Sulforaphane alleviates psoriasis by enhancing antioxidant defense through KEAP1-NRF2 Pathway activation and attenuating inflammatory signaling

Abstract

Psoriasis is a chronic inflammatory skin disease that affects millions of people worldwide. Sulforaphane (SFN) has been shown to have anti-inflammatory and antioxidant properties. In this study, we investigated the effects of SFN on a mouse model of psoriasis induced by imiquimod (IMQ) and its underlying molecular mechanism. Mice treated with SFN showed significant improvement in psoriatic symptoms, including reduced erythema, scales, and cutaneous thickness. Histopathological analysis and immunohistochemical staining revealed decreased expression of K16, K17, and Ki67 in SFN-treated mice, indicating reduced abnormal differentiation of keratinocytes and cutaneous inflammation. SFN treatment also reduced the activation of STAT3 and NF-κB pathways and downregulated pro-inflammatory cytokines IL-1β, IL-6, and CCL2. In vitro experiments using HaCaT cells demonstrated that SFN inhibited IL-22 and TNF-α-induced activation of inflammatory pathways and keratinocyte proliferation. Network pharmacology analysis suggested that the KEAP1-NRF2 pathway might be involved in the protective effects of SFN on psoriasis. We observed reduced NRF2 expression in human psoriatic lesions, and subsequent experiments showed that SFN activated KEAP1-NRF2 pathway in vivo and in vitro. Importantly, NRF2-deficient mice exhibited aggravated psoriasis-like symptoms and reduced response to SFN treatment. Our findings indicate that SFN ameliorates psoriasis symptoms and inflammation through the KEAP1-NRF2 pathway, suggesting a potential therapeutic role for SFN in the treatment of psoriasis.

Fig. 1. Sulforaphane (SFN) ameliorates psoriatic symptoms in IMQ-induced psoriatic mouse model.

a Mice were treated with IMQ and sulforaphane daily for 7 consecutive days and sacrificed on day 8. n = 5. b The macroscopic appearance and H&E staining (magnification × 100) of the skin tissue. c The PASI scores of the skin tissue. d Spleen weight index, which was measured by taking spleen weight to body weight. e The average epidermal thickness (μM), derived from 18–20 random measurements. Statistical significance was determined by one-way ANOVA analysis. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 2. Psoriatic mice treated with SFN exhibit decreased keratinocyte differentiation and reduced cutaneous inflammation.

a The contents of Ki67, keratin 16 (K16) and keratin 17 (K17) in the dorsal epidermis were measured using immunohistochemical staining (magnification × 200). b -STAT3, STAT3, p-IκBα, κBα, K16 and K17 expression was detected using Western blotting. c The relative expression levels of p-STAT3, p-IκB, K16 and K17 were analyzed by normalization to GAPDH. d Quantitative real-time polymerase chain reaction (qRT–PCR) was performed to evaluate the effects of SFN on the mRNA expression of Il1b, Il6 and Ccl2 in psoriatic mouse skin. n = 3. Statistical significance was determined by one-way ANOVA analysis. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 3. SFN mitigates TNF-a and IL-22-induced activation of inflammatory pathways and proliferation in HaCaT cells.

a The expression of p-STAT3, STAT3, K16, and K17 in response to SFN and IL-22 (25 ng/ml) treatment was determined by western blotting. b The relative expression levels were analyzed by normalization to GAPDH. c Western blot analysis of the protein expression of p-IκBα, IκBα, IL-1β, K16 and K17. d The relative protein levels were assessed by ImageJ software. Statistical significance was determined by one-way ANOVA analysis. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 4. Target analysis of SFN in psoriasis.

a Chemical structure of SFN. b PPI network of SFN targets predicted by SuperPred(https://prediction.charite.de/) and OMIM (https://www.omim.org/). The yellow nodes represent the large hub nodes, and the blue nodes represent the other nodes. The node size is proportional to the target degree in the network. c Target genes were retrieved by taking the intersection of targets of SFN and genes related to psoriasis, and the PPI network of the target genes (d) was constructed by the STRING database. e Interrelation analysis of pathways via assessment of Reactome processes in ClueGO.

Fig. 5. NRF2 expression is decreased in psoriatic skin compared to normal skin.

a NRF2 protein expression was elevated in skin tissue (arrows) from the Human Protein Atlas website. The orange arrow indicates the levels of NRF2 in skin tissue. b NFE2L2 mRNA levels of normal skin (NN), normal skin in patients with psoriasis (PN) and psoriatic skin in patients with psoriasis (PP) expressed in skin tissue from GEO datasets (GSE119087). c RT–PCR was performed to evaluate the mRNA level of NFE2L2 in psoriatic human skin (PSO) and skin from healthy tissue (NC). d The protein level of p-NRF2 and NRF2 in PSO and NC was determined by immunofluorescence analysis. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 6. SFN increases the expression of NRF2 and p-NRF2 in IMQ-induced psoriatic mouse model.

a Protein expression of NRF2 and p-NRF2 evaluated using Western blot. b The relative expression levels were analyzed by normalization to GAPDH. ***p < 0.001. c Immunofluorescence staining for NRF2 and p-NRF2 on skin sections of flaky mice was performed.

Fig. 7. NRF2 pathway was involved in the cytoprotection of SFN against IL-22 and TNF-α.

a The expression of NRF2 and p-NRF2 in response to SFN and IL-22 treatment was determined by western blotting. b The expression of NRF2 and p-NRF2 in response to SFN and TNF-α treatment was determined by western blotting. c HaCaT cell superoxide measured by staining with DHE (magnification × 400). d HaCaT cells were transduced with lentivirus carrying shRNA against a scramble sequence (sh-C) or against NRF2(sh-NRF2-a and sh-NRF2-b). Expression levels of NFE2L2 were determined by qRT–PCR and normalized to Actb levels. e The Western Blot results of SFN and IL-22 treatment in the NRF2 knockdown group (sh-NRF2-b) and the control group (sh-C). f The Western Blot results of SFN and TNF-α treatment in the NRF2 knockdown group (sh-NRF2-b) and the control group (sh-C). *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 8. NRF2 depletion exacerbates IMQ-induced psoriasis-like symptoms and negates the therapeutic effect of SFN.

a Wild-type (WT) and NRF2^−/− mice were topically treated with IMQ for seven consecutive days and sacrificed on day 8. Images were taken on day 4 and day 8. n = 5. b The PASI scores of the skin tissue. c p-STAT3, STAT3, p-P65, P65, p-IκBα, IκB, K16 and NQO1 expression was detected using Western blotting. d The relative expression levels of protein were analyzed by normalization to GAPDH. e H&E staining (magnification × 100) and immunohistochemical staining of Ki67, K17 and K16 (magnification × 200) in skin sections of mice were performed. f The average epidermal thickness (μM), derived from 18–20 random measurements. g qRT–PCR was performed to evaluate the mRNA expression of Il17a and Il23a in mouse skin. n = 3. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 9. The mechanism driving the therapeutic impact of SFN in combating psoriasis.

Diminished expression of NRF2 plays a role in oxidative stress injury in psoriasis. By activating the NRF2 signaling pathway, SFN mitigates oxidative stress injury and reduces the inflammatory response associated with psoriasis.