| Toll-like receptors are a large class of pattern recognition receptors (PRRs). The members in this family are membrane-bound proteins, distributing on cell membranes and endosome surfaces, structurally similar to interleukin-1 (IL-1). The biological function of TLRs is that when they combines with the specific ligands, they act a series of downstream protein cascades through signal transduction by MyD88-dependent or independence pathway, and transduce the signals to receptors of cell nucleus, finally lead to the activation of nuclear factor kappa enhancer binding protein (NF-κB), IL-1 receptor kinase (IRAK), activating protein-1(AP-1) and induce synthesis and release of IL-6, TNF-αand IFN. Although immune response/inflammation induced by certain cytokines such as IL-6 has been shown to have the inhibitory effects on virus replication, however, an over-activated immune response/inflammation would be harmful to animals.It is known that Oxymatrine has the effects of anti-inflammatory, anti-virus, anti-oxidation. In this study, we focused on the regulatory effects of oxymatrine on immune response. It has been shown that oxymatrine down-regulated the expression of TLR4, TLR2, MyD88 and NF-κB. However, the molecular basis of oxymatrine involved in immune regulation has not been fully elucidated. Whether oxymatrine has a inhibitory effect on the expression of toll-like receptor 7 (TLR7) and its downstream molecules MyD88 and TRAF-6 has not yet been reported. Studies on the detailed mechanisms of immune regulation of oxymatrine will provide useful information for prevention and treatment of autoimmune diseases.The effects of oxymatrine on TLR7 mRNA and its downstream cytokine were examined. (1) Analysis of cytotoxicity of oxymatrine on RAW264.7 macrophages. RAW264.7 cells were treated with oxymatrine at different concentrations ranging from 0.63 mg/mL to 20 mg/mL. (2) Evaluation of the effects of oxymatrine on the transcription of TLR7 and IL-6 in RAW264.7 cells, RAW264.7 cells were treated with 100μg/mL oxymatrine. The expression of TLR7 mRNA was determined by Real-time PCR analysis at 3, 6, 12, 24h post-treatment. The expression of IL-6 was analyzed by ELISA at 12,24h post-treatment. (3) To further confirm the results obtained, mouse splenic B lymphocytes that express TLR7 at high level were treated with oxymatrine to evaluate the effects of oxymatrine on the transcription of TLR7, MyD88, TRAF-6 and IL-6. Splenic B lymphocytes were incubated with oxymatrine of 100μg/mL and 500μg/mL, and then the expression of TLR7 and MyD88, TRAF-6 in the transcriptional level were determined by Real-time PCR analysis, the expression of IL-6 was determined by ELISA at 24h post-treatment.In response to oxymatrine treatment, TLR7 was found to be down-regulated at the transcriptional level in a time- and dose-dependent manner in RAW264.7 cells. The effect of oxymatrine at 24h post-treatment became weaker, it may due to the decreased concentration of oxymatrine. The secretion of IL-6 in RAW264.7 cells was inhibited by oxymatrine treatment. This inhibition was found to be in a time- and dose-dependent manner. The expression of TLR7, MyD88 and TRAF-6 at mRNA level was also found to be in a dose-dependent manner in splenic B lymphocytes. The secretion of IL-6 in splenic B lymphocytes was suppressed by oxymatrine in a dose-dependent manner at 24h post-treatment.In summary, we found that oxymatrine was able to down-graduate the expression of TLR7 in the transcription level and IL-6 expression in RAW264.7 cells, and to down-graduate the expression of TLR7, MyD88 and TRAF-6 in mRNA level and IL-6 expression in splenic B lymphocytes. The results imply that the immunosuppressive effects of oxymatrine may be related with the down-regulation of the expression of TLR7 and its downstream molecules.
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