The Role of Transient Receptor Potential A1 and G Protein-Coupled Receptor 39 in Zinc-Mediated Acute and Chronic Itch in Mice

Abstract

Itching is a common symptom of many skin or systemic diseases and has a negative impact on the quality of life. Zinc, one of the most important trace elements in an organism, plays an important role in the regulation of pain. Whether and how zinc regulates itching is largely unclear. Herein, we explored the role of Zn²⁺ in the regulation of acute and chronic itch in mice. It is found that intradermal injection (i.d.) of Zn²⁺ dose-dependently induced acute itch and transient receptor potential A1 (TRPA1) participated in Zn²⁺-induced acute itch in mice. Moreover, the pharmacological analysis showed the involvement of histamine, mast cells, opioid receptors, and capsaicin-sensitive C-fibers in Zn²⁺-induced acute itch in mice. Systemic administration of Zn²⁺ chelators, such as N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), pyrithione, and clioquinol were able to attenuate both acute itch and dry skin-induced chronic itch in mice. Quantitative polymerase chain reaction (Q-PCR) analysis showed that the messenger RNA (mRNA) expression levels of zinc transporters (ZIPs and ZnTs) significantly changed in the dorsal root ganglia (DRG) under dry skin-induced chronic itch condition in mice. Activation of extracellular signal-regulated kinase (ERK) pathway was induced in the DRG and skin by the administration of zinc or under dry skin condition, which was inhibited by systemic administration of Zn²⁺ chelators. Finally, we found that the expression of GPR39 (a zinc-sensing GPCR) was significantly upregulated in the dry skin mice model and involved in the pathogenesis of chronic itch. Together, these results indicated that the TRPA1/GPR39/ERK axis mediated the zinc-induced itch and, thus, targeting zinc signaling may be a promising strategy for anti-itch therapy.

Keywords: GPR39; Itch; TRPA1; Zn2+; p-ERK.

Figure 1

Zn²⁺ evoked a scratching behavior in the neck and cheek models of mice. (A,D) The total number (A) and time course (D) of the scratching behavior induced by intradermal (i.d.) injection of ZnCl₂ (1–150 mM) in the nape of the neck in mice. (B,E) The total number (B) and time course (E) of scratching behavior induced by i.d. injection of Zn(CH₃COO)₂ (0.3–150 mM) in the nape of the neck in mice. (C,F) The total number (C) and time course (F) of the scratching behavior induced by i.d. injection of ZnSO₄ (1–150 mM) in the nape of the neck in mice (*P < 0.05, ***P < 0.001 vs. Saline, ^###P < 0.001 vs. 50 mM ZnCl₂, ^#P < 0.05 vs. 50 mM Zn(CH₃COO)₂, one-way AVOVA following post-hoc Bonferroni's test; n = 6–8 per group). (G,H) The total number of wiping (G) and scratching behavior (H) induced by i.d. injection capsaicin (10 μg) in the cheek in mice. (I,J) The total number of wiping (I) and scratching behavior (J) induced by i.d. injection ZnCl₂ (3 and 30 mM) in the cheek in mice (***P < 0.001 vs. Saline, unpaired Student's t-test; n = 6–8 per group). All data are expressed by means ± SEM. n.s., not significant.

Figure 2

Histamine, mast cells, opioid receptors, and capsaicin-sensitive C-fibers participate in ZnCl₂-induced itch in mice. (A) The effects of coadministration of a histamine H1 receptor antagonist chloropheniramine (300 μg) on histamine (800 μg)-induced itch in mice. (B) The effects of coadministration of chloropheniramine (300 μg) on ZnCl₂ (3 mM)-induced itch in mice. (C–E) The effects of coadministration of ZnCl₂ (3 mM) on histamine [800 μg; (C)]-, compound 48/80 [25 μg; (D)]-, chloroquine [50 μg; (E)]-induced itch in mice. (F) Pretreatment of compound 48/80 (200 μg) on ZnCl₂ (3 mM)-induced itch in mice. (G) The effects of systemic administration of morphine (1 mg/kg) and naloxone (1 mg/kg) on ZnCl₂ (3 mM)-induced itch in mice. (H) The effects of systemic administration of resiniferatoxin (RTX) on the latency time of tail flick response to hot water (52°C). (I) The effects of C-fiber depletion on ZnCl₂ (3 mM)-induced itch in mice. (J) After intraplantar injection of ZnCl₂ (50 mM, 20 μl) in the hind paw, the Von Frey test was used to detect the mechanical pain threshold in mice (*P < 0.05, **P < 0.01, ***P < 0.001 vs. Saline, unpaired Student's t-test; n = 6–9 per group). All data are expressed by means ± SEM. n.s., not significant.

Figure 3

Transient receptor potential A1 (TRPA1) is required for Zn²⁺-induced itch in mice. (A,B) The effects of coadministration of Ruthenium Red on ZnCl₂ (3 mM)-induced (A) and Zn(CH₃COO)₂ (3 mM)-induced itch (B) in mice (***P < 0.001 vs. Vehicle, one-way AVOVA following post-hoc Bonferroni's test; n = 6–9 per group). (C,D) The effects of coadministration of a TRPA1 blocker, A967079 on ZnCl₂ [(C); 3 mM]-induced and Zn(CH₃COO)₂ [(D); 3 mM]-induced itch in mice (***P < 0.001 vs. Vehicle, unpaired Student's t-test; n = 6–8 per group). (E,F) The effects of coadministration of a TRPA1 blocker, HC030031 (50 and 100 μg) on ZnCl₂- [(E); 3 mM] and Zn(CH₃COO)₂- [(F); 3 mM] induced itch in mice (*P < 0.05, ***P < 0.001 vs. Vehicle, one-way AVOVA following post-hoc Bonferroni's test; n = 6–9 per group). (G,H) The effects of coadministration of a TRPV1 blocker capsazepine on ZnCl₂ [(G); 3 mM]- and Zn(CH₃COO)₂ [(H); 3 mM]-induced itch in mice. (I,J) The effects of coadministration of a TRPV4 blocker, HC067047 on ZnCl₂ [(I); 3 mM]- and Zn(CH₃COO)₂ [(J); 3 mM]-induced itch in mice (Unpaired Student's t-test; n = 6–8 per group). All data are expressed by means ± SEM. n.s., not significant.

Figure 4

Transient receptor potential A1 (TRPA1) was involved in Zn²⁺ induced itch in mice, but TRPV1 and TRPV4 were not involved. (A,B) ZnCl₂ [(A); 3 mM]-evoked and Zn(CH₃COO)₂ [(B); 3 mM]-evoked acute itch were reduced in Trpa1^−/− mice. (C,D) ZnCl₂ [(C); 3 mM]-evoked and Zn(CH₃COO)₂ [(D); 3 mM]-evoked acute itching were not expressed in Trpv1^−/− mice. (E,F) ZnCl₂ [(E); 3 mM]-evoked and Zn(CH₃COO)₂ [(F); 3 mM]-evoked acute itch were also not expressed in Trpv4^−/− mice (***P < 0.001 vs. WT mice, unpaired Student's t-test; n = 6–8 per group). All data are expressed by means ± SEM. n.s., not significant.

Figure 5

Zinc chelators attenuated acute itching behavior in mice. (A–C) The effects of intraperitoneal (i.p.) injection of TPEN (1–10 mg/kg) on ZnCl₂ [(A); 3 mM], compound 48/80 [(B); 100 μg], and chloroquine-induced itch [(C); 200 μg] in mice. (D–F) The effects of pyrithione (5–10 mg/kg; i.p.) on ZnCl₂ [(D); 3 mM], compound 48/80 [(E); 100 μg], and chloroquine-induced itch [(F); 200 μg] in mice. (G–I) The effects of clioquinol (5–10 mg/kg; i.p.) on ZnCl₂ [(G); 3 mM], compound 48/80 [(H); 100 μg], and chloroquine-induced itch [(I); 200 μg] in mice (*P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle, one-way AVOVA following post-hoc Bonferroni's test; n = 6 per group). (J) Open field test was performed after systemic administration of TPEN (10 mg/kg), pyrithione (10 mg/kg), and clioquinol (10 mg/kg) in mice. (K) Rotarod test was performed after i.p. injection of TPEN (10 mg/kg), pyrithione (10 mg/kg), and clioquinol (10 mg/kg) (*P < 0.05, ***P < 0.001 vs. Vehicle, unpaired Student's t-test; n = 6–7 per group). All data are expressed by means ± SEM. n.s., not significant.

Figure 6

The effects of the administration of three Zn²⁺ chelating agents on dry skin chronic model. (A) The establishment of dry skin-induced chronic itch model. (B) The three Zn²⁺ chelators, such as TPEN (3 mg/kg), pyrithione (5 mg/kg), and clioquinol (5 mg/kg) can significantly inhibit the chronic itching behavioral response of mice induced by dry skin (***P < 0.001 vs. Ctrl, ^##P < 0.01, ^###P < 0.001, ^$$$ P < 0.001, ^&&&P < 0.001 vs. AEW + Vehicle, two-way ANOVA following post-hoc Bonferroni's test; n = 6–10 per group). (C) The mRNA expression levels of Trpa1, Trpv1, and Trpv4 in the DRG of dry skin induced chronic itch model (*P < 0.05, **P < 0.01 vs. Ctrl, unpaired Student's t-test; n = 5 per group). (D) The effects of treatment with Zn²⁺ chelators on the epidermal thickness of the dry skin induced chronic itch model. (*P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctrl, unpaired Student's t-test; n = 5 per group). (E) The epidermal thickness statistics of the dry skin model group and the treatment group (***P < 0.001 vs. Ctrl, ^###P < 0.001 vs. AEW + Vehicle, unpaired Student's t-test; n = 4 per group). (F) Zn²⁺ chelators pyrithione (5 mg/kg) and clioquinol (5 mg/kg) can decrease Trpa1 mRNA level, but not for TPEN (3 mg/kg) (*P < 0.05, ***P < 0.001 vs. AEW, unpaired Student's t-test; n = 5 per group). All data are expressed by means ± SEM. Ctrl, control; n.s., not significant.

Figure 7

The expression of ZIPs/Slc39as and ZnTs/Slc30as in the DRG of dry skin induced chronic itch model. (A) The gene expression of ZIPs and ZnTs family and itch marker in the DRG neuron of mouse based on previous published single cell RNA-seq database. (B,C) The mRNA expression levels of (B) Slc39as and (C) Slc30as in the DRG of WT mice. (D,E) The mRNA expression levels of (D) Slc39as and (E) Slc30as in the DRG of dry skin induced chronic itch model (*P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctrl, unpaired Student's t-test; n = 5 per group). All data are expressed by means ± SEM. Ctrl, control; n.s., not significant.

Figure 8

Activation of extracellular signal-regulated kinase (ERK) signaling was involved in Zn²⁺-mediated acute and chronic itch in mice. (A,B) Western blots (upper panel) and quantification (lower panel) shows that p-ERK expression is significantly increased at 10 and 30 min in DRG (A) and skin (B) after i.d. injection of ZnCl₂(3 mM) (*P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctrl, unpaired Student's t-test; n = 4). (C) Intrathecal injection of U0126 (1 nmol) decreased the ZnCl₂-induced scratching behavior in mice by 3 mM (**P < 0.01 vs. Saline, unpaired Student's t-test; n = 6). (D,E) Western blots (upper panel) and quantification (lower panel) show that the expression of p-ERK in DRG (D) and skin (E) of dry skin induces chronic itch model (**P < 0.01, ***P < 0.001 vs. Ctrl, ^##P < 0.01, ^###P < 0.001 vs. AEW + Vehicle, unpaired Student's t-test; n = 4 per group). All data are expressed by means ± SEM. Ctrl, control; n.s. not significant.

Figure 9

GPR39 was possibly involved in dry skin-induced chronic itch in mice. (A) The RT-PCR results showed that GPR39 is expressed in the spinal cord and the skin of mice, and only little in the DRG. (B) A GPR39 agonist, TC-G-1008 (1–100 μg) was not able to evoke acute itch in mice (one-way AVOVA following post-hoc Bonferroni's test; n = 6–8). (C) TC-G-1008 (25 μg) significantly increased AEW-induced chronic itch in mice (***P < 0.001 vs Ctrl, ^##P < 0.01 vs. AEW, two-way ANOVA following post-hoc Bonferroni's test; n = 8). (D) The expression of Gpr39, Il-6, Il-33, and Tslp in the skin of dry skin induced chronic itch model (*P < 0.05, ***P < 0.001 vs. Ctrl, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. AEW + Vehicle, unpaired Student's t-test; n = 5 per group). (E) In HaCaT cells, the mRNA expression levels of Gpr39 were significantly upregulated after ZnCl₂ (100 μM) incubated for 0.5 and 1 h. (F) In HaCaT cells, the protein expression levels of p-ERK were significantly upregulated after ZnCl₂ (100 μM) incubated for 0.5 h (*P < 0.05, ***P < 0.001 vs. Ctrl, one-way AVOVA following post-hoc Bonferroni's test; n = 5–6 per group). All data are expressed by means ± SEM. Ctrl, control; n.s. not significant.