meanwhile,time in the interaction zone with target of susceptible mice that received LV-CR3 sh RNA-injected was incread when compared with GFP-sh RNA group(P < 0.05).(5)Microglia in the mPFC was significantly activated by LPS injection.(6)LPS decreased synaptophysin density in mPFC of LPS-treated mice,infusions of C3 inhibitor reversed the decrease of synaptophysin density induced by LPS stress.(7)Antidepressants fluoxetine treatment reversed the behavioral deficiency induced by CSDS.Fluoxetine treatment decreased the immobility time of CSDS mice in TST(P < 0.001),and the interaction ratio of CSDS mice was increased by fluoxetine treatment(P < 0.001).Time in the interaction zone with target of CSDS mice was increased after fluoxetine treatment(P < 0.01).Western blotting detected the expression of CR3 and C3 showed that fluoxetine treatment could significantly reverse the expression level of C3(P < 0.01)and CR3(P < 0.001)in CSDS mice.Conclusion: The mRNA and protein level of CR3 in peripheral blood and mPFC of susceptible mice were increased significantly.Specific Knockdown CR3 in mPFC reversed depressive-like behaviors induced by CSDS.LPS activated microglia and decreased synaptophysin density in mPFC of LPS-treated mice,infusions of C3 inhibitor reversed the decrease of synaptophysin density induced by LPS stress.Antidepressants fluoxetine treatment of the susceptible mice significantly reversed depressive-like behaviors and reversed the expressions of C3 and CR3.All results implied that C3/CR3 signaling pathway was involved in the pathophysiology of major depression by C3-mediated synaptic pruning,provided new strategy for the treatment of depression.