The Effect And Mechanism Of Sinomenine On Chemokines And Their Receptors Of Dendritic Cells In Patients With Rheumatoid

BackgroundRheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks the joints producing a inflammatory synovitis that often progresses to destruction of the articular cartilage and ankylosis of the joints. Rheumatoid arthritis can also produce diffuse inflammation in the lungs, pericardium, pleura, and sclera, and also nodular lesions, most common in subcutaneous tissue under the skin. Although the cause of rheumatoid arthritis is unknown, autoimmunity plays a pivotal role in its chronicity and progression. About 1% of the world's population is afflicted by rheumatoid arthritis, women three times more often than men. Onset is most frequent in 40 to 50 years, but no age is immune. It can be a disabling and painful condition, which can lead to substantial loss of functioning and mobility.The activation of T cells by as yet unknown antigens in the immunogenetically susceptible host is most probably the event that initiates the rheumatoid process. T cell activation subsequently leads to multiple effects including activation and proliferation of synovial lining and endothelial cells, recruitment and activation of additional proinflammatory cells from the bone marrow and circulation, secretion of cytokines and proteases by macrophages and fibroblast-like synovial cells, and autoantibody production. Dendritic cells are the most potent subset of antigen presenting cells. They are derived from bone marrow stem cells and reside in peripheral tissues or blood. Upon exposure to antigens and cytokines the peripheral DC s, express high amounts of peptide-MHC, and upregulate their costimulatory molecules, migrate to draining lymph nodes, and interact with T cells to stimulate or tolerize them. Dendritic cells have been found in synovium and joint fluid in rheumatoid arthritis, often at the center of a cluster of T cells. These DC s express MHCⅡ, the costimulatory molecules CD40, CD80, CD86, adhesion molecules such as DC-SIGN and chemokine receptors.According to Traditional Chinese Medicine, this condition is called Bi Zheng, which is typically divided into four types: Wind-Cold Bi, Cold-Bi, Dampness-Bi and Heat-Bi. Through a thorough examination and consultation, including an assessment of the pulse and tongue, a diagnosis is made. Therapeutic principles For incipient bi-syndrome, expelling pathogenic factors should be the main therapeutic principle, including dispelling wind, dispersing cold, clearing away heat, eliminating dampness and dredging meridians and collaterals. For a weak patient suffering from bi-syndrome or a chronic case with deficiency of healthy qi, in addition to the therapy for expelling pathogenic factors, tonifying the spleen, liver, kidneys and blood should also be employed. For cases complicated by phlegm and blood stasis, activating blood circulation, dissipating blood stasis and masses and eliminating phlegm should be applied. Sinomenine, an alkaloid extracted from the Chinese medicinal plant, Sinomenium acutum, has been utilized to treat RA in China for over 2000 years. A wide range of pharmacological actions which includes anti-inflammatory and anti-rheumatic effects can be mediated by sinomenine. Although sinomenine exhibits a wide range of pharmacological activities, mechanistic study on sinomenine is very limited yet. In order to further illustrate the immunosuppressive action of sinomenine, we deeply investgated the effect and mechanism of sinomenine on chemokines and their receptors of DCs in patients with RA.Objectives 1.To explore the relationship between disease activity and expression of chemokine receptors on the surface of dendritic cells in patients with RA;2. To build up detection system of real time PCR for measuring chemokines and their receptors of dendritic cells;3. To investigate the effect of sinomenine on expression of chemokines and chemokines receptors of dendritic cells in patients with RA;4. To evaluate the effect of sinomenine on the expression of ubiquitin and ubiquitin-protein ligase of dendritic cells in patient with RA.Methods1. Culture of monocyte-derived DCs in patients with RA in vitroPeripheral blood was collected and blood mononuclear cells were isolated. DCs were stimulalted with GM-CSF and IL-4 by the methods built in our past research.2. Determining the relationship between expression of chemokine receptors of RA patients and disease activityTweenty eight patients and ten healthy control were chosen and disease activity score 28 were calculated. DCs from patients with RA were divided into four groups according to the disease activity of patients or healthy control. The expression of CCR5 and CCR7 were detected by flow cytometer. The level of serum rheumatoid factor, C reactive protein, anti-CCP antibody were observed. The correlation of expression of CCR5, CCR7 and disease activity of RA patients were analyzed.3. Detecting the effect of sinomenine on the expression of chemokines and their receptors of DCs in patients with RADCs harvested from patients with RA were exposed to sinomenine 1mM, 2mM, 5mM sinomenine and medium for 12h, respectively. After treated with sinomenine, total RNA of DCs in patients with RA was isolated. cDNA of CCR5, CCR7, CXCL9, CXCL10, CXCL11 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 CCR5, CCR7, CXCL9, CXCL10, CXCL11 and GAPDH mRNA. Expression of CCR5, CCR7, CXCL9, CXCL10, CXCL11 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^{-△△t} and expressed as the fold increase compared to the untreated cells. Then, expression of CXCL9, CXCL10, CXCL11 were detected by ELISA.4. Observing the effect of sinomeine on expression ubiquitin and E3 ligase of DCsAfter exposed to sinomenine, DCs were collected and total RNA and protein was extracted. Expression of ubiquitin and E3 were analyzed by real time PCR and western blotting, respectively.5. Statistical analysisAll data are expressed as means±SD. Differences among one-way designed groups were analyzed by one-way ANOVA followed by Dunnett test. Linear relationship between two random variables were tested by Pearson correlation. A value of p<0.05 was considered statistically significant.Result1. Monocytes were cultured in the presence of GM-CSF and IL-4. Cells became nonadherent and clustered, exhibiting protruding veils typical of DCs. In contrast to healthy control, higher expression of CCR5 and CCR7 were observed in low, middle and high activity group.2. There are linear correlation relationship between CCR5 expressed on DCs and disease activity RF, CRP, anti-CCP antibody of RA patients in low activity(RF, r=0.775, P=0.014; CRP, r=0.802, P=0.009), middle activity(RF, r=0.0854, p=0.002; CRP, r=0.818, P=0.004) and high activity(RF, r=0.773, P=0.015; CRP, r=0.699, p=0.036) group, respectively. And the same relationship were also observed in low activity(RF, r=0.754, P=0.019; CRP, r=0.968, P=0.000), middle activity(RF, r=0.0854, P=0.002; CRP, r=0.949, P=0.000) and high activity(RF, r=0.938, P=0.000; CRP, r=0.968, P=0.000) group for CCR7.3. Significant differences of expression of CCR5(F=89.236, P=0.000) and CCR7(F=94.731, P=0.000) were observed in DCs exposed to 1mM, 2mM, 5mM sinomenine or control. Compared with control, CCR5 and CCR7(P<0.05 or P<0.01) were down-regulated in DCs treated with sinomenine at dose of 2mM or 5mM. Standard curves were obtained by real time PCR. Perfect linear correlations between different multiproportion dilution template for quantitation and cycle number (0.9943 for GAPDH, 0.9955 for CCR5, 0.9926 for CCR7, 0.9981 for CXCL9, 0.9938 for CXCL10, 0.9994 for CXCL11) were found in each group. Sinomenine appeared to effectively inhibit the expression of CCR5, CCR7, CXCL9, CXCL10, CXCL11 mRNA in DCs. Compared with control, significant differences were found in DCs treated with 2mM and 5mM sinomenine (P0.05 or P<0.01). Significant down-regulation of CXCL9, CXCL10, CXCL11 secretion were noticed in DCs exposed to sinomenine comparing to that treated with medium alone.4. There were significant differences at expression of ubiquitin(F=41.222, P=0.000) and E3 ligase(F=59.657, P=0.000) mRNA between control and sinomenine groups. In contrast to control, DCs treated with 2mM, 5mM sinomenine expressed lower ubiquitin and E3 ligase. The similar result was also observed in the expression of ubiquitin and E3 ligase in protein level detected by Western Blot assay.Conclusion1. Chemokine receptors are expressed on DCs in RA patients with different disease actitivy. Chemokine receptors can be used as a index to monitor the disease activiy of RA.2. Sinomenine can effectively suppress the expression of chemokines and their receptors of DCs in patients with RA at mRNA and protein level. Sinomenine exhibits the effect of relieving RA through inhibiting the expression of chemokines and their receptors, which can lead to DCs migration.3. DCs can effectively decrease the expression of ubiquitin and E3 ligase of DC. The influence of sinomenine on DCs may relate to inhibit the ubiquitin system, which account for the maturation and migration of DCs.