A Novel HDAC6 Inhibitor Ameliorates Imiquimod-Induced Psoriasis-Like Inflammation in Mice

Abstract

Psoriasis is a chronic inflammatory skin disease characterized by abnormal proliferation of keratinocytes and infiltration of inflammatory cells. Significant challenges remain in developing effective and safe targeted therapies for psoriasis. Here, we reported the discovery of novel cystamine derivatives for the treatment of psoriasis. These compounds effectively attenuated LPS-induced inflammation in vitro, and the optimal candidate CS1 ameliorated imiquimod-induced psoriasis-like inflammation in mice. Mechanistically, CS1 bound and inhibited the deacetylase HDAC6, subsequently inhibited the AKT, MAPK, and STAT3 pathways, attenuated the hyperproliferation and altered differentiation of keratinocytes and reduced the infiltration of immune cells. These findings suggest that HDAC6 may serve as a potential target for drug development in the treatment of psoriasis.

Keywords: HDAC6; MAPK; STAT3; inflammation; psoriasis.

Figure 1

Cystamine-based compounds effectively inhibit LPS-induced inflammation. (A) mRNA levels of iNOS in RAW264.7 cells were examined by qPCR. (B) CCK8 assay was performed to determine the cell viability of RAW264.6 cells treated with 4 hits. (C,D) CCK8 assays were performed to determine the cell viability of RAW264.7 and HACAT cells incubated with CS1 for 24 h. The values are presented as the mean ± SEM (n = 3 independent samples). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. CS1 (B) or CTRL (C,D). p-values were calculated using ordinary one-way ANOVA (B) and unpaired two-tailed Student’s t-test (C,D).

Figure 2

CS1 suppresses inflammatory stimuli-mediated cytokines and chemokine levels. (A–I) mRNA levels of inflammatory cytokines in RAW264.7 cells were examined by qPCR. (J–L) The levels of IL1β, IL6, and NO in the culture supernatant of RAW cells. The values are presented as the mean ± SEM (n = 3 independent samples). ## p < 0.01, ### p < 0.001, and #### p < 0.0001 vs. CTRL. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. LPS. p-values were calculated using ordinary one-way ANOVA.

Figure 3

CS1 intervention attenuates Imiquimod (IMQ)-induced psoriasis in Balb/c mice. (A) Mice were subjected to a daily dose of 62.5 mg of IMQ cream on the shaved back skin for one week. From day 8, mice were treated with PEG400 or CS1 for 3 days. At the end of the experiment, all mice were sacrificed for following assay. (B) Mice were photographed, and the representative photos were presented. (C) Mice were weighted every two days during days 1–7 and weighted daily during days 8–11. (D) The psoriasis area and severity index (PASI) score was recorded every two days during days 1–7 and calculated daily during days 8–11. (E) Hematoxylin and eosin (H&E) staining of mice back skin. (F,G) The epidermal thickness and dermal infiltrating cells were measured by Image J software. (H–J) mRNA levels of inflammatory cytokines in mice back skins were examined by qPCR. (K–O) The levels of cytokines and chemokines in the skin tissues. The values are presented as the mean ± SEM (n = 6 mice per groups). # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001 vs. CTRL. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. vehicle. p-values were calculated using unpaired two-tailed Student’s t-test (C,D) and ordinary one-way ANOVA (F–O).

Figure 4

CS1 reduces infiltration of macrophages and TCRγ/δ cells in skin. (A) Skin sections were stained with antibodies against F4/80 (green) and DAPI (blue). (B) Skin sections were stained with antibodies against TCRγ/δ (red) and DAPI (blue). Scale bar: 100 μm. The values are presented as the mean ± SEM (n = 4 mice skin samples per groups). #### p < 0.0001 vs. CTRL. *** p < 0.001 and **** p < 0.0001 vs. Vehicle. p-values were calculated using ordinary one-way ANOVA.

Figure 5

CS1 suppresses the expression of proliferation and differentiation markers in IMQ mice. (A–C) Immunofluorescence was performed to analyze the expression of KI-67, K17, and p-AKT in skin tissue sections. Scale bar: 100 μm. The values are presented as the mean ± SEM (n = 4 mice skin samples per groups). ## p < 0.01 and #### p < 0.0001 vs. CTRL. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. vehicle. p-values were calculated using ordinary one-way ANOVA.

Figure 6

CS1 inhibits the protein expression of the inflammatory signaling cascade. (A) Immunofluorescence was performed to analyze the expression of p-AKT in skin tissue sections. (B) Immunoblots of p38 and downstream signaling markers with CS1 treatment observed in skin tissues. The values are presented as the mean ± SEM (A) n = 4 mice skin samples per groups. (B) n = 3 mice skin samples per groups). ### p < 0.001 and #### p < 0.0001 vs. CTRL; * p < 0.5, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. CTRL. p-values were calculated using ordinary one-way ANOVA.

Figure 7

CS1 inhibits the activity of HDAC6. (A) Fluorescent probe for HDAC6 and HDAC1 enzyme activity. (B) Immunoblots of acetylated tubulin in RAW264.7 cells (left) and HACAT cells (right). (C) Cellular thermal shift assay was performed to determine the binding of CS1 and HDAC6. (D) Immunoblots of HDAC6 and acetylated-tubulin with CS1 treatment observed in skin tissues. The values are presented as the mean ± SEM (A) n = 3 independent samples; (D) n = 3 mice skin samples per groups). ## p < 0.01 and #### p < 0.0001 vs. CTRL; * p < 0.5, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. HDAC (A) or vehicle (D). Immunoblot results in (B,C) are representative of three independent experiments. p-values were calculated using ordinary one-way ANOVA (A,D) and unpaired two-tailed Student’s t-test (C).