The Mechanism Of CLCA1 Mediates Inflammatory Pain Via Regulating The Expression Of CaMKⅡα In LHb

Background:Inflammatory pain is one of the most common clinical complaints,which seriously affects the quality of life and work of patients.The molecular mechanism of inflammatory pain is still not fully understood,and the exploration of effective therapeutic targets for inflammatory pain is still a major challenge.The lateral habenula（LHb）is one of the brain regions involved in the regulation of pain.It receives direct and indirect pain signals from the spinal cord and is a hotspot of current research.Calcium-activated Chloride channel regulator 1（CLCA1）,a member of the CLCA family,is a metalloproteinase that activates Ca2⁺dependent Cl^-channels in epithelial tissues.Calcium/calmodulin-dependent protein kinase Ⅱα（CaMKⅡα）is a multifunctional serine/threonine protein kinase.CaMKⅡαis a major subtype of Ca MKⅡ and it is crucial in synaptic plasticity,which is the core of synaptic signaling and morphological regulation.CaMKⅡαplays an important role in the transmission of nociceptive information,and the up-regulation of CaMKⅡαexpression at the synaptic site promotes the formation of nociceptive sensitization.In this study,we established a complete Fredrickson adjuvant（CFA）inflammatory pain model and found that CLCA1is involved in pain regulation in the LHb region of mice.In addition,we found that CaMKⅡαexpression in this region is upregulated during inflammatory pain.Along this line of thought,we shifted our research focus to the association between CLCA1 and CaMKⅡαand its mechanism in inflammatory pain.Objective:To explore the relationship between CLCA1 and CaMKⅡαin LHb and its mechanism in inflammatory pain.Methods:C57BL/6J mice（8 weeks of age）were divided into Sham operation group（Sham）,inflammatory pain group（CFA）,inflammatory pain+lentivirus group（CFA+CLCA1-LV）and inflammatory pain+negative control virus group（CFA+NC-LV）,with 5 mice in each group.Mice in CFA+CLCA1-LV group and CFA+NC-LV group were microinjected with lentivirus and negative control virus in the right LHb region one week before the experiment.On the beginning day of the experiment,CFA inflammatory pain model was established in all mice except the Sham operation group,and CFA model was replaced by 0.9%sodium chloride solution in Sham group.Paw withdrawal mechanical Threshold（PWT）was measured before model built and 1,3,5 and 7 days after model establishment.On the 7th day after model establishment,tissues needed for the experiment were sampled,and the expression changes of CLCA1 and CaMKⅡαin LHb region of mice in each group were detected by western blot and RT-PCR.Combined with behavioral experiment,CLCA1 and CaMKⅡαwere confirmed to be involved in the formation of inflammatory pain.The association between CLCA1 and CaMKⅡαwas confirmed by IHC.Results:1.The PWT of inflammatory pain mice was decreased,CLCA1 expression in LHb region was significantly down-regulated while the expression of CaMKⅡαwas significantly increased;2.The CLCA1 and CaMKⅡαwere co-localized in neurons of the LHb region;3.After overexpression of CLCA1 in the LHb region of mice with lentivirus,PWT was significantly increased compared with the negative control group,and the expression of CaMKⅡαwas significantly down-regulated with the up-regulation of CLCA1.Conclusions:1.CLCA1 is involved in the development of CFA-induced inflammatory pain;2.CLCA1 mediates the formation and development of inflammatory pain by regulating the expression of CaMKⅡα.