The Effects And Mechanisms Of Chemokine CCL2 To Learning Memoryand Cognition In Rats

ObjectiveTo explore the effects of Chemokine CC motif ligand 2（CCL2）to learning memory and cognition in rats and the involvements in oxidative stress,inflammation,excitotoxicity and apoptosis.Our study provided a underlying lethal role of CCL2 in CNS pathogenesis.Methods1.200 male SD rats were equally divided into 4 batches（50 SD rats/batch）.Each batch was subsequently divided into 5 groups（10 SD rats/group）including control,sham,0.5ng CCL2,5ng CCL2 and 50ng CCL2.Except control group,each group was received stereotaxic surgery.The drug injection volume was2.5μL in one side,the injection speed was constantly 0.3μL·min^-1.Sham group was given the equal volume of Sterile saline.2.50 SD rats in one batch were used to behavior exploration.Morris water maze test was employed to assess the learning and memory from 3^rdd to8^thh days after surgery,then novel object recognitive test was performed to evaluate the cognitive level on 9^thh and 10^thh days,then quickly dissected the brain and removed the hippocampus above ice for further biochemical detection.3.The other three batches with a total of 150 SD rats were killed on day1,3 and 6 after CCL2 intra-hippocampal injection respectively for further biochemical detection.4.The oxidative commercial kits were used to assess the activity of SOD,GSH-Px and the content of MDA in hippocampus on different days.5.qRT-PCR was performed to detect the mRNA expression of following indexes on different days:（1）Inflammatory cytokines IL-1β,CXCL-10 and IL-6;（2）Excitatory glutamate metabolistic indexes GLAST,GLT-1 and PAG;（3）Apoptotic-associated protein caspase-8,caspase-3,Bax,Bcl-2.Results1.The results of navigation test of morris water maze showed that compared to sham group,the average swimming speeds among each CCL2group had no significance（P>0.05）,the average escape latency and average swimming distances were significantly higher（P<0.05）.In probe test,the percentage of distances in target quadrant,the percentage of time in target quadrant and crossing times among CCL2 groups were significantly lower than sham group（P<0.05）.2.In novel object recognition test,the discrimination indexes in each CCL2 group decreased which 5ng CCL2 and 50ng CCL2 groups showed significance in comparision to sham group（P<0.05）.3.The oxidative stress results showed that compared to sham group,the activity of SOD in 0.5ng CCL2 group significantly decreased on day6（P<0.05）while the content of MDA and the activity of GSH-Px failed to have any significance on different days.5ng CCL2 treatment groups showed significant descending SOD activity on day 1,3 and 6（P<0.05）,the content of MDA were significantly increased on day 3 and day 10（P<0.05）,the activity of GSH-Px were significantly ascended on day 6（P<0.05）.50ng CCL2 declined SOD activity among all time points（P<0.05）and significantly rose MDA content on day 3,6 and 10（P<0.05）while the activity of GSH-Px was increased on day 1,3 and 6 with significance（P<0.05）.4.The results of inflammatory-associated genes expression revealed that compared with the sham group,0.5ng CCL2-treated rats significantly increased IL-1βand CXCL-10 mRNA on day 3（P<0.05）while the expression of IL-6showed no significance among 4 time points（P>0.05）.5ng CCL2 group exhibited significant up regulation of IL-1βmRNA on day 3 and 6（P<0.05）as well as CXCL-10 mRNA on day 6（P<0.05）.The expression of IL-6 on day 1was down regulated with significant difference（P<0.05）.The IL-1βmRNA expression was ascended on day 3,6 and 10 with difference（P<0.05）and CXCL-10 expression was increased on day 6 and 10（P<0.05）in 50ng CCL2group.IL-6 expression was significantly declined on day 1（P<0.05）.5.The results of excitatory glutamate-associated genes showed that in comparision to sham group,0.5ng CCL2 group revealed an significant ascending expression of GLAST on day 3（P<0.05）,the expression of GLT-1and PAG failed to show any significant difference among 4 different days（P>0.05）.5ng CCL2 treatment rats presented high GLAST mRNA expression on day 3（P<0.05）while the expression of both GLT-1 and GLAST were decreased on day 6（P<0.05）,PAG mRNA expression was up regulated on day 6（P<0.05）.The results in 50ng CCL2 revealed an increasing expression of GLAST and GLT-1 on day 3（P<0.05）and decreasing expression on day 6（P<0.05）,PAG mRNA expression was increased on day 3,6 and 10（P<0.05）.6.The results of apoptosis-associated genes expression showed that 0.5ng CCL2 significantly induced caspase-8 and Bax on day 3（P<0.05）while caspase-3 was increased on day 6（P<0.05）,Bcl-2 and Bax/Bcl-2 ration exhibited no significance among 4 different days（P>0.05）.Both of caspase-8and caspase-3 were ascended on day 3,6 and 10 elicited by 5ng CCL2（P<0.05）,Bax mRNA was raised on day6（P<0.05）,yet Bcl-2 and Bax/Bcl-2 ration had no significance among 4 different days（P>0.05）.50ng CCL2 increased caspase-8and caspase-3 mRNA expression on day 3,6 and 10 with significance（P<0.05）,the Bax and Bax/Bcl-2 ration were up regulated on day 3 and 6（P<0.05）.Bcl-2failed to show significance on all different time points（P>0.05）.Conclusion:1.0.5ng-50ng CCL2 injured learning and memory of rats in a dose-dependent manner.2.0.5ng-50ngCCL2impairedcognitiveabilityofratswith dose-dependent manner.3.CCL2 induced oxidative stress in hippocampus with the prominent effects on 3^rdd to 6^thh days after CCL2 injection.4.CCL2 obviously induce the inflammatory cytokines IL-1βand CXCL-10 genes expression to participated inflammation on 3^rdd to 6^thh days which the results are consistent to oxidative stress outcomes.5.CCL2 down regulated the mRNA expression of GLAST,GLT-1 and increased PAG mRNA expression on day6 which linked to excitotoxicity.6.CCL2 promoted apoptotic-associated genes caspase-8,caspase-3,Bax expression and inhibited Bcl-2 expression with the prominent effects on day 6.