Triptolide alleviates psoriasis through inhibiting the Wnt5a/β-Catenin signaling pathway

Abstract

Background: Psoriasis, an immune-mediated chronic inflammatory skin disease, is characterized by keratinocyte proliferation and inflammatory cell infiltration. T ripterygium wilfordii is a potential treatment option for psoriasis, and triptolide (TP) is one of its active components. TP may possess the potential to treat psoriasis; however, its mechanism of action remains unknown.

Objective: The research aims to explore the therapeutic effect of TP on psoriasis and elucidate its potential targets.

Methods: The imiquimod-induced psoriasis-like lesion mouse model was used to identify the mechanism underlying the therapeutic effect of TP.RNA-seq strategy was utilized to forecast the targets and mechanisms of TP in the context of psoriasis.Finally, we verify the effect of TP in the IL-17A-induced keratinocyte hyperproliferation and inflammation model.

Results: TP reduced epidermal hyperplasia as well as psoriasis area and severity index scoring. Moreover, treatment with TP inhibited IMQ-induced splenomegaly and T-helper 17 cell differentiation in the psoriatic mice. Additionally, the treatment reduced the serum levels of pro-inflammatory cytokines such as interleukin (IL)-17A, IL-22, IL-23, IL-6, and tumor necrosis factor-α in the mice. The sequencing of RNA obtained from skin lesions of the psoriatic mice indicated that treatment with TP significantly downregulated Wnt5a RNA levels. Moreover, the Wnt5a/β-catenin pathway upregulated by IMQ was downregulated by treatment with TP. Additionally, IL-17A induced and upregulated Wnt5A and β-catenin mRNA expression, and TP inhibited this upregulated expression in HaCaT cells. Furthermore, TP inhibited proliferation, promoted apoptosis, and arrested the cell cycle in the IL-17A-induced keratinocyte hyperproliferation and inflammation model, thereby exhibiting its anti-inflammatory properties.

Conclusion: TP alleviated psoriasis in mice by exerting anti-inflammatory effects and inhibited keratinocyte proliferation, which was partly achieved by regulating the Wnt5a/β-catenin signaling pathway.

Keywords: Wnt5a/β-catenin; inflammation; interleukin-17A; psoriasis; triptolide.

FIGURE 1

TP alleviated the IMQ-induced psoriasis-like mouse skin lesion. (A) Macroscopic presentation of mouse back skin after 7 days of treatment. (B) Daily body weight of mice (n = 7). (C) The scales, erythema and thickness of the back skin were evaluated daily from the first day according to the Psoriasis Area Severity Index (PASI), with a score ranging from 0 to 4, and the total score was calculated: scales + erythema + thickness (n = 7). (D) Haematoxylin and eosin (H&E) staining of back skin lesions from different groups of mice (scale bar = 200 μm, n ≥ 3). (E) Immunohistochemical analysis of PCNA in the epidermis (scale bar = 200 μm, n ≥ 3). All Data are represented as the mean ± SEM. ^#### p < 0.0001 versus CON; *p < 0.05, **p < 0.01, ****p < 0.001,****p < 0.0001 versus IMQ; ns, not significant. IMQ, imiquimod; MTX, methotrexate 1 mg/kg; L-TP, triptolide 75 μg/kg; H-TP, triptolide 150 μg/kg.

FIGURE 2

TP shows significant anti-inflammatory effect on psoriasis in vivo. (A) Macroscopic presentation of spleens of mice in each group. (B) Spleen index (spleen weight/body weight) was calculated (n = 7). (C) The frequency of Th17 cells was in spleen of each group analyzed by flow cytometry (n ≥ 3). (D) Statistical plots of Th17 cell frequency in spleen of each group (n ≥ 3). (E) The protein expression of IL-17A, IL-22, IL-23, IL-6 and TNF-α in serum of each group were analyzed by ELISA (n ≥ 3). All Data are represented as the mean ± SEM. ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 versus CON; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus IMQ; ns, not significant.

FIGURE 3

Increased Wnt5a expression in IMQ-induced psoriasis mice was inhibited by TP treatment (A) GO enrichment analysis of back skin of mice in the IMQ group and H-TP group (n = 3). (B) Heat maps showing differentially expressed genes in the classification of skin development in the back skin of IMQ and H-TP groups mice (n = 3). Increased transcript levels are colored red and decreased levels are colored green. (C)The expression of Wnt5a was evaluated by immunohistochemical staining of mouse back skin (Scale bar = 50 μm, n = 3). (D) Total RNA was isolated from back skin tissues, and RT-PCR was used to investigate the mRNA levels of Wnt5a (n = 3). (E) The expression of Wnt5a and β-Catenin in the back skin of different groups were measured with Western blot. The column figures of quantitative Wnt5a and β-Catenin protein expression data are shown on the right (n = 3). All Data are represented as the mean ± SEM. ^## p < 0.01, ^### p < 0.001 versus CON; *p < 0.05, **p < 0.01, ***p < 0.001 versus IMQ; ns, not significant.

FIGURE 4

TP suppresses IL-17A-induced upregulation of the Wnt5a/β-catenin signaling pathway in HaCaT cells. (A) The mRNA levels of Wnt5a induced by IL-17A in HaCaT cells were analyzed by qRT-PCR. (B) The Wnt5a and β-Catenin protein levels induced by IL-17A in HaCaT cells were detected by Western blot. (C) The mRNA levels of Wnt5a by TP-treated in HaCaT cells were analyzed by qRT-PCR. (D) The Wnt5a and β-Catenin protein levels by TP-treated in HaCaT cells were detected by Western blot. (E) The mRNA levels of Wnt5a treated by IL-17A and/or TP in HaCaT cells were analyzed by qRT-PCR. (F) The Wnt5a and β-Catenin protein levels treated by IL-17A and/or TP in HaCaT cells were detected by Western blot. All Data are represented as the mean ± SEM, n = 3. ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 versus CON or 0h; **p < 0.01, ***p < 0.001, ****p < 0.0001 versus IL-17A; ns, not significant. IL-17A, interleukin-17A 100 ng/mL; TP20, triptolide 20 nM; TP40, triptolide 40 nM; TP80, triptolide 80 nM.

FIGURE 5

TP effectively inhibited the proliferation, promoted the apoptosis and arrested the cell cycle of HaCaT cells that stimulated by IL-17A (A) After HaCaT cells were treated with IL-17A and/or TP for 24 h, the proliferation of HaCaT cells was detected by MTT assay. (B) 5-ethynyl20-deoxyuridine (EdU) staining assayed the number of proliferating HaCaT cells that had been treated with IL-17A and/or TP for 24 h. Positive cells were stained by EdU (red) and total cell nucleus was stained with Hoechst (blue) (scale bar = 100 μm, n = 3). (C) Representative scatter plots of the flow cytometry analysis of the fraction of apoptotic HaCaT cells that had been treated with IL-17A and/or TP for 24 h. (D) The apoptotic characteristics in HaCaT cell nucleus presented by Hoechst Staining assay. After treated with IL-17A and/or TP for 24 h before stained with Hoechst 33,342 staining, the nucleus was observed and photographed (scale bar = 100 μm, n = 3). (E) The analysis of cell cycle phase of HaCaT cells treated with IL-17A and/or TP for 24 h. Cells were stained with propidium iodide (PI) and determined with Flow cytometry. (F) After HaCaT cells were treated with IL-17A and/or TP for 24 h, Western blot analysis showed the levels of proteins involved apoptosis, proliferation and cell cycle. All Data are represented as the mean ± SEM, n = 3. ^### p < 0.001 versus CON; **p < 0.01, ***p < 0.001, ****p < 0.0001 versus IL-17A.

FIGURE 6

TP affected the expression of chemokines and cytokines in HACAT cells stimulated with IL-17A. After HaCaT cells were treated with IL-17A and/or TP for 24 h, the mRNA levels of chemokines and cytokines were determined by qPCR. (A) CXCL1; (B) CXCL2; (C) CXCL8; (D) CCL20; (E) IL-1β; (F) IL-6; (G) IL-19. All Data are represented as the mean ± SEM, n = 3. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 versus CON; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus IL-17A; ns, not significant.

FIGURE 7

The mechanism of TP in the treatment of psoriasis. TP can effectively alleviate imiquimod-induced psoriasis symptoms in mice, such as epidermal thickening, Th17 cell differentiation, macrophage activation, and the release of inflammatory factors like IL-17A in serum. In keratinocytes, TP targets the Wnt5A/β-catenin signaling pathway to suppress the excessive proliferation of keratinocytes.