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The Roles and Mechanisms of Methotrexate or Simvastatin on Th17/IL-17 in Peripheral Blood Mononuclear Cells from Rheumatoid Arthritis in Vitro Study

Author: LiYanShan
Tutor: JiangLinZuo
School: Fudan University
Course: Internal Medicine
Keywords: IL-17 Methotrexate Arthritis, rheumatoid Mononuclear Cells Simvastatin BATF
CLC: R593.22
Type: Master's thesis
Year: 2011
Downloads: 97
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Abstract

First part: different concentrations of methotrexate for rheumatoid and healthy people in peripheral blood mononuclear cells Th17/IL-17 effects of background: rheumatoid arthritis (class Leifengguan) is a systemic, inflammatory autoimmune disease recurrent synovitis can cause joint destruction, progressive disorder, severe disability. Methotrexate (Methotrexate, MTX) clinical widely used and preferred change disease modifying antirheumatic drugs (disease-modifying anti-rheumatic drugs DMARDs), its mechanism of action remains unclear. Th17 cells as an important immune regulatory cells, the secretion of IL-17 is an important inflammatory factor involved in rheumatoid inflammatory reaction. MTX as a valid class wind off therapy, its anti-inflammatory effects and Th17/IL-17 relationship is not clear. Purpose: in vitro observation of peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs) of Th17 cell differentiation and IL-17 synthesis effects of different concentrations of MTX explore MTX may be effective in treating rheumatoid mechanism. Method: separation of rheumatoid patients and healthy people in PBMCs, to different concentrations of MTX pretreatment, stimulation of anti-human CD3 and anti-CD28 mAb, 37 ℃ 5% CO2 incubator. Were cultured at different stages of the collection of cells or supernatant, using reverse transcription by RT-PCR analysis of the expression level of IL-17mRNA; ELISA assay IL-17 concentration in the liquid cell culture medium; fluorescent antibody-labeled cell surface antigens CD4 and extracellular within the protein IL-17, flow cytometry analysis of the proportion of cells in CD4 IL-17. Results: rheumatoid and healthy human PBMCs after anti-CD3 and anti-human CD28 mAb activation, IL-17mRNA expression level (RA: vs.0.54 ± 0.15, 0.78 ± 0.14, p = 0.02: HC: 0.71 ± 0.17 vs. 0.34 ± 0.14, p lt; 0.01) and IL-17 protein secretion (RA: 436.16 ± 205.08 vs.15.36 ± 6.57, p <0.01: HC: 271.83 ± 145.11 vs.11.48 ± 7.12, p lt; 0.01) was significantly liter high, and different concentrations of MTX group to varying degrees weakened the IL-17mRNA and IL-17 secretion of protein levels, 5.0 and 25.0μg/mlMTX significantly inhibit the the of IL-17mRNA IL-17 protein production (p <0.05). Rheumatoid PBMCs produce higher levels of IL-17, while the MTX more rheumatoid PBMCs expression and secretion of IL-17 is significantly suppressed compared with healthy human PBMCs. MTX dose-to-value (lgMTX) expression levels of IL-17mRNA negative correlation (RA: R2 = 0.96, p = 0.02; HC: R2 = 0.94, p = 0.03), the relationship did not show this effect in the IL-17 protein secretion levels. MTX did not significantly reduce the proportion of CD4 IL-17 T cells in PBMCs. Conclusion: MTX inhibition of rheumatoid and healthy human PBMCs synthesis and secretion workers L-17, such a role in rheumatoid PBMCs more pronounced inhibition of IL-17 generated may be one of the mechanisms of MTX effective treatment of rheumatoid. Part II: simvastatin mononuclear cells of rheumatoid and healthy people synthetic work L-17 and Thl7 transcription factor BATF impact background: class wind off as a chronic inflammatory disease, in the destruction of the synovial membrane leading to joint function The loss also increases the risk of cardiovascular disease. The statin class of lipid-lowering drugs, cardiovascular drugs, were found in recent years has immunomodulatory effects have significant anti-inflammatory effect in rheumatoid treatment. IL-17 is an important inflammatory factor, B-cell activating transcription factor (B-cell activating transcription factor, BATF) involved in rheumatoid inflammatory reaction is important transcription regulation of the IL-17 gene transcription factor which belongs to the activated protein -1 (activator protein-1, AP-1) family. Statins affect the generation of inflammatory cytokines by AP-1 pathway, will play an unclear role in the treatment of rheumatoid inhibit IL-17 synthesis by acting on the BATF. Objective: To study simvastatin rheumatoid PBMCs synthesis of the role of IL-17 and B-cell activation of the transcription factor BATF. : The health and Leifengguan PBMCs with or without simvastatin or mevalonate conditions to PMA and ionomycin stimulation training. By RT-PCR analysis of IL-17, BATF and of NFAT2 the mRNA expression levels of 3 - glyceraldehyde phosphate dehydrogenase (GAPDH) as internal reference; ELISA assay IL-17 concentration in the culture medium of the cells in each group; Western Blot analysis of BATF protein expression changes after simvastatin. Results: rheumatoid PBMCs 20μM simvastatin role after the the IL-17mRNA level induced by PMA and ion neomycin inhibition (0.60 ± 0.18 vs.0.3l ± 0.17, p = 0.04), IL-17 protein secretion also weakened (79.64 ± 12.67 vs.44.61 ± 3.90, p9-0.01). Rheumatoid PBMCs by the BATF mRNA level was significantly higher than that in healthy people (vs.0.25 ± 0.07, 0.39 ± 0.05, p = 0.04), but after simvastatin BATFmRNA expression level of change is not obvious, BATF protein levels were also no significant changes. Mevalonate and simvastatin join PBMCs, the effect of simvastatin on the role of IL-17 produced significantly lower. Conclusion: Simvastatin is possible to suppress the activation of PBMCs synthesis and secretion of IL-17, this effect is not produced by inhibiting the BATF the expression of the transcription factor which, despite the BATF in the Leifengguan PBMCs in high expression.

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CLC: > Medicine, health > Internal Medicine > Systemic disease > Autoimmune diseases > Autoimmune diseases, connective tissue disease > Rheumatoid arthritis
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