The Role And Mechanism Of G Protein Coupled Receptor MrgprD In Promoting Lipopolysaccharide (LPS)-induced Inflammatory Pain

Aims:Bacterial infection or injection of lipopolysaccharide(LPS),a component of bacterial cell wall,can cause inflammatory pain.Although infection-induced pain is very common,but it has been received much less attention.The pathological mechanisms underlying such pain are not yet clear.At present,clinical treatments are not satisfactory due to significant side effects caused by commonly used analgesic drugs.Therefore,an unmet need exists for unveiling the mechaisms underlying the bacterial infection-induced pain and identifing new targets for the development of drugs.MrgprD is one member of the Mas-related G-protein-coupled receptor(Mrgpr)family,which is highly expressed in small-diameter sensory neurons of DRG Recently,MrgprD has been suggested to be involved in mechanical pain.However,the relationship between MrgprD and inflammatory pain remains elusive.In this study,we investigated the potential role of MrgprD in the LPS-induced neuro-inflammation and inflammatory pain.We also determined whether MrgprD is linked to the activation of NF-?B during the inflammatory process.Methods:In this study,we investigated the potential role of MrgprD in inflammation following the systemic inflammation generated by intraperitoneal injection of LPS(2.5 mg/kg).Using animal behavioral tests,Western blotting,immunohistochemistry and ELISA,we compared the differences of pain-associated behaviors and inflammatory responses in DRG tissues between wild type(WT)and Mrgprd knockout(Mrgprd-/-)mice.In addtion,we determined the effects of MrgprD overexpression on the activation of NF-?B signaling pathway.We also detected the impacts of MrgprD on the transcriptional activity of NF-?B subunit p65 by chromatin immunoprecitation(ChiP)assay and ELISA.Results:Intraperitoneal injection of LPS(2.5 mg/kg)induced an evident peripheral neuro-inflammation,the mechanical hyperalgesia as well as the increased expression of MrgprD in DRG.The LPS-induced hypersensitivities to mechanical and cold stimuli were substantially suppressed in Mrgprd knockout mice compared to WT littermates.The depletion of MrgprD also attenuated the LPS-induced production of pro-inflammatory cytokines and infiltration of macrophages in DRG tissues.Ectopic expression of MrgprD promoted the LPS-triggered activation of NF-?B,but not MAPKs.In addition,MrgprD overexpression significantly enhanced the transcriptional activity of NF-?B subunit p65 and the LPS-induced production of pro-inflammatory cytokine,TNF-?,in HEK293 cells stably overexpressed with TLR4.In summary,this study suggests that MrgprD actions as a novel regulator in NF-?B signaling and enhances the LPS-triggered inflammatory response in DRG neurons,thereby facilitating the development of inflammatory pain.