The Study Of Anti-inflammatory Effects And Mechanisms Of Lipoxin A4 In Psoriasis By Regulating HMGB1

Objective: Psoriasis is a chronic inflammatory skin disease and there is still no known possibility of a cure.Lipoxin A4(LXA4)and BML-111(5(S)-6(R)-7-trihydroxyheptanoic acid methyl ester)have potent dual pro-resolving and anti-inflammatory properties.High mobility group box 1(HMGB1)serves as an inflammatory cytokine which is highly expressed in peripheral blood and extracellularly in psoriatic lesions and is involved in the development of psoriasis.Therefore,we investigated the effects of LXA4 and BML-111 on the HMGB1 signaling cascade and inflammation in lipopolysaccharide(LPS)-induced keratinocytes and imiquimod(IMQ)-induced psoriasiform dermatitis in mice.Methods: 1.IMQ-induced psoriasiform dermatitis model,BALB/c mice were randomized into three groups:(1)IMQ + BML-111 group: mice received intraperitoneal injection of BML-111 at a dose of 1 mg / kg / day followed by the application of IMQ cream for 7 days.(2)IMQ group: IMQ-treated mice received injection of the same volume of saline as in the IMQ+BML-111 group for 7 days.(3)Normal group: mice received intraperitoneal injection of saline and were topically treated with Vaseline.2.To score the severity of the skin lesion inflammation in mice,an objective scoring system was developed based on the clinical Psoriasis Area and Severity Index(PASI).3.Immunofluorescence,Western blot,and real-time quantitative polymerase chain reaction(q RT-PCR)were used to detect the expression of HMGB1 in skin lesions of mice.4.Western blot and q RT-PCR were used to detect the expression and activation of HMGB1 downstream receptors such as advanced glycation end products receptor(RAGE)and toll like receptor 4(TLR4)and signaling pathways in the skin from protein and m RNA levels respectively.5.q RT-PCR revealed the expression of inflammatory cytokines(IL-1β,TNF-α,IFN-γ,IL-6,IL-17 a,IL-17 c,IL-23 and IL-22)from m RNA levels.The expression levels of inflammatory cytokines(IL-1β,TNF-a,IL-17 and IL-23)in the serum of mice were determined by enzyme-linked immunosorbent assay(ELISA).6.Keratinocytes were divided into four groups:(1)NC group: Keratinocytes were normally cultured;(2)LXA4 group: Keratinocytes were exposed to LXA4;(3)LPS group: Keratinocytes were exposed to LPS;(4)LXA4+LPS group: Keratinocytes were exposed to LPS with preincubation with LXA4 for 30 minutes.7.The expression of HMGB1 in cytoplasm and nucleus of keratinocytes were subjected to western blotting analysis.The expression level of HMGB1 in keratinocytes was assessed by q RT-PCR;The culture supernatant of keratinocytes was obtained 24 hours after LPS challenge to determine HMGB1 concentration by ELISA;Co-Immunoprecipitation(Co-IP)showed the acetylation level of HMGB1 in cells.8.Western blot and q RT-PCR were performed to analyze the activation of RAGE and TLR4 and their signaling pathways(p-ERK 1/2 and NF-κB p65)in keratinocytes.Results: In vivo study,we found that BML-111 intervention significantly alleviated the morphological and histological changes of IMQ-induced psoriasiform dermatitis in mice.BML-111 inhibited the translocation and expression of HMGB1 from the nucleus to the cytoplasm in the skin lesions.BML-111 down regulated the HMGB1 receptor TLR4 and RAGE and their downstream signaling pathways(p-ERK1/2 and NF-κB p65),and BML-111 significantly inhibited the expression of proinflammatory cytokines(IL-1β,TNF-α,IL-6,IL-17 a,IL-17 c,IL-23 and IL-22)in the skin tissue induced by IMQ.Lower levels of IFN-γ in the IMQ + BML-111 group,but the results were not statistically significant.The serum levels of IL-1β、TNF-α、IL-17 and IL-23 were increased by IMQ treatment,but the increase levels of TNF-α,IL-17 and IL-23 in the IMQ group were notably decreased by BML-111 administration.In vitro,our study showed that LXA4 suppressed the translocation and expression of HMGB1 in LPS-induced keratinocytes,and this inhibitory effect may be attributed to the acetylation level of HMGB1.LXA4 inhibited the HMGB1/RAGE and HMGB1/TLR4 signaling in keratinocytes.Conclusion: Our findings indicate that LXA4 and its analog may be potential therapeutic candidates for psoriasis because of their ability to modulate the translocation and expression of HMGB1.