The effect of kinin B1 receptor on chronic itching sensitization

Abstract

Background: Altered kallikrein-related peptidase activity and bradykinin are associated with skin disorders in humans and mice under chronic inflammation conditions. The bradykinin B1 receptor (B1R), also known as one of the G-protein-coupled receptor family and usually absent in intact tissues and upregulated during tissue injury, is responsible for vasodilation, capillary permeability, nociceptor sensitization, and pain; it is indispensable for physiopathological progress in chronic inflammation conditions, but its roles and effectors in the itching sensation of the allergic contact dermatitis model are poorly defined.

Results: We focused on incurable itching in a diphenylcyclopropenone (DCP) chronic inflammation experimental model. Preventive treatment with the B1R antagonist R892 significantly suppressed spontaneous scratching, while the B2R selective antagonist did not. B1R expression in the skin tissues of this model was detected using a quantitative, real-time polymerase chain reaction, Western blotting, and immunohistochemistry; B1R mRNA and protein levels were increased compared with a sham-treated control group. A higher B1R IHC staining signal was observed in the keratinocytes in DCP-treated mice compared with a vehicle-treated group, so we studied the B1R function when superimposed on a protease-activated receptor 2 (PAR2) background, establishing B1R as a pivotal mediator of PAR2 function in HaCaT cell lines.

Conclusion: Our data provide evidence that B1R facilitates the chronic itching sensation related to keratinocytes in a DCP-treated chronic inflammation experimental model.

Fig. 1

Scratching behaviors in 30 min of DCP-treated mice. Test substances were administered 30 min before observation. Graph showing increased spontaneous scratching of animal model in DCP-treated group (a). Preventive treatment with B1R antagonist R892 (b), kallikrein inhibitor NFM (c) significantly suppressed spontaneous scratching compared with sham-treated group (n = 8), and was significantly different from the vehicle group, while B2R antagonist HOE-140 (d) did not. *P < 0.05 as determined by the Student t test

Fig. 2

B1R is up regulated in skin tissues of DCP-treated group. Western blot (a) and RT-PCR (b) analysis of B1R in skin tissues obtained from sham (control) and DCP-treated mice. Protein expression (a) and PCR product (b) obtained with the specific B1R and β-tubulin antibodies or B1R and GAPDH primers. Insets (a upper panels) show a representative band of the protein expressed in both groups. Data were normalized against β-tubulin or GAPDH and expressed as the mean ± SEM of five independent mice. **P < 0.01 by the Student t test

Fig. 3

Immunofluorescence images of skin tissues in DCP-treated mice. Serial sections of post-fixed skin tissues were used (a) showing representative IF images with antibodies to B1R (green) and the neuronal marker PGP9.5 (red), litter level of B1R was detected in the PGP9.5 positive areas, B1R expression alone (green) in keratinocytes or some dermis areas (b). A representative IF image with antibodies to PGP9.5 was used to observe morphology of skin

Fig. 4

HE and IHC in mouse skin tissue. Sections of post-fixed mice skin tissues in DCP and vehicle-treated group immunostained with antibodies to B1R (a, b) showing that B1R staining signals were markedly increased in keratinocytes in DCP-treated mice. In photos of both groups, H&E staining was used to observe morphological and pathological changes

Fig. 5

B1R expressed in HaCaT cells on a PAR2 background. Positive immunocytochemistry reaction for B1R was observed and displaying membrane and cytosolic localization in HaCaT keratinocytes after 24-h incubation with vehicle (control), PAR2 agonist SLIGKV-NH2 (100 mM) (a). Representative images displaying nuclear localization of p65 (green) and DAPI (blue) in HaCaT keratinocytes after 24-h incubation with vehicle, SLIGKV-NH2 (100 mM) (c). Western blot of HaCaT cells following treatment with vehicle (control), SLIGKV-NH2 (100 mM). Samples were probed with antibodies against B1R and tubulin (b). B1R mRNA levels were also significantly augmented (d). Pretreatment with the NF-κB inhibitor PDTC (25 μM) prevented SLIGKV-NH2-induced B1R expression (b, d). Values represent mean ± SEM; *P < 0.05; **P < 0.01 by the Student t test