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The Effect of Sinomenine on Dendritic Cells in Patients with Rheumatoid Arthritis and Its Mechanism

Author: ZhaoYi
Tutor: LiJuan
School: First Military Medical University
Course: Traditional Chinese Medicine
Keywords: Sinomenine Rheumatoid arthritis Dendritic cells
CLC: R259
Type: PhD thesis
Year: 2007
Downloads: 292
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BackgroundRheumatoid arthritis (RA) is the most common inflammatory joint disease, affecting about 1% of the population with a female: male ratio of 3:1. RA has a worldwide distribution, but the reported prevalence rates are at least three times higher in the industrialized countries than elsewhere. Although the disease can occur at any age, prevalence increases with age and the peak incidence is between the forth and sixth decade. RA has a wide clinical spectrum, ranging from self-limiting disease to progressive chronic disease, with extra articular manifestations. The disease is characterized by persistent and progressive synovitis of peripheral joints, leading to destruction of cartilage and subchondral bone. The pathogenesis of RA is still a dynamic area of research. The etiology of the early events in RA remains illusive and is most likely multifactorial. In RA, chronic inflammation is localized in the synovial tissue; this is what causes the clinical signs of arthritis. Tissue hyperplasia (fibroblast proliferation), hypervascularization and accumulation of white blood cells including T cells, B cells, plasma cells, monocytes/macrophages and granulocytes characterize the rheumatoid synovium. It has been proposed that T cells play a major role in the immune processes in RA. One of the strongest arguments for the T cell-dependent nature of the disease comes from clinical studies where susceptibility to RA and disease progression was linked to the expression of HLA-DR4 and HLA-DR1. RA can be seen as a disease driven by activated T cells responding to as yet unknown antigens. These molecules present antigens to the T cell receptor of CD4 positive T cells as a first step in T cell activation. After this antigen stimulus, the inflammatory event then depends on many different mediators operating in synergistic inflammatory cascades. Dendritic cells (DCs) are the most effective or ’professional’ of the antigen-presenting cells (APC) that initiate primary immune responses. They are located at surveillance sites where they capture and process antigens. Once DCs have arrived in draining lymph nodes, they push T cells into one of the direction followed by their interaction: polarize them toward a Th1 or a Th2 response, or induce T regulatory cells. They then initiate and regulate T- and B-cell responses by expressing lymphocyte costimulatory molecules, migrating to lymphoid organs and secreting biologically active molecules. Dendritic cells not only activate lymphocytes to induce the immune response, but they also minimize autoimmune reactions by tolerizing T cells to self-antigens. So, DCs play a critical role in RA.In traditional Chinese medicine (TCM), the condition that is congruent with arthritis is called "Bi syndrome." Bi syndrome manifests as pain, soreness, or numbness of muscles, tendons and joints, and is the result of the body being "invaded" by the external climatological factors of Wind, Cold, Heat, and/or Dampness. The symptoms manifested by the individual depend on which external pathogenic factor is strongest. The confluence of these pathogens in the affected areas also can lead to the generation of Heat, manifesting as inflammation. Long-term Bi syndrome can lead to the formation of Phlegm and the deformation of bones and joints. Pain and reduced range of motion in TCM indicates the presence of a blockage; conditions such as RA, therefore, are always treated by the methods of removing Wind-Dampness and smoothing meridians. Sinomenine is an alkaloid extracted from the Chinese medicinal plant, Sinomenium acutum, which has been utilized to treat RA in China for over 2000 years. Sinomenine has been shown to mediate a wide range of pharmacological actions which includes anti-inflammatory and anti-rheumatic effects. Although sinomenine exhibits a wide range of pharmacological activities such as anti-inflammatory, anti-rheumatic, immunosuppressive, analgesic and anti-arrhythmic, mechanistic study on sinomenine is very limited yet. In order to further illustrate the immunosuppressive action of sinomenine, we deeply investgated the effect of sinomenine on the immune function of DCs in patients with RA and the exact mechanism is also approached.Objectives1. To establish cultural system of human monocyte-derived dendritic cells in vitro;2. To investigate the effect of sinomenine on the immune function of dendritic cells in patients with RA;3. To build up detection system of real time PCR for measuring TLR2 mRNA and TLR4 mRNA;4. To evaluate the effect of sinomenine on the expression of TLR2, TLR4 mRNA and protein of dendritic cells in patient with RA;5. To observe the effect of sinomenine on the activity and translocation of nuclear factor-kappa B (NF-κB) of dendritic cells in patient with RA;6. To provide further insight into the mechanism of the immunesuppression of sinomenine, and, moreover, provide evidences and pathways of sinomenine to treat RA.Methods1. Culture and identification of human monocyte-derived DCsPeripheral blood was collected and blood mononuclear cells were isolated. After monocyte-enriched cells were cultured with GM-CSF and IL-4 in RPMI-1640 for 8 days, immature DCs were harvested. To obtain mature DCs, immature DCs were cocultured with LPS (1μg/ml) for 12h before cell collection. Morphology, phenotypes and ability to stimulate T cells were measured by optical microscope, electron microscope, flow cytometer and mixed lymphocyte reaction (MLR), respectively.2. Determining the effect of sinomenine on immune function of DCs in patiets with RAImmature DCs from patients with RA were obtained as previously described. Before harvested, DCs were exposed to 1mM, 2mM, 5mM sinomenine and medium for 12h, respectively. MTT assay was performed to confirm whether sinomenine has cytotoxity on DCs. IL-12p70 in cultural supernatant, phenotypes, ability to stimulate T cells were detected by ELISA, flow cytometer and MLR. Then, total RNA of DCs treated with sinomenine was extracted and expression of IL-12p35 mRNA, CD80 mRNA, and CD86 mRNA was analyzed by reverse transcription PCR (RT-PCR).3. Detecting the effect of sinomenine on the expression of TLR2 and TLR4 of DCsAfter treated with sinomenine, total RNA of DCs in patients with RA was isolated. cDNA of TLR2, TLR4 and GAPDH was amplified by PCR and products were purified. Templates were diluted in different concentration. Real time PCR was performed to build up standard curves for TLR2 mRNA, TLR4 mRNA and GAPDH mRNA. Expression of TLR2 mRNA, TLR4 mRNA and GAPDH mRNA for each sample was detected by real time PCR. The mRNA level of each sample for each gene was normalized to that of the GAPDH mRNA. The relative mRNA level was represented as 2-ΔΔt and expressed as the fold increase compared to the untreated cells. Then, protein of DCs exposed to sinomenine was extracted and western blotting was executed to detected expression of TLR2, TLR4 and GAPDH of DCs.4. Observing the effect of sinomeine on activity and translocation of NF-κB in DCsAfter exposed to sinomenine, DCs were collected and total RNA and protein was extracted. Expression of RelB, phosphorylation IκBαand phosphorylation p38MAPK was analyzed by RT-PCR and western blotting, respectively. Nuclear protein was also extracted and electrophoretic mobility shift assay (EMSA) was performed to measure the activity of NF-κB. Finally, laser confocal microscope was used to analyze the nuclear translocation of RelB.5. Statistical analysisAll data are expressed as means±SD. Differences among two-way designed groups were analyzed by factorial ANOVA, followed by analyzing main effect, separated effect and interaction. Differences among one-way designed groups were analyzed by one-way ANOVA followed by Dunnett test. Difference among two independent groups was analyzed by t-test. A value of p<0.05 was considered statistically significant.Result 1. Monocytes were cultured for 8 days in the presence of GM-CSF and IL-4. Cells became nonadherent and clustered, exhibiting protruding veils typical of DCs. After stimulated with LPS, DCs presented the typical mature morphous. In contrast to control DCs, CD1a (P<0.05), CD40 (P<0.01), CD80 (P<0.01), CD83 (P<0.01), CD86 (P<0.01) and HLA-DR (P<0.01) were higher in LPS-DCs. Stronger ability to activate T cell was also observed in LPS-DC at ratio of 5:1 (P<0.01), 10:1 (P<0.01), 20:1 (P<0.05), 50:1 (P<0.05).2. Significant differences of cell viability were not observed at the concentration of 1mM, 2mM, 5mM, 10mM comparing with the control, showing over 90% cell viability after sinomenine treatment. But the cell viability was markedly reduced at the concentration of 15mM and 20mM. Compared with control, CD40, CD80, CD83, CD86 and HLA-DR (P<0.05 or P<0.01) were down-regulated in DCs treated with sinomenine at dose of 2mM or 5mM. At ratio of 5:1, 10:1, 20:1, 50:1 the significant lower cpm values were observed in DCs treated with 2mM and 5mM sinomenine (P<0.01). A significant down-regulation of IL-12 secretion was noticed in DCs exposed to 5mM (P<0.01) sinomenine comparing to that treated with medium alone. Similar results were also observed using RT-PCR, DCs exposed to medium and 1mM sinomenine express higher level IL-12p35 mRNA than it in DCs treated with 2mM and 5mM sinomenine. In contrast to control, expression of CD80 mRNA and CD86 mRNA was significantly decreased in DCs exposed to 2mM and 5mM sinomenine.3. Standard curves were obtained by real time PCR. Perfect linear correlations between different multiproportion dilution template for quantitation and cycle number (0.9985 for GAPDH, 0.9945 for TLR2, and 0.9912 for TLR4) were found in each group. Sinomenine appeared to effectively inhibit the expression of TLR2 mRNA and TLR4 mRNA in DCs. Compared with control, significant differences were found in DCs treated with 2mM and 5mM sinomenine (P<0.01). Meanwhile, sinomenine also can decrease the expression of TLR2 and TLR4 in DCs at protein level. Compared with control, lower expression of TLR2 and TLR4 were also observed in DCs exposed to 2mM and 5mM sinomenine (P<0.01).4. No significant difference at expression of RelB mRNA was found in control and sinomenine groups (P>0.05). To assess the activation of NF-κB, EMSA was performed. Satisfactory specificity was observed in the result and capacity of binding DNA probes tagged with biotin declined after treated with sinomenine. Compare with control, lower gray scale values were found in 1mM (P<0.05), 2mM (P<0.01), 5mM (P<0.01) sinomenine groups. There were significant differences at expression of phosphorylation IκBα, between control and sinomenine groups. In contrast to control, DCs treated with 2mM, 5mM sinomenine expressed lower phosphorylation IκBα(P<0.01). But the similar result was not observed in the expression of phosphorylation p38 (P>0.05). To evaluate localization of intracellular RelB in DCs exposed to sinomenine, DCs strained with FITC-conjugated antibody and Hoechst 33342 were examined under a laser confocal microscope. NF-κB RelB immunofluorescent staining of DCs showed significant nuclear staining in the control cells, only few cytoplasmic staining. However, when treated with sinomenine, the nuclei staining became lesser with augment of sinomenine concentration.Conclusion1. Human monocytes can be differentiated into immature DCs by stimulated with GM-CSF and IL-4. The mature DCs can be induced by LPS from the immature DCs. Cultural system of human monocyte-derived DCs in vitro is successful and this may be helpful for subsequent experiments.2. Sinomenine can effectively suppress the immune function of DCs in patients with RA. The pharmacological actions of immunosuppression of sinomenine may be (1) inhibiting the expression of MHC II molecules on DCs blocks the antigen presenting function of DCs and the ability to activate T cells; (2) inhibiting the expression of costimulatory molecules on DCs blocks the costimulatory signal pathway, which may result in T cells anergy; (3) inhibiting the section of IL-12 in DCs blocks T cells differentiation and activation. The therapeutic effect of sinomenine on RA may be related to the pharmacological action of suppressing the immune function of DCs.3. The effect of sinomenine on decreasing the expression of TLR2 and TLR4 of DCs may block recognition of DCs for pathogens and antigens and also may block the activation signals for DCs. Sinomenine exhibits the effect of relieving RA through inhibiting the expression of TLR2 and TLR4, which can lead to DCs inactivation.4. DCs can effectively decrease the binding activity of NF-κB and RelB migration from cytoplasm to nucleus of DC, but no effect to RelB expression. The influence of sinomenine on DCs may not correlate to the level of NF-κB but its binding activity and translocation. Sinomenine mediated decrease of IκB phosphoralation induces the down-stream activation and translocation of NF-κB, and may account for the inhibition of the maturation of DCs.

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