Spinal GRPR And NPRA Contribute To Chronic Itch In A Murine Model Of Allergic Contact Dermatitis

Background:Recurrent and refractory chronic pruritus has been a worldwide problem,but the underlying mechanisms of chronic pruritus,especially neural mechanisms,are still unclear,and the lack of a pathogenesis of persistent and recurrent pruritus hinders the emergence of effective therapies.Current treatments for chronic pruritus are limited to the use of antihistamines,local and systemic corticosteroids,or certain antiallergic drugs.However,the efficacy of these drugs is extremely limited,and systemic use of corticosteroids is also associated with many serious side effects.Therefore,there is an urgent need for a more effective and safe method of treating chronic pruritus.Recent research advances in itching have found that many cells,molecules,and neural circuits are involved in the development of chronic itching at multiple levels,from the skin to the spinal cord.At present,there are at least two peptides,gastrin-releasing peptide(GRP)and natriuretic peptide b(Nppb,also known as BNP),which are itch-transmitting specific neuropeptides.The cells expressing these two neuropeptide receptors are gastrin-releasing peptide receptor(GRPR)and natriuretic peptide receptor A(NPRA or NPR1),which are pruritus-specific neurons in the spinal cord.However,it is unclear whether and how they work in persistent and recurrent itching.Studies have shown that pruritus and pain have similar neural pathways,but the cellular and molecular mechanisms that mediate itching and pain are different.Therefore,in-depth exploration of the unique mechanisms related to pruritus can develop more reasonable and effective treatments for corresponding clinical treatments.Objective: To investigate the role of spinal GRPR and NPRA in the occurrence and development of chronic itch in allergic contact dermatitis model mice,and identify new targets for the development of drugs and other strategies to treat the myriad forms of chronic itch.Methods: 1.Establish a mouse model of allergic contact dermatitis(ACD)using dibutyl orthoester(SADBE),a small molecule hapten,to assess its itching and skin pathological changes and related immunological analysis.2.Quantitative PCR and multi-factor analysis of skin in SADBE mice.3.Compare the differences of chronic pruritus induced by SADBE in mice with intrathecal GRPR inhibitor group and intrathecal selective GRPR+ cell killing toxin(Bombesin-saporin)group with control mice.4.Compare the differences in chronic itch caused by SADBE between mice injected with intrathecal NPRA antagonists,selective NPR1+ cytotoxic toxin(BNP-saporin),and Npr1 KO mice compared with control mice.The levels of cervical DRG Nppb m RNA and spinal Npr1 m RNA in SADBE mice were analyzed total scratching bouts were counted and analyzed 5.Through RNA-scope and immunofluorescence techniques,analyze and compare the expression of GRPR and NPRA in the spinal cord of ACD model group and control group mice,and explore the correlation between spinal GRPR and NPRA activation on SADBE-mediated chronic itching pathway.6.RNAscope analysis of the expression of genes encoding histamine receptors Hrh1 and Hrh4 in the skin and DRG of ACD model mice;acute and chronic pruritus behavioral evaluation after H1 R antagonist and H4 R antagonist drug treatment.Results:1.Spontaneous scratching until 5 weeks after last SADBE treatment.Elevated serum Ig E and increased spleen weights.Quantitative analysis of enhanced epidermal thickness and increased mast cell infiltration in the dermis.Representative H&E staining,immunostaining of the epidermal marker KRT14,and toluidine blue staining of the neck skin sections.q PCR analysis showed m RNA levels of the indicated target genes in skin.*P < 0.05,**P < 0.01,***P < 0.001,compared with day 0.2.Increased Grpr levels 7 days after the last SADBE painting in C57 mice detected by RNAscope ISH or q PCR or in GRPR-e GFP mice detected with anti-GFP antibody.SADBE-induced prolonged itch in C57 mice decreased after i.t.GRPR antagonist or in Grpr KO or C57 mice after BB-sap(400 ng,i.t.).RNAscope ISH assay showed abolishment of Grpr+ neurons in BB-sap mice.Double RNAscope ISH of Grpr+ with Vglut2+ or Vgat+ in cervical spinal cord.**P < 0.01,***P < 0.001,t-test.3.Increased m RNA levels of Nppb in DRG and Npr1 in spinal cord at 3,7,and 21 days after the last SADBE treatment detected by q PCR analysis.RNAscope ISH assays of Nppb on frozen cervical DRG sections in ACD mice at 21 days after the last SADBE painting.Impaired SADBE-induced prolonged itch in C57 ACD mice after injection of the NPRA antagonist anantin,Npr1 KO mice or BNP-sap treated mice.Npr1 neurons were significantly ablated in the cervical spinal cord of BNP-sap treated mice,RNAscope ISH double staining for Npr1+ with Vglut2+ or Vgat+.4.It.injection of BNP induced scratching in mice 14 days after BB-sap or blank-sap treatment.i.t.injection of BNP,GRR induced scratching in mice 14 days after BNP-sap and blank-sap treatment.RNAscope staining showed that Grpr+ rarely colocalized with Npr1 neurons in the cervical spinal cord.Npr1 neurons were not significantly ablated in the BB-sap,and Grpr neurons were not significantly ablated in the BNP-sap mice.5.Representative immunostaining against histamine in the neck skin sections.RNAscope ISH assays of Hrh1 and Hrh4 on nape skin paraffin sections in the mice at 21 days after the last SADBE painting.RNAscope assays on frozen sections of cervical DRG with Hrh1 probes in SADBE mice at 21 days after the last SADBE painting.Double RNAscope staining of Hrh1 with Grp or Nppb or Grp with Nppb on frozen cervical DRG sections.(P < 0.05).6.Acute itch in C57 mice induced by histamine(500 mg,i.d.)but not by(b)GRP(0.1 nmol,i.t.)was significantly reduced after treatment with H1 R antagonist olopatadine(i.p.10 mg/kg)and H4 R antagonist JNJ7777120(i.p.30 mg/kg)or their coinjection.Chronic itch induced by SADBE in C57 mice or Grpr KO mice was significantly reduced by olopatadine and JNJ7777120 or their coinjection.Conclusions: 1.The activation of SADBE leads to the prolongation of chronic itching and the promotion of inflammation.2.GRPR relays SADBE-induced persistent itch in the spinal cord.3.NPRA relays SADBE-induced persistent itch in the spinal cord.4.GRPR+ and NPR1+expressing neurons in the spinal cord are two distinctive populations for itch signal processing.5.Both histaminergic and non-histaminergic pathways contribute to SADBE-induced prolonged itch.