Hippocampal CA1 ?CaMK? Mediates Neuroinflammatory Responses Via COX2-PGE2 Signaling Pathways In Depression

Background:Depression is a neurological disorder characterized by persistent low mood and is one of the neurological and psychiatric diseases that seriously endanger human health.The onset of depression is affected by multiple factors such as mental health?genetics?biology?and society.Recent studies have found that when the body is stimulatedbyexternal stress,it usually occurs with the occurrence of neuroinflammation in specific brain regions,but the mechanism of neuroinflammatory reaction in the pathogenesis of depression is still unresolved.Therefore,this study intends to construct a chronic unpredictable mild stress stimuli(CUMS)rat model,using behavioral,molecular biology and morphological methods to study whether ?CaMKII in hippocampal CA1 region participates in the regulation of COX2-PGE2 signaling pathway.Neuroinflammatory response and explore its role and mechanism in the pathogenesis of depression.Methods:(1)Male Wistar rats(140g-160g)were randomly allocated to one of the following groups:Control(non-stressed group),CUMS,CUMS treated with COX-2 inhibitorcelecoxib(Celecoxib+CUMS,20mg/kg),CUMS treated with fluoxetine(Fluoxetine +CUMS,40 mg/kg).Except for the control group,the other threegroups were given a chronic unpredictable mild stress for 5 weeks.Each time,the rats in the drug pretreatment group were administered via an intraperitoneal(i.p.)injectionat 30 min prior to CUMS procedure daily with Celecoxib and Fluoxetine.Behavioral tests such as forced swimming test and sucrose preference test were performed after the 5 weeks of cums expoure.(2)The expression of COX-2 and ?CaMKII and the phosphorylationlevel of ?CaMK? protein were detected by Western Blot.The content of PGE2 was detected by ELISA.The number and morphology of microglia and astrocytes were detected by laser confocal microscope.(3)The expression of Nrf-2 and HO-1 was detected by Western Blot;the enzyme activity of SOD,CAT and T-AOC was detected by kit,the content of product was detected by MDA and NO kit,and the change of enzyme content was determined by LDH kit;The levels of ROS and DNA oxidative damage of DHE.?4-HNE and 8-OHdG were detected by laser confocal microscope.(4)We constructed overexpression or knockdown of adeno-associated virus CaMKIItargeting the hippocampal CA1 region for stereotactic injection.After behavioral test,part of the rats from the non-stressed group and depressed group were randomlyallocated to one of the following groups:(a)Wild type(WT);(b)CUMS;(c)CUMS+AAV-Control;(d)CUMS+AAV-?CaMK? RNAi;(e)WT+AAV-control;(f)WT+AAV-?CaMK?.Two weeks after virus infection,behavioral tests such as forced swimming test and sucrose water preference test were performed;Western Blot was used to detect the expression of p3 8?p-p3 8?p-CREB?p-ATF2?COX-2;qPCR was used to detect COX-2mRNA levels;Concentrations of PGE2 were measured using Elisa kits.(5)The depressed group were randomlyallocated to one of the following groups:(a)Control(non-stressed group);(b)CUMS;(c)SB203580 treatment followedby CUMS(SB203580+CUMS,10 ?g/kg);(d)DMSO treatmentfollowed by CUMS(DMSO+CUMS,1%).Rats were injected with SB203580 or 1%DMSO in the lateral ventricle 30 minutes before each stress stimulation.After 5 weeks,behavioral tests such as forced swimming test and sucrose preference test were performed.Western Blot,qPCR and other techniques were used to detect changes in related indicatorsResults:(1)Behavioral test results showed that after 5 weeks of CUMS exposure,there are increased immobility times anddecreased swimming times as compared to thenon-stressed control group(P<0.05),and the sucroseconsumption wassignificantly reduced in the sucrose preference test(P<0.05),suggesting that the rats produce depression-like behaviors such as behavioral despair and loss of pleasure.(2)Western Blot results showed that compared with the control group,the levels of COX-2 and?CaMK?phosphorylation and PGE2 content in hippocampal CA1 area of CUMS group were increased;Laser confocal microscope results showedthat the number of microglia and astrocytes cells in the CA1 region was significantly increased,CUMS exposure significantly increased mRNA levels of IL-1?,TNF-? and IFN-?.Celecoxib treatment effectively inhibited the occurrence of neuroinflammation and significantly improved the depression-like behavior in rats.(3)Compared with the blank control group,the results of Western Blot showed that the expression of Nrf-2 and HO-1 in the CUMS group was decreased,and the enzyme activities of SOD,CAT and T-AOC was decreased.At the same time,the production of MDA and NO was increased,and the content of LDH was increased.The results of laser confocal microscopy showed that DHE and 4-HNE staining were enhanced,ROS levels were significantly increased,and DNA damage was enhanced in 8-OHdG,suggesting that oxidative stress was enhanced in depression-like rats.(4)After the AAV-CaMK? overexpression was injected into the hippocampal CA1 regions of control rats,Western Blot results showed that ?CaMK?overexpressionproduced an overall statistically significant increase in the expression of p38?p-p38?p-CREB?p-ATF2?COX-2.(5)After injection of RNA interference(RNAi)form of ?CaMKII(AAV-?RNAi)in hippocampal CA1 area of depression rats,Western Blot results showed that AAV-?RNAiproduced a significant decrease in the expression of p38?p-p38?p-CREB?p-ATF2?COX-2.(6)Compared with the CUMS group,pretreatment with SB203580 significantly reduced theexpressionof p-CREB?p-ATF2?COX-2 and other related proteins,and significantly inhibited the occurrence of depression-like behavior in rats.Conclusion:(1)CUMS can effectively induce the depression-like behavior in rats,accompanied by an increase in the expression of ?CaMK? in the hippocampal CA1 region of rats.(2)?CaMK? inhibits neuroinflammatory and oxidative stress induced by chronic stress stimulation via regulating p38-COX2-PGE2 signaling pathway,thereby improving the occurrence of depressive symptoms.