Up-regulation Of NR2B Involved In Cathepsin C-induced Microglia Toward To M1 Activation Status

Background:Microglia?MG?are the primary immune competent and phagocytic cells inside the central nervous system?CNS?,which are broadly viewed as CNS counterparts to peripheral monocyte and macrophages.As the protector of CNS,MG is the earliest responder to CNS injury to brain injury and disease,which switch to activation status quickly.It is well known that MG can alter their phenotypes and functions in response to micro-environmental disturbances.Depending on the predominance of secreted factors,MG have been characterized to two activation phenotypes: pro-inflammatoryM1 activation phenotype or the anti-inflammatory-M2 activation phenotype."Classically activated" M1-phenotype MG can resists environmental changes by secreting pro-inflammatory cytokines against pathogens and tumor cells in the host defense process and may cause tissue and cellular damage.The "alternative-activated" M2-phenotype MG can alleviates inflammatory response by releasing antiinflammatory cytokines.And M2-phenotype MG to diminished injury and promote repair by generating repair-related factors.Normally,the pro-and anti-inflammatory responses need to be balanced to prevent the potential detrimental effects of a prolonged,unregulated inflammation.Hence,controlling MG activation status is important to alleviate neuroinflammation.Cathepsin C?CatC?,also known as dipeptidase I?DPPI?,is a member of lysosomal cysteine protease.CatC has dipeptidyl peptidase activity and can participate in the peripheral inflammatory response by activating serine protease,cathepsin G and protease 3.However,it has been gradually confirmed that CatC is expressed in the CNS and participants in neuroinflammatory diseases.We found that CatC,expressed in activated MG in the brain,is involved in LPS intraperitoneal injection and proinflammatory factors-induced neuroinflammation.Then,we found that CatC OE aggravates neuroinflammation in LPS-induced WT,CatC OE?CatC Overexpression?and CatC KD?CatC Knockdown?genotype mice.And it is achieved by promoting MG to M1-activation status,which was found in further in vitro experiments.However,the mechanisms of CatC promotes MG switch to M1 activation status are still unclear.Objective:This article aims to elucidate the molecular mechanisms of CatC promotes MG switch to M1 activation status.This not only provides further insight into the genetic function of CatC,but also provides a new target for the treatment of CNS neuroinflammation.Methods: In the present study,we used Microarray-Based Gene Expression Analysis to screen and analyze differentially expressed genes in primary cultured MG with active CatC monomer stimulation.According to the results,NR2 B was selected as the main molecule.Then,the expression of NR2 B,change of NR2B-mediated intracellular Ca²⁺ concentration and activation of Ca²⁺-dependent PKC/p38 MAPK/NF-?B signal transduction pathway were analyzed by q RT-PCR,flow cytometry and Western blot.All data are expressed as Means ± SEM of triplicate experiments.For all tests,p? 0.05 was considered statistically significant.Result:1.Microarray-Based Gene Expression Analysis showed that,there were 254 differentially expressed genes in CatC-stimulated primary culture MG,including 103 up-regulated genes and 151 down-regulated genes,compared with unstimulated group.The q RT-PCR results validated that six selected up-regulated genes were up-regulated in CatC-stimulated primary culture MG.2.CatC stimulation increased level of mRNA,protein expression and phosphorylation of NR2 B both in primary culture MG and BV2 cells.NR2B expression and phosphorylation level were decreased after cathepsin inhibitor E-64 stimulation.3.The concentrations of Ca²⁺ were increased in CatC-stimulated BV2 cells.And the level of Ca²⁺ was decreased in BV2 cells with E-64 co-stimulation and NMDA inhibitor MK-801 pre-treatment.4.PKC was phosphorylated in CatC-stimulated primary culture MG and BV2 cells.The level of PKC phosphorylation was decreased in CatC-stimulated MG after E-64 co-stimulation and NMDA inhibitor MK-801 pre-treatment.5.The key molecules of MAPKs/NF-?B signaling pathway?p38,I?B?,p65?were activated in both CatC-stimulated primary cultured MG and BV2 cells.And the ratio of phosphorylation/total p38,I?B?,p65 were increased;the bands of p38,I?B?,p65 phosphorylation were reduced and the ratio of phosphorylation/total p38,I?B?,p65 were decreased in the groups with E-64 co-stimulation and MK-801 pretreatment.Conclusions: CatC induces NR2 B up-regulation and increases cytoplasmic Ca²⁺ concentration in MG followed by the activation of intracellular Ca²⁺-dependent PKC/p38 MAPK/NF-?B signaling pathway which promote MG towards M1 polarization.