Datura Metel L. Ameliorates Imiquimod-Induced Psoriasis-Like Dermatitis and Inhibits Inflammatory Cytokines Production through TLR7/8-MyD88-NF-κB-NLRP3 Inflammasome Pathway

Abstract

Background: Psoriasis is a chronic, immune-mediated inflammatory skin disease, and the inflammatory response plays an important role in its development and progression. Datura metel L. is a traditional Chinese medicine that exhibited a significant therapeutic effect on psoriasis in our previous study due to its remarkable anti-inflammatory effect. Meanwhile, the mechanism underlying its effects on psoriasis is still unclear.

Methods: An imiquimod-induced psoriasis-like dermatitis mouse model was constructed to evaluate the protective effect of the effective part of Datura metel L. (EPD), which was verified by evaluations of the Psoriasis Area and Severity Index (PASI) score. Hematoxylin and eosin (H&E) staining, immunohistochemical examination, enzyme-linked immunosorbent assay (ELISA), and Western blot were used to measure the inflammatory cytokines and the protein expression associated with the Toll-like receptor 7- myeloid differentiation primary response gene 88-nuclear Factor-κB-nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3 (TLR7/8-MyD88-NF-κB-NLRP3) inflammasome pathway.

Results: EPD significantly decreased the PASI, reduced epidermal thickness, and decreased the proliferation and differentiation of epidermal cells in psoriasis-like dermatitis C57BL/6 mice induced by imiquimod (IMQ). Furthermore, EPD reduced the infiltration of CD3+ cells to psoriatic lesions, as well as ameliorated the elevations of intercellular adhesion molecule 1 (ICAM-1) and inhibited the production of imiquimod-induced inflammatory cytokines, including IL-1β, IL-2, IL-6, IL-10, IL-12, IL-17, IL-22, IL-23, tumor necrosis factor-α (TNF-α), monocyte chemotactic protein 1 (MCP-1), and interferon-γ (IFN-γ). Besides, EPD decreased the imiquimod-induced expression levels of TLR7, TLR8, TRAF6, MyD88, p-IKKα, p-IKBα, p-NF-κB, NLRP3, apoptosis-associated speck-like protein contained a caspase recruitment domain (ASC), cysteinyl aspartate specific proteinase 1 (caspase-1), and IL-1β.

Conclusion: This study demonstrated that EPD exhibited a protective effect on an imiquimod-induced psoriasis mice model by inhibiting the inflammatory response, which might be ascribed to the inhibition of the TLR7/8-MyD88-NF-κb-NLRP3 inflammasome pathway.

Keywords: Datura metel L.; imiquimod; inflammatory cytokines; psoriasis; toll-like receptor 7/8.

Figure 1

Effective part of Datura metel L. (EPD) improved the morphological and histological features of imiquimod (IMQ)-induced psoriasis dermatitis in mice. (A) Representative macroscopic views of the dorsal skin of C57BL/6 mice following continuous treatment for seven days. (B) Scaling, thickness, and erythema of the back skin was scored daily on a scale from 0 to 4. Additionally, the cumulative score (scaling plus thickness plus erythema) was depicted. (C) Histological evaluation of the back skin of IMQ-induced psoriasis-like mice (staining with hematoxylin and eosin, i.e., H&E; magnification 200×, scale bars indicate 50 μm). (D) Epidermal thickness of the dorsal skin on day 8. Data are represented as mean ± SD, n = 8. ** P < 0.01 vs. control group, ^## P < 0.01 vs. model group.

Figure 2

EPD inhibited the epidermal cell proliferation and differentiation of IMQ-induced psoriasis dermatitis mice models. (A) Expression of proliferating cell nuclear antigen (PCNA) and Ki-67 in each group were detected by immunohistochemistry (200×). (B,C) Average optical density (AOD) of PCNA and Ki-67 in each group. (D) The protein expressions of involucrin were detected using Western blot assay. (E) Quantification of protein levels of involucrin. Values are presented as the means ± SD (n = 3). ** P < 0.01 vs. control group, ^## P < 0.01 vs. model group.

Figure 3

EPD inhibited inflammatory cell infiltration in IMQ-induced psoriasis-like mouse mode. (A) The expression of intercellular adhesion molecule 1 (ICAM-1) and CD3 in each group were detected by immunohistochemistry (200×). (B) AOD of PCNA in each group. (C) Quantification of CD3-positive cells in skin tissues. Values are presented as the means ± SD (n = 3). ** P < 0.01 vs. Control group, ^## P < 0.01 vs. Model group.

Figure 4

The concentrations of inflammatory cytokines in the dorsal skin of IMQ-induced psoriasis dermatitis in mice were measured with the corresponding ELISA kits. Vales are mean ± SD (n = 8 mice per group). ** P < 0.01 vs. control group, ^# P < 0.05, ^## P < 0.01 vs. model group.

Figure 5

EPD suppressed the activation of the Toll-like receptor 7–myeloid differentiation primary response gene 88–nuclear factor-κB (TLR7/8–MyD88–NF-κB) signaling pathways. (A) The protein expressions of TLR7, TLR8, TRAF6, MyD88, p-IKKα, IKKα, p-IKBα, IKBα, p-NF-κB, and NF-κB were detected using Western blot assay. (B–H) The quantification of protein levels of TLR7, TLR8, TLAF6, MyD88, p-IKKα, IKKα, p-IKBα, IKBα, p-NF-κB, and NF-κB. Values are presented as the means ± SD of three independent experiments. ** P < 0.01 vs. control group, ^# P < 0.05, ^## P < 0.01 vs. model group.

Figure 6

EPD inhibited the IMQ-activated nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome signaling pathway. (A) The protein expressions of NLRP3, apoptosis-associated speck-like protein contained a caspase recruitment domain (ASC), cysteinyl aspartate specific proteinase 1 (caspase-1), and IL-1β were detected using immunohistochemistry (200×). (B–E) AOD of NLRP3, ASC, caspase-1 and IL-1β. Values are presented as the means ± SD (n = 6). (F) The protein expressions of NLRP3, ASC, caspase-1, and IL-1β were detected using Western blot assay. (G–J) Quantification of protein levels of NLRP3, ASC, caspase-1, and IL-1β. Values are presented as the mean ± SD of three independent experiments. ** P < 0.01 vs. control group, ^# P < 0.05, ^## P < 0.01 vs. model group.

Figure 7

Schematic representation of the animal experiment protocol.

Figure 8

Schematic diagram of the mechanism by which EPD attenuates IMQ-induced psoriasis by inhibiting the inflammation responses via TLR7/8–MyD88–NF-κB–NLRP3 inflammasome signaling pathways.