The Complement C3/C3aR Axis Regulates Rotenone-Induced Learning And Memory Impairments In Mice

BackgroundRotenone(Rot)is a plant insecticide widely used worldwide,mainly used to control insects,lice,and for lake and reservoir management.Studies have shown that long-term exposure to Rot will produce strong dopaminergic neurotoxicity,cause motor dysfunction,and increase the risk of Parkinson’s disease(PD),which is a potential environmental risk factor for PD.PD is the second largest neurodegenerative disease in the world,and its harm is serious.In addition to motor impairment,most patients with PD also have non-motor symptoms such as decreased learning and memory ability.Learning and memory ability are mainly regulated by the pruning of neural synapses by microglia,which is a key process that promotes synapse formation and regulates neuronal activity and synaptic plasticity.Recent research by our group found that Rot exposure can damage hippocampal neurons in mice,activate microglia-mediated neuroinflammatory responses,disrupt synaptic plasticity,and then impair the learning and memory ability of mice.However,the underlying mechanism by which Rot induces neuroinflammation and how neuroinflammation mediates neurodegeneration downstream are still unclear.The complement activation pathway plays a key role in the immune system.C3 is one of the intrinsic components of complement and the highest content of complement in serum.The lysate of C3,C3 a,can be coupled with C3 a receptor(C3aR)to activate microglia,induce the activation of immune mediators and the release of pro-inflammatory cytokines,and then participate in the neuroinflammatory reaction.Several studies have shown that the complement C3/C3 a R signaling pathway mediates microglial activation and neuroinflammatory responses,and is involved in neurodegeneration.However,whether the C3/C3 a R pathway is involved in Rot-induced neuroinflammation and learning and memory impairment has not been reported.ObjectivesIn this study,the role and mechanism of complement C3/C3 a R pathway in Rot induced neuroinflammation and learning and memory impairment were explored by combining C3 knockout(C3 Ko)mice and C3 a R inhibitor SB290157(C3aR inhibitor)in a Rot infected mouse model,which provided new ideas for elucidating the molecular mechanism of Rot neurotoxicity and related PD,and developing new therapeutic strategies.Methods1.Rot infected mouse model: adult male SPF-level C57BL/6 mice and C3 Ko mice were randomly divided into control group(Con)and Rot infected group(15animals/group each).Rot was infected with the virus by intraperitoneal injection at a dose of 1.5mg/kg,once a day for 21 days,and the mice in the Con group received the same amount of solvent.2.C3 a R inhibitor intervention model: adult male SPF-level C57BL/6 mice were randomly divided into four groups: Con,Rot,Rot+C3aR inhibitor and C3 a R inhibitor,with 15 animals in each group,and the Rot infection method was the same as above.C3 a R inhibitor was injected by intraperitoneal injection 30 minutes after Rot exposure,once a day(1mg/kg),for 21 consecutive days.Con group mice received the same amount of solvent treatment.3.C3 and C3 a R expression detection: The expression level of C3 and C3 a R in mouse hippocampus was detected by protein blotting and immunofluorescence double staining;The m RNA levels of C1 q,C3 and C3 a R in mouse hippocampus were detected by q RT-PCR.4.Mouse behavior test: Morris water maze experiment was used to detect changes in learning and memory function in mice.5.Detection of neurodegeneration and synaptic damage: immunohistochemistry and western blot analysis were carried out by anti-Neu N and PSD95 antibodies to detect neurodegeneration and synaptic damage in the hippocampus of mice.6.Detection of neurodegeneration and synaptic damage: immunohistochemistry experiments were performed using Iba-1 and GFAP antibodies to detect microglia and astrocytes activation,respectively;q RT-PCR was used to detect m RNA levels of inflammatory factors i NOS,TNF-αand IL-1β.7.Blood brain barrier(BBB)permeability detection:Fibrinogen protein blotting test was used to detect the changes of BBB permeability in mice,and the expression level of tight junction proteins(ZO-1,Occludin and Claudin-5)in mouse hippocampus was detected by protein blotting.8.Detection of apoptosis and necrotic apoptosis: Tunel staining was used to detect apoptosis;The protein expression of Caspase3,BCL-2 and Bax,as well as the phosphorylation levels of necrotizing apoptosis-specific indicators RIPK1,RIPK3 and MLKL,were detected by western blotting.Results1.Effect of Rot infection on C3 expression in the hippocampal region of mice:Western blot results showed that compared with the Con group,the expression of C3 protein in the hippocampal region of Rot infected mice was significantly increased,and the difference was statistically significant(p < 0.01);the results of ELISA kit showed that compared with the Con group,the expression of C3 a protein in the hippocampal region of Rot infected mice was significantly increased(p<0.05);the immunofluorescence double staining results showed that the C3 level of astrocytes in the hippocampal region of Rot infected mice was significantly increased;The q RT-PCR results showed that the m RNA levels of C3 and C1 q of Rot-infected mice were significantly increased compared with the Con group(p < 0.01).The above results showed that Rot infection increased the expression of C3 and C3 a proteins in the hippocampal of mice.2.Effects of C3 Ko on Neuronal Damage,Synaptic Loss and α-Synuclein Expression in Rod-induced mice: The immunohistochemical results showed that compared with the wild-type Con group,the number of neurons in the hippocampal region of the wild-type Rot infected group was significantly reduced,and the synapses were reduced.Compared with the wild-type Rot group,the number of neurons in the hippocampal region of mice in the C3 Ko Rot infected group increased significantly(p<0.01)and the synapses increased(p<0.05).The results of western blotting showed that the expression levels of Neu N and PSD-95 proteins in the wild-type Rot group decreased significantly.Compared with the wild-type Rot group,the expression of Neu N(p<0.01)and PSD-95(p<0.01)in the hippocampal region of mice in the C3 Ko Rot infected group was significantly increased.Compared with the wild-type Con group,the fluorescence intensity of Ser129-α-synuclein in the hippocampal area of the wild-type Rot infected group was significantly enhanced.Compared with the wild-type Rot group,the fluorescence intensity of Ser129-α-synuclein in the hippocampal region of the C3 Ko Rot infected group was significantly reduced(p<0.01).The results of western blot experiments showed that the expression of seapocampal Ser129-α-synuclein in the wild-type Rot infected group was significantly increased compared with the wild-type Con group.Compared with the wild-type Rot group,the expression of hippocampal Ser129-α-synuclein in the C3 Ko Rot infected group was still significantly reduced(p<0.01).3.Effect of C3 Ko on Rot induced learning and memory impairment in mice: The results of water maze test showed that the escape latency of mice in the Rot group was significantly longer than that in the C3 Ko Rot group(p<0.01);The distance traveled by the positioning platform increased significantly(p<0.01);The time of crossing the platform for the first time was significantly prolonged(p<0.05);The total number of crossing platforms decreased significantly(p<0.05);The results showed that C3 Ko could alleviate the learning and memory impairment in the hippocampus of mice induced by Rot.4.Effect of Rot on the expression of C3 a R in mice: Western blot showed that compared with Con group,the expression level of C3 a R in the hippocampus of mice exposed to Rot increased significantly(p<0.01);The results of q RT-PCR showed that compared with Con group,the content of C3 a R in Rot group increased(p < 0.01);Compared with Con group,the C3 a R intensity of microglia in hippocampus of mice exposed to Rot increased significantly(p<0.01),and the C3 a R intensity of neurons also increased significantly(p< 0.05).The above conclusions showed that exposure to Rot increased the expression of C3 a R in microglia and neurons in the hippocampus of mice.5.Inhibition of C3 a R on Rot-induced neuronal damage,synaptic loss and α-Effect of synaptic nucleoprotein expression: Immunohistochemical staining showed that compared with C3 a R inhibitor+Rot group,the number of neurons and synapses in the hippocampus of mice exposed to Rot decreased significantly(p<0.05);Western blot showed that the expression of Neu N and PSD-95 in the hippocampus of mice exposed to Rot was significantly lower than that in the C3 a R inhibitor+Rot group(p<0.05);The immunofluorescence results showed that compared with the Rot group,in the hippocampus of the C3 a R inhibitor+Rot group The fluorescence intensity of Ser129-α-synuclein decreased significantly(p < 0.01);Western blotting showed that compared with C3 a R inhibitor+Rot group,Rot group the expression of Ser129-α-synuclein in the mice hippocampussignificantly were still increased(p <0.05).6.Effects of C3 a R inhibitors on learning and memory impairment in Rot induced mice: The results of water maze test showed that the escape latency of mice in the Rot exposure group was significantly longer than that in the C3 a R inhibitor + Rot exposure group(p < 0.01);The travel distance of the positioning platform also increased significantly(p < 0.01);The time of crossing the platform for the first time was significantly prolonged(p<0.05);The total number of crossing platforms decreased significantly(p<0.05);The results showed that C3 a R inhibition alleviated the learning and memory impairment in the hippocampus of mice induced by Rot.7.Effect of C3/C3 a R axis on the activation of microglia and astrocytes in the hippocampus of mice induced by Rot: Immunohistochemical staining results showed that compared with Con group,the activation level of microglia and astrocytes in the hippocampus of mice exposed to Rot increased;Compared with the C3 Ko Rot group,the activation level of microglia and astrocytes in the hippocampus of mice exposed to Rot still increased(p < 0.05);Compared with the C3 a R inhibitor + Rot group,the activation level of microglia and astrocytes in the hippocampus of mice exposed to Rot still increased(p < 0.05).The results of q RT-PCR showed that the m RNA level of inflammatory factors in the hippocampus of mice exposed to Rot was significantly higher than that in Con group;Compared with the C3 Ko Rot group,the m RNA level of inflammatory factors in the hippocampus of mice exposed to Rot also increased significantly(p<0.01);Compared with the C3 a R inhibitor + Rot group,the m RNA level of inflammatory factors in the hippocampus of mice exposed to Rot also increased significantly(p<0.01).The above results indicate that inhibition of C3 Ko or C3 a R can improve the activation of microglia and astrocytes in the hippocampus of mice induced by Rot.8.Effect of C3/C3 a R axis on Rot induced increased hippocampal blood-brain barrier permeability in mice: Western blotting showed that compared with wild-type Rot infected group,hippocampal fibrinogen content in C3 Ko Rot infected group was also significantly decreased(p < 0.01);Compared with Rot infected group,hippocampal fibrinogen expression level of mice in C3 a R inhibitor + Rot infected group was also significantly increased(p < 0.05).Compared with Con group,the expression level of tight junction protein of blood-brain barrier in hippocampus of mice exposed to Rot decreased;Compared with the C3 Ko Rot group,the expression level of tight junction protein in the hippocampus of mice exposed to Rot increased significantly(p < 0.05);Compared with the C3 a R inhibitor + Rot group,the expression level of tight junction protein in the hippocampus of mice exposed to Rot increased significantly(p<0.05).These results suggest that regulating the C3/C3 a R axis mitigated Rot induced increased hippocampal BBB permeability in mice.9.Role of C3/C3 a R axis in neuronal apoptosis and programmed necrosis in Rot infected mice: Immunohistochemical staining showed that compared with wild-type Rot infected group,the number of apoptotic cells in hippocampus of C3 Ko Rot infected group was significantly decreased(p<0.01);Compared with Rot poisoning group,the number of apoptotic cells in hippocampus of mice in C3 a R inhibitor + Rot poisoning group was also significantly decreased(p<0.05).The detection of apoptosis by Western blot showed that compared with Con group,the expression levels of caspase3,BCL-2 and Bax in the hippocampus of mice exposed to Rot were significantly increased;The detection of cell necrotic apoptosis by Western blot showed that compared with the Rot group,the expression levels of P-RIPK1,P-RIPK3 and P-MLKL in the hippocampus of mice exposed to C3 Ko Rot were significantly lower.The results showed that the regulation of C3/C3 a R axis could reduce the occurrence of rot-induced neuronal apoptosis and necrotic apoptosis in mouse hippocampus.Conclusions1.Rot induced learning and memory dysfunction in mice with C3/C3 a R axis participation.2.The involvement of C3/C3 a R axis in Rot induced learning and memory impairment in mice may be related to activation of glial cells and induction of neuroinflammatory response.3.Rot regulated by C3/C3 a R axis induces apoptosis and necrotic apoptosis of mouse hippocampal neurons.