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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.
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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.
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Objective:To investigate the efficacy and safety of infliximab in the treatment of severe plaque psoriasis and its effect on the expression of programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1) in psoriatic lesions.Methods:A total of 17 patients with severe plaque psoriasis were enrolled from Shanghai Skin Disease Hospital from February 2019 to April 2019, and were treated with intravenous drips of infliximab at a dose of 5 mg/kg at weeks 0, 2, 6, 14, 22, 30, 38 and 46. Efficacy was evaluated by using the psoriasis area and severity index (PASI) score at weeks 2, 6, 10, 14, 22, 30, 38, 46 and 52, and adverse events were recorded during the trial. Real-time PCR was performed to determine the expression of PD-1 and PD-L1 in skin tissues of 8 volunteer controls, as well as in skin lesions of 14 patients with plaque psoriasis before treatment and 5 patients with plaque psoriasis after 10-week treatment, and immunofluorescence assay to measure the expression of PD-1 and PD-L1 in skin tissues of 5 volunteers and 5 patients with psoriasis. The independent sample t-test was used to compare the expression of PD-1 and PD-L1 in skin tissues between the patients with plaque psoriasis and controls, and paired t-test to compare the expression of PD-1 and PD-L1 in the skin lesions of patients before and after infliximab treatment. Results:After 2, 6, 10, 14, 22, 30, 38, 46 and 52 weeks of infliximab treatment, the proportion of patients with plaque psoriasis achieving PASI75 was 1/17, 6/16, 9/16, 10/16, 15/15, 14/15, 13/14, 11/13 and 10/11, respectively. Antinuclear antibody staining turned positive in 12 patients, which was the most common adverse reaction, and 1 patient experienced an infusion reaction, which was the most severe adverse reaction. Before the treatment, the expression of PD-1 and PD-L1 (1.111 ± 0.391, 0.902 ± 0.169, respectively) was significantly higher in the skin lesions of patients with psoriasis than in the skin tissues of controls (0.620 ± 0.225, t=3.116, P=0.007; 0.474 ± 0.360, t=3.208, P=0.006, respectively) ; after infliximab treatment, the expression of PD-1 and PD-L1 (0.570 ± 0.230, 0.150 ± 0.050, respectively) in the improved skin lesions was significantly lower than that in the corresponding lesions before the treatment (1.238 ± 0.414, t=3.107, P=0.036; 0.966 ± 0.184, t=8.423, P=0.001, respectively) . Conclusions:Infliximab is effective and safe for the treatment of plaque psoriasis, but monitoring is necessary during treatment. The expression of PD-1 and PD-L1 is aberrantly upregulated in plaque psoriasis lesions, and decreased after infliximab treatment, suggesting that PD-1/PD-L1 may be involved in inflammation regulation in psoriasis.
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Aim To observe the retinal angiogenesis and detect the altered expression of genes related with angiogenesis and oxidative stress during the develop-ment of oxygen-induced retinopathy ( OIR) in newborn mice. Methods OIR was established in newborn mice according to the protocol of Smith et al. Newborn mice at 7 days old were placed into 75 . 5% oxygen for up to 5 days, and then they were put in room air for another 5 days. Retinal neovascularization was ob-served by immunofluorescence staining with cluster of differentiation 31 ( CD31 ) . Gene expression was de-tected using Real-time PCR analysis. Retinal CD31 immunofluorescence staining assay showed that relative hypoxia induced retinal neovascularization in OIR mice after hyperoxia-induced subside of retinal microvascu-lar. Results Real-time PCR analysis showed that vas-cular endothelial growth factor ( VEGF) and its recep-tor ( VEGFR) such as VEGFA, VEGFD, VEGFR1, VEGFR2 gene expression were increased in OIR mouse as compared to control. Platelet-derived growth factor ( PDGF) and its receptor ( PDGFR) such as PDGFA, PDGFB, PDGFRa, PDGFRb gene expression was also increased in OIR mouse as compared to control. Matrix metalloproteinases ( MMPs ) such as MMP2 gene ex-pression were increased in OIR mouse as compared to control. Gene expressions of nuclear factor-related fac-tor ( Nrf2 ) and its downstream genes such as the two subunits of glutamate-cysteine ligase ( GCL):the cata-lytic subunit ( GCLC) and regulatory subunit ( GCLM) were both decreased in OIR mouse as compared to con-trol. Conclusion Our research demonstrates that the expression of genes related with angiogenesis is in-creased in retinas in the development of OIR in mice, whereas the expression of Nrf2 and its downstream genes is all decreased.
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Objective To built up the ERP model,measure mode and P300 potential reference standard in mice with vascular dementia(VD),and characterize the P300 potential in mice with VD.Methods Fortyeight mice were randomly divided into a normal group.sham operation group and a VD group.The mice in the Vd group were subject to repetitive ischemia and reperfusion by using the ligation of bilateral common carotid arteries so as to establish the VD model.The behavioral abnormalities were investigated by step-down test and water maze test.The N2 and P3 components of P300 potentials were also recorded.Results It was shown that the learning and memory abilities as reflected by the step down test and water maze test scores were decrease in mice in the VD group when compared with those in the normal group and sham operation group(P<0.05).The N2 and P3 latencies significantly prolonged(P<0.01)and P3 amplitudes decreased(P<0.05)in VD group as well.Conclusions In VD mice,there is a significant prolongation of the P300 potential latency and a significant decrease of learning and memory abilities.Recordings of P300 from unanesthetized mice could be an objective,non-invasive,quantitative and valuable electrophysiological method for studying the cognitive function of VD mice.