Paeonol Ameliorates Chronic Itch and Spinal Astrocytic Activation via CXCR3 in an Experimental Dry Skin Model in Mice

Abstract

Paeonol is a bioactive phenol presents mainly in Paeonia suffruticosa Andr. (Paeoniaceae), Paeonia lactiflora Pall., and Dioscorea japonica Thunb. (Dioscoreaceae), harboring various pharmacological activities including anti-inflammatory, antioxidant, immune regulatory activity and reverse chemoresistance. Recent reports revealed paeonol exhibited good effects on chronic dermatitis, such as atopic dermatitis (AD) and psoriasis. However, whether paeonol is effective for dry skin disease and its mechanism of action still remain unclear. In this study, we analysed the effects of paeonol on a mouse model of dry skin treated with acetone-ether-water (AEW), which showed impressive activities in reducing scratching behavior and skin inflammation. To elucidate the underlying molecular targets for the anti-pruritic ability of paeonol, we screened the expression of possible chemokine pathways in the spinal cord. The expression of CXCR3 was significantly alleviated by paeonol, which increased greatly in the spinal neurons of AEW mice. In addition, treatment of paeonol significantly inhibited AEW-induced expression of astrocyte activity-dependent genes including Tlr4, Lcn2 and Hspb1 et al. The inhibitory effects of paeonol on scratching behavior and astrocytic activation in the spinal cord induced by AEW were abolished when CXCR3 was antagonized or genetically ablated. Taken together, our results indicated that paeonol can ameliorate AEW-induced inflammatory response and itching behavior, and reduce the expression of spinal astrocyte activity-dependent genes induced by AEW, which are driven by CXCR3.

Keywords: AEW; CXCR3; anti-pruritic; astrocyte; inflammation; paeonol.

FIGURE 1

Paeonol alleviated AEW-induced scratching behavior of dry skin mice. (A) The chemical structure of paeonol. (B) Schematic diagram of the animal experimental protocol. Spontaneous scratching of mice was videotaped and analysed. (C) The behavior results of different groups on day 7. Data were expressed as mean ± SEM. ^### p < 0.001, compared with the blank group. ^** p < 0.01, ^*** p < 0.001, compared with the AEW model (n = 6–7 each group). Pae-H, paeonol-high dose; Pae-M, paeonol-middle dose; Pae-L, paeonol-low dose; PC, positive control drug, HC-030031. The relevant labels in the following figures are the same unless otherwise mentioned.

FIGURE 2

Paeonol improved the pathological changes of dorsal neck skin in AEW mice. (A) The representative images of dorsal neck skin of each group on day 7. (B) The representative images of Haematoxylin and eosin (H&E) staining and the thickness of epidermis were measured. (C) The representative images of toluidine blue staining and the mast cell number were counted. Data were expressed as mean ± SEM. ^### p < 0.001, compared with the blank group; ^** p < 0.01, ^*** p < 0.001, compared with the AEW model group (n = 3 each group).

FIGURE 3

Paeonol reduced protein levels of pro-inflammatory cytokines in the serum of AEW mice. (A–C) Administration of paeonol decreased the levels of IL-1β (A), IL-4 (B), IL-6 (C) in the serum of AEW-induced mice. Data were expressed as mean ± SEM. ^# p < 0.05, ^## p < 0.01, ^#### p < 0.0001, compared to the blank group; ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, ^∗∗∗ p < 0.0001, compared to the AEW model group (n = 4–7 each group).

FIGURE 4

The effects of paeonol on the expression of chemokine receptors and ligands in the spinal cords. The increased mRNA levels of chemokine receptor Cxcr3 (A) and chemokines including Ccl6, Ccl9, and Ccl17 (B) in the spinal cord of AEW mice were decreased by paeonol application. Data were expressed as mean ± SEM. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, compared to the blank group; ^∗ p < 0.05, ^∗∗ p < 0.01, compared to the AEW model group (n = 4–8 each group).

FIGURE 5

The expression profile of CXCR3 in the spinal cord in the AEW-stimulated mice. The mRNA (A) and protein (B) level of CXCR3 in the spinal cord increased as stimulation of AEW went on (n = 3–7 each group). (C) Immunofluorescence co-staining images of CXCR3 with neuron marker (NeuN), microglial marker (CD11b) and astrocyte marker (GFAP) in the spinal dorsal horn of AEW mice (n = 4 each group). Data were expressed as mean ± SEM. ^∗∗ p < 0.01, compared to the Day 0 group.

FIGURE 6

Paeonol decreased the expression of CXCR3 in the spinal neurons of AEW group. (A) The results of immunofluorescence indicated paeonol downregulated the CXCR3⁺NeuN⁺ double-positive cell ratio in the AEW spinal cord (n = 4 each group). (B) The results of western blot demonstrated paeonol reduced the protein level of spinal CXCR3 in AEW model group (n = 4 each group). Data were expressed as mean ± SEM. ^## p < 0.01, ^### p < 0.001, compared to the blank group; ^∗∗ p < 0.01, compared to the AEW model group.

FIGURE 7

The effects of paeonol on chronic itch in AMG487 pre-treated WT and Cxcr3 ^−/− mice. (A) Schematic protocol of CXCR3 inhibition and induction of AEW-induced dry skin model. (B) Schematic protocol of AEW-induced dry skin model in Cxcr3 ^−/− mice. (C) The effects of paeonol on AEW-induced spontaneous scratching in WT mice pre-treated with AMG487. (D) The effects of paeonol on AEW-induced spontaneous scratching in Cxcr3 ^−/− mice (n = 7–9 each group). Data were expressed as mean ± SEM. ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, compared to the WT-AEW model group.

FIGURE 8

Paeonol suppressed the expression of astrocyte activity-dependent genes in the spinal cord of AEW mice. (A–H) Administration of paeonol inhibited the mRNA levels of genes related to reactive astrocytes (n = 5–9 each group). Data were expressed as mean ± SEM. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, compared to the blank group; ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, compared to the AEW model group.

FIGURE 9

The effects of paeonol on astrocyte activity-dependent genes in WT mice and Cxcr3 ^−/− mice. (A–H) The effects of paeonol on mRNA levels of genes related to reactive astrocytes in WT and Cxcr3 ^−/− mice (n = 5–9 each group). Data were expressed as mean ± SEM. ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, ^∗∗∗ p < 0.0001, compared to the WT-AEW model group.