Psoriasis is a disease with a proinflammatory base, in which an increased expression of leptin, tumor necrosis factor alpha (TNF-α), interleukin (IL) IL-12/23, IL-6, is observed. A drug used in the treatment of psoriasis of moderate and acute strength is the monoclonal antibody anti-TNF–adalimumab. The goal of this study was to evaluate the influence of adalimumab on changes in the expression profile of leptin-related genes in human keratinocyte cells exposed to lipopolysaccharide A and analyze if adalimumab acts via leptin pathways. The evaluation of changes of the pattern of genes connected with leptin and proteins coded by them was marked in a culture of human keratinocytes (HaCaT) exposed to 1 µg/mL lipopolysaccharide A (LPS) for 8 h in order to induce the inflammatory process, then to 8 µg/mL of adalimumab for 2.8 and 24 h in comparison with the control (cells not treated with the substances). The techniques used were mRNA microarray, Real-Time Quantitative Reverse Transcription Reaction (RTqPCR), Enzyme-Linked Immunosorbent Assay (ELISA), as well as transfections of HaCaT culture with leptin small interfering RNA (siRNA) in order to see whether adalimumab works through pathways dependent on leptin. A statistically lower expression of leptin and its receptors was observed under the influence of the drug, independent of the exposition time of keratinocytes to adalimumab. In the cells transfected with leptin siRNA, a lower concentration of JAK2 and STAT3 proteins was observed, which confirms that adalimumab works through pathways dependent on leptin. Adalimumab has a modulatory effect on the gene expression pattern and the proteins coded by them connected with leptin in keratinocytes treated with LPS in vitro.
Previous studies have not completely elucidated the role of the histaminergic system in the pathogenesis of psoriasis. This study aimed to evaluate the effects of adalimumab and cyclosporine A on the expression of histaminergic system-related genes and miRNAs regulating these genes in bacterial lipopolysaccharide A (LPS)-stimulated human keratinocyte (HaCaT) cells. HaCaT cells were treated with 1 µg/mL LPS for 8 h, followed by treatment with 8 µg/mL adalimumab or 100 ng/mL cyclosporine A for 2, 8, or 24 h. Untreated cells served as controls. The cells were subjected to ribonucleic acid (RNA) extraction and microarray, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay analyses. Statistical analysis was performed using the Statistica 13.0 PL (StatSoft, Cracow, Poland) and the Transcriptome Analysis Console programs (Affymetrix, Santa Clara, CA, USA) ( p < 0.05). The differential expression of the following two miRNAs was not affected in LPS-stimulated cells upon treatment with cyclosporine A or adalimumab: hsa-miR-583 (downregulated expression), involved in the regulation of histamine receptor 1 – HRH1 (overexpression); has-miR-1275 (downregulated expression), involved in the regulation of histamine receptor 1 – HRH3 (overexpression) and Solute carrier family 22 member 3 – SLC23A2 (downregulated expression)). Adalimumab and cyclosporine A modulated the histaminergic system in HaCaT cells in vitro . However, further studies are needed to elucidate the underlying mechanisms. Abbreviations: (-) – downregulated in comparison to the control, (+) – overexpression in comparison to the control, ACTB - β-actine, ADA - Adenosine deaminase, ADCYAP1 - Adenylate Cyclase Activating Polypeptide 1, BMP - bone morphogenetic protein, bp - base pair, cAMP - adenosine 3’ 5’-cyclic monophosphate, CBX7 - Chromobox protein homolog 7, cDNA - double-stranded complementary DNA, CSA - cyclosporine A DAG – diacylglycerol, DIAPH - Diaphanous related formin 1, DNMT - DNA methyltransferases, DRD2 - Dopamine receptor D2, EDN1 - Endothelin 1, EDNRA - Endothelin receptor type A, ELISA - Enzyme-linked immunosorbent assay, EZH2 - Enhancer of zeste homolog 2, FC - fold change, GABRB1 - Gamma-aminobutyric acid (GABA) A receptor, alpha 1, GABRB2 - Gamma-aminobutyric acid (GABA) A receptor, alpha 2, GABRB3 - Gamma-aminobutyric acid (GABA) A receptor, alpha 3, HaCaT - Human adult, low-calcium, high-temperature keratinocytes, HIS - Human Histamine, HLAs - human leukocyte antigens, HNMT - Histamine N-methyltransferase, HNMT - Histamine N-Methyltransferase, HRH1 – histamine receptor 1, HRH2 – histamine receptor 2, HRH3 – histamine receptor 3, HRH4 – histamine receptor 4, HTR6 - 5-Hydroxytryptamine Receptor 6, IGF1 - Insulin-like growth factor 1, IL10 -interleukin 10, IL12 -interleukin 12, IL...
Background: Increased levels of phosphorylated ERK and p38 MAPK proteins have been observed in psoriatic skin biopsies compared to controls, which may be associated with an impaired expression pattern of dual activity protein phosphatase (DUSP). Objective: The purpose of this study was to assess changes in the expression profile of mRNA DUSP 1-7 and miRNA regulating their expression in human keratinocyte cells (HaCaT) had exposed to the liposaccharide A (LPS). Methods: HaCaT was exposed to 1 µg/ml LPS and next adalimumab by 2,8,24h compared to untreated cells. The microarray method was used to analyze expression pattern of mRNAs, miRNAs, and ELISA to evaluate changes in the level of the proteins. RTqPCR was used to validate the microarray data. Transcriptome Analysis Console and Statistica Software 13 PL were used in statistical analysis (p<0.05). Results: The highest changes in expression was observed for DUSP2 (FC +11.12) and DUSP5 (FC +5.53) in HaCaT culture after 2 hours exposition on adalimumab. It was observed that miR-1275 (FC -2.39) and miR-34a (FC +6.52) might regulate level of DUSP2, and miR-27a (FC +3.55), miR-27b (FC +2.87) are involved in DUSP5 expression. Conclusion: The results obtained suggest that DUSP2 and DUSP5 may be considered as complementary molecular markers in the diagnosis and monitoring of the effectiveness of psoriasis therapy. It was confirmed that hsa-miR-34a, hsa-miR-1275, hsa-miR-3188, hsa-miR-382, hsa-miR-27a, hsa-miR-27b, hsa-miR-16 have the highest influence on the expression pattern of DUSP1-7.
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