RESUMO
Objective:To investigate changes in circadian gene cryptochrome 2 (CRY2) expression in mouse models of psoriasis and HaCaT cells, and to explore underlying mechanisms.Methods:Imiquimod-induced mouse model experiment: 12 C57BL/6 female mice were randomly and equally divided into imiquimod group receiving topical imiquimod treatment for 5 consecutive days and control group receiving no treatment; these mice were sacrificed on day 6, skin tissues were resected from the back of mice, and immunofluorescence staining was performed to determine the CRY2 expression in the epidermis. HaCaT cell transfection experiment: HaCaT cells with small interfering RNA (siRNA) -mediated knockdown of CRY2 served as siRNA-CRY2 group, and siRNA-NC group as control group; 5-ethynyl-2′-deoxyuridine (EdU) staining was performed to evaluate the proliferative activity of the HaCaT cells, real-time fluorescence-based quantitative PCR (qPCR) to determine the mRNA expression of chemokines in the HaCaT cells, and Western blot analysis to determine phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2) . Tumor necrosis factor-α (TNF-α) -stimulated animal and cell experiments: 12 C57BL/6 female mice were randomly and equally divided into TNF-α group subcutaneously injected with TNF-α solution in the ear for 6 days, and phosphate buffered saline (PBS) group subcutaneously injected with the same amount of PBS; the mice were sacrificed on day 7, skin tissues were resected from the ear of mice, and immunofluorescence staining was conducted to determine the CRY2 expression in the epidermis; CRY2-knockdown HaCaT cells stimulated with 50 ng/ml TNF-α for 12 hours served as siRNA-CRY2 + TNF-α group, and siRNA-NC + TNF-α group as control group; qPCR was performed to determine the mRNA expression of chemokines in HaCaT cells in the above groups. Statistical analysis was carried out by using two-independent-sample t test. Results:Immunofluorescence staining showed that the CRY2 protein expression was significantly lower in the mouse dorsal epidermis in the imiquimod group (0.94 ± 0.23) than in the control group (2.30 ± 0.25, t = 3.99, P = 0.016) . Compared with the siRNA-NC group, the siRNA-CRY2 group showed significantly increased proportions of EdU-positive cells (48.13% ± 10.97% vs. 38.23% ± 0.81%, t = 5.00, P = 0.007) , mRNA expression levels of chemokines CXCL1 and CXCL8, as well as significantly increased phosphorylated (p) -ERK1/2 protein expression levels (all P < 0.05) , while there were no significant differences in the CCL20 mRNA expression or ERK1/2 protein expression between the two groups (both P > 0.05) . Immunofluorescence staining showed significantly decreased CRY2 protein expression level in the mouse ear epidermis in the TNF-α group (0.37 ± 0.34) compared with the PBS group (2.04 ± 0.17, t = 4.38, P = 0.012) ; the relative mRNA expression levels of chemokines CXCL1, CXCL8, and CCL20 in HaCaT cells were significantly higher in the siRNA-CRY2 + TNF-α group than in the siRNA-NC + TNF-α group (all P < 0.05) . Conclusion:CRY2 was markedly underexpressed in psoriasis, which might promote the proliferation of keratinocytes and expression of chemokines CXCL1, CXCL8 and CCL20, and TNF-α might be an upstream cytokine that could downregulate CRY2 expression.
RESUMO
Objective:To investigate the expression of fatty acid desaturase 2 (FADS2) in psoriatic skin lesions, as well as its regulatory factors.Methods:FADS2 expression in psoriatic skin lesions was analyzed by using the dataset GDS4602 in Gene Expression Omnibus (GEO) database. Skin tissues were obtained from the back of 5 C57BL/6 mouse models of imiquimod-induced psoriasis, normal skin of 4 patients without psoriasis or other immune skin diseases, lesions of 4 patients with psoriasis before and after 10-week treatment with infliximab, as well as lesions of 3 patients with psoriasis before and after 12-week treatment with secukinumab in Shanghai Skin Disease Hospital. FADS2 expression was determined by both immunohistochemical staining and Western blot analysis in the epidermis of mouse skin tissues, and by immunohistochemical staining in that of human skin tissues. In vitro cultured human immortalized keratinocytes (HaCaT) were divided into several groups to be treated with 50 ng/ml tumor necrosis factor-α (TNF-α) alone for 0, 6, 12 and 24 hours respectively, 200 ng/ml interleukin-17A (IL-17A) alone for 0, 6 and 12 hours respectively, or treated with 50 ng/ml TNF-α and 5 μmol/L BAY 11-7082 (a nuclear factor-κB pathway inhibitor) for 6 hours (TNF-α+ BAY 11-7082 6 h group) , and the cells receiving normal culture served as the control group. After the above treatment, real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were conducted to determine the mRNA and protein expression of FADS2 respectively. Statistical analysis was carried out by using one-way analysis of variance and t test. Results:Analysis of the dataset GDS4602 showed that the FADS2 mRNA expression was significantly lower in the lesional and non-lesional skin tissues from the patients with psoriasis (0.656 ± 0.475, 1.503 ± 1.062, respectively) than in the normal skin tissues (2.035 ± 1.226; F = 55.17, 3.07, P < 0.001, = 0.012, respectively) , and was significantly lower in the lesional skin tissues than in the non-lesional skin tissues from the patients with psoriasis ( F = 26.27, P < 0.001) . Western blot analysis and immunohistochemical staining both showed significantly decreased FADS2 protein expression in the mouse skin tissues in the imiquimod group (gray-value ratio: 0.463 ± 0.172; fluorescence intensity: 21.840 ± 3.125) compared with the normal control group (gray-value ratio: 1.000, t = 7.00, P = 0.002; fluorescence intensity: 30.720 ± 6.850, t = 3.15, P = 0.035) . Compared with the skin lesions before treatment, the FADS2 protein expression significantly increased in the skin lesions from the patients with psoriasis after 10-week treatment with infliximab (43.775± 3.342 vs. 27.950 ±1.218, t = -6.95, P = 0.006) , but was not significantly changed in the skin lesions from the patients with psoriasis after 12-week treatment with secukinumab (28.667 ± 3.402 vs. 31.933 ± 2.987, t = 2.72, P = 0.113) . qPCR revealed that the FADS2 mRNA expression significantly decreased in HaCaT cells in the TNF-α 6 h group and TNF-α 12 h group compared with the TNF-α 0 h group ( P = 0.002, 0.003, respectively) , while there was no significant change in the FADS2 mRNA expression in the IL-17A 6 h group and IL-17A 12 h group compared with the IL-17A 0 h group ( P = 0.849, 0.961, respectively) . The FADS2 mRNA expression significantly decreased in HaCaT cells in the TNF-α 6 h group (0.682 ± 0.132) compared with the control group (1.000, t = 4.82, P = 0.017) , but significantly increased in the TNF-α + BAY 11-7082 6 h group (1.541 ± 0.525) compared with the TNF-α 6 h group ( t = -3.58, P = 0.037) . Western blot analysis revealed significantly decreased FADS2 protein expression in HaCaT cells in the TNF-α 24 h group compared with the TNF-α 0 h group ( F = 6.24, P = 0.013) . Conclusion:FADS2 expression was downregulated in psoriatic lesions, which may be related to TNF-α.