Objective:To investigate the expression of ephrin type-A receptor 2 (EPHA2) in psoriatic lesions and its effect on the proliferation and differentiation of normal human epidermal keratinocytes (NHEKs) .Methods:The GDS4602 dataset from the Gene Expression Omnibus (GEO) database was analyzed to determine EPHA2 gene expression changes in psoriatic lesions. Skin tissue samples were collected from 3 psoriasis patients and 3 healthy controls, and EPHA2 expression was determined in the skin tissues by immunofluorescence staining. Twelve female BALB/c mice were randomly divided into 3 groups (4 mice in each group) : a normal control group (receiving no treatment), an imiquimod group (topically treated with 62.5 mg of imiquimod 5% cream), and an imiquimod + ALWⅡ-41-27 group (topically treated with 62.5 mg of imiquimod 5% cream, followed by intraperitoneal injections of the EPHA2 inhibitor ALWⅡ-41-27 at a dose of 20 mg·kg -1·d -1) ; after 6 days of treatment, dorsal skin samples were harvested for hematoxylin-eosin (HE) staining, immunofluorescence staining was performed to determine the expression of EPHA2 and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), and real-time fluorescence-based quantitative PCR (qPCR) was conducted to determine the mRNA expression of the nuclear proliferation antigen Ki67, involucrin (Ivl), loricrin (Lor), and keratin 10 (Krt10). In vitro cultured NHEKs were divided into a control group (receiving no treatment), an M5 group (treated with 10 ng/ml M5 cytokines [including interleukin-17A, interleukin-22, interleukin-1α, oncostatin M and tumor necrosis factor-α]), an ALWⅡ-41-27 group (treated with 1 μmol/L ALWⅡ-41-27), and an M5 + ALWⅡ-41-27 group (treated with 10 ng/ml M5 and 1 μmol/L ALWⅡ-41-27) ; after 24 hours of treatment, the 5-ethynyl-2′-deoxyuridine (EdU) assay was performed to assess cellular proliferative activity, Western blot analysis to determine the expression of EPHA2, ERK and their phosphorylated proteins, and qPCR to determine the mRNA expression of KI67, IVL, LOR, and KRT10. One-way analysis of variance, Dunnett's T3 test, two-independent-sample t test, and paired t test were used for statistical analysis. Results:GEO database analysis revealed upregulated EPHA2 expression in psoriatic lesions compared with normal skin tissues from healthy controls ( t = 21.07, P < 0.001). Immunofluorescence staining showed increased EPHA2 expression in skin tissues from psoriasis patients and mouse models of psoriasis compared with those from healthy controls and normal control mice, respectively (both P < 0.01). In the animal experiments, the imiquimod group showed thicker epidermis, increased Ki67 mRNA expression, decreased mRNA expression of Ivl, Lor, and Krt10, and elevated p-ERK1/2 expression compared with the normal control group and imiquimod + ALWⅡ-41-27 group (all P < 0.05). In the cell experiments, the M5 group showed an increased proportion of EdU-positive cells (35.61% ± 1.18% vs. 24.83% ± 0.60% and 12.49% ± 1.52%, t = 8.12, 12.00, P = 0.015, 0.001, respectively), increased KI67 mRNA expression, and decreased mRNA expression of IVL, LOR, and KRT10 compared with the control group and M5 + ALWⅡ-41-27 group (all P < 0.05) ; Western blot analysis revealed that the expression levels of EPHA2, p-EPHA2, and p-ERK1/2 in NHEKs were significantly higher in the M5 group than in the control group and M5 + ALWⅡ-41-27 group (all P < 0.05), but there was no significant difference in the ERK1/2 protein expression among groups ( P > 0.05) . Conclusion:EPHA2 expression was upregulated in psoriatic lesions, which may promote keratinocyte proliferation and inhibit its differentiation, possibly via the ERK pathway.

Objective:To investigate the expression of ephrin type-A receptor 2 (EPHA2) in psoriatic lesions and its effect on the proliferation and differentiation of normal human epidermal keratinocytes (NHEKs) .Methods:The GDS4602 dataset from the Gene Expression Omnibus (GEO) database was analyzed to determine EPHA2 gene expression changes in psoriatic lesions. Skin tissue samples were collected from 3 psoriasis patients and 3 healthy controls, and EPHA2 expression was determined in the skin tissues by immunofluorescence staining. Twelve female BALB/c mice were randomly divided into 3 groups (4 mice in each group) : a normal control group (receiving no treatment), an imiquimod group (topically treated with 62.5 mg of imiquimod 5% cream), and an imiquimod + ALWⅡ-41-27 group (topically treated with 62.5 mg of imiquimod 5% cream, followed by intraperitoneal injections of the EPHA2 inhibitor ALWⅡ-41-27 at a dose of 20 mg·kg -1·d -1) ; after 6 days of treatment, dorsal skin samples were harvested for hematoxylin-eosin (HE) staining, immunofluorescence staining was performed to determine the expression of EPHA2 and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), and real-time fluorescence-based quantitative PCR (qPCR) was conducted to determine the mRNA expression of the nuclear proliferation antigen Ki67, involucrin (Ivl), loricrin (Lor), and keratin 10 (Krt10). In vitro cultured NHEKs were divided into a control group (receiving no treatment), an M5 group (treated with 10 ng/ml M5 cytokines [including interleukin-17A, interleukin-22, interleukin-1α, oncostatin M and tumor necrosis factor-α]), an ALWⅡ-41-27 group (treated with 1 μmol/L ALWⅡ-41-27), and an M5 + ALWⅡ-41-27 group (treated with 10 ng/ml M5 and 1 μmol/L ALWⅡ-41-27) ; after 24 hours of treatment, the 5-ethynyl-2′-deoxyuridine (EdU) assay was performed to assess cellular proliferative activity, Western blot analysis to determine the expression of EPHA2, ERK and their phosphorylated proteins, and qPCR to determine the mRNA expression of KI67, IVL, LOR, and KRT10. One-way analysis of variance, Dunnett's T3 test, two-independent-sample t test, and paired t test were used for statistical analysis. Results:GEO database analysis revealed upregulated EPHA2 expression in psoriatic lesions compared with normal skin tissues from healthy controls ( t = 21.07, P < 0.001). Immunofluorescence staining showed increased EPHA2 expression in skin tissues from psoriasis patients and mouse models of psoriasis compared with those from healthy controls and normal control mice, respectively (both P < 0.01). In the animal experiments, the imiquimod group showed thicker epidermis, increased Ki67 mRNA expression, decreased mRNA expression of Ivl, Lor, and Krt10, and elevated p-ERK1/2 expression compared with the normal control group and imiquimod + ALWⅡ-41-27 group (all P < 0.05). In the cell experiments, the M5 group showed an increased proportion of EdU-positive cells (35.61% ± 1.18% vs. 24.83% ± 0.60% and 12.49% ± 1.52%, t = 8.12, 12.00, P = 0.015, 0.001, respectively), increased KI67 mRNA expression, and decreased mRNA expression of IVL, LOR, and KRT10 compared with the control group and M5 + ALWⅡ-41-27 group (all P < 0.05) ; Western blot analysis revealed that the expression levels of EPHA2, p-EPHA2, and p-ERK1/2 in NHEKs were significantly higher in the M5 group than in the control group and M5 + ALWⅡ-41-27 group (all P < 0.05), but there was no significant difference in the ERK1/2 protein expression among groups ( P > 0.05) . Conclusion:EPHA2 expression was upregulated in psoriatic lesions, which may promote keratinocyte proliferation and inhibit its differentiation, possibly via the ERK pathway.

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.