The Study Of Anti-inflammatory Effects And Molecular Mechanisms Of Lipoxin A4in Keratinocyte And Psoriasis

Lipoxin A4inhibits the production of inflammatory cytokines induced by lipopolysaccharide in keratinocytes through up-regulation of SOCS2and degradation of TRAF6Objective:To study the effect of LXA4on the inflammatory cytokines production induced by lipopolysaccharide (LPS) and the possible upstream mechanism in normal human epidermal keratinocytes (NHEKs).Method:NHEKs were isolated, cultured. The expression of toll-like receptor4(TLR4), LXA4receptor (ALXR) and aryl hydrocarbon receptor (AhR) in NHEKs were detected by reverse transcription polymerase chain reaction (RT-PCR) firstly. NHEKs were stimulated by LPS (lOug/ml) with or without preincubation with LXA4(100nmol/L) for30min. After incubation for appropriate time, changes in the mRNA and protein levels of tumor necrosis factor-alpha (TNF-a) and interleukin-1β (IL-1β) produced by NHEKs were determined by real-time quantitative polymerase chain reaction (real-time qPCR) and enzyme-linked immunosorbent assay (ELISA). mRNA expressions of TRAF6and SOCS2, protein expressions of TRAF6, SOCS2and nuclear translocation of NF-kB-p65were measured by real-time qPCR and western blotting, respectively.Results:NHEKs expressed TLR4, ALXR and AhR. LXA4significantly inhibited mRNA and protein expressions of TNF-a, IL-1β and TRAF6induced by LPS in NHEKs, and LXA4obviously increased the expression of SOCS2in mRNA and protein levels. The nuclear NF-kB-p65protein expression induced by LPS was inhibited after preincubation with LXA4in NHEKs. Conclusion:LXA4inhibited the production of TNF-a and IL-1β induced by LPS in NHEKs through up-regulation of SOCS2and degradation of TRAF6. Part Ⅱ Lipoxin A4inhibits proliferation and inflammatory cytokine/chemokine production of human epidermal keratinocytes associated with the ERK1/2and NF-kB pathwaysObjective:The effects of LXA4on cell proliferation and inflammatory cytokines, chemokines production were definited, and the molecular mechanisms about cell cycle and anti-inflammatory of signal pathway of LXA4were determined, both in the inactived and LPS-stimulated NHEKs.Method:NHEKs were stimulated with or without LPS (lOug/ml) in the absence or presence of LXA4(100nmol/L). The effects of LXA4on the proliferation of NHEKs were examined by WST-8and carboxyfluorescein diacetate succinimidyl ester (CFSE) assay, cell cycle phase was detected by DNA staining. Expressions of mRNA and proteins of inflammatory cytokines were measused by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The expressions of cyclin D1and P16INK4Awere determined by real-time PCR and western blotting, ERK1/2and NF-kB-p65were assessed by western blotting.Results:Cell proliferation and inflammatory cytokine/chemokine production of NHEKs were suppressed by LXA4, which caused G0/G1phase cell cycle arrest in NHEKs. The expression of cyclin D1was down-regulated by LXA4, contrary to the results of P16INK4A. The ERK1/2phosphorylation and NF-kB-p65nuclear translocation of NHEKs were both suppressed by LXA4.Conclusion:Cell growth and inflammatory cytokine/chemokine production of NHEKs were inhibited by LXA4, and the inhibitory effects might be associated with the mechanisms of cyclin D1/P16INK4A, ERK1/2and NF-kB signal transduction pathway. Part Ⅲ Lipoxin A4inhibits skin lesions in imiquimod-inducedpsoriasis-like mice by downregulating inflammationObjective:To investigate the effect of lipoxin A4in imiquimod-induced psoriasis-like inflammation in mice.Method:The mice were equally randomly divided into blank group, model group and LXA4group. The changes of lesional skin in mice were assessed according to the psoriasis area and severity index (PASI); the thickness of epidermis was measured and the morphological changes of the skin tissues were observed under light microscope through HE staining; the changes of Th1/Th17in the splenic cells of mice were detected by flow cytometry.Results:Compared with the model group, the cutaneous symptoms including scales, erythema and skin thickening in LXA4group were alleviated, with PASI scores decreased; on the aspect of tissue pathology, the epidermal neutrophils microabcesses were almost resolved, the epidermal over-proliferation relived, the numbers of dermal inflammatory cells reduced significantly, and Th17cells in splenic were decreased.Conclusion:LXA4inhibits the skin lesions in imiquimod-inducedpsoriasis-like mice by pro-resolving neutrophils microabcesses, inhibiting the epidermal over-proliferation and regulating the differentiation of T lymphocyte.