| Background: Indoleamine 2, 3-dioxygenase(IDO) is a rate-limiting enzyme which is responsible for tryptophan metabolism outside the liver, it degrades the essential amino acid tryptophan and generates a series of metabolites. The abnormal IDO activity induces the dysregulation of neuroimmune, involved in the occurrence of neuropsychiatric diseases. In addition to its effects on the immune system, tryptophan catabolism also affects the central nervous system(CNS). IDO can transform tryptophan, the precursor for the neurotransmitter serotonin, into kynurenine metabolism, leading to production of series of toxic metabolites. Within the CNS, 95% of the tryptophan is catabolized through the kynurenine pathway, which can be activated by pro-inflammatory cytokines via IDO.Pro-inflammatory and anti-inflammatory cytokines influence the activity of IDO in opposite ways. Pro-inflammatory molecules such as IFN-γ, TNF-α, IL-1β, synergistically increase IDO activity and tryptophan consumption. In contrast, anti-inflammatory cytokines IL-4 and IL-10 inhibit the IDO-mediated tryptophan catabolism in T-cell, dendritic cells, and macrophages. But in CNS, the effects on IDO by anti-inflammatory cytokines are not consistent. In neuroendocrine cells, IL-10 can potentially provide a protective effect by modulating IDO expression, while anti-inflammatory cytokines IL-4 and IL-13 greatly enhanced IDO expression in microglia.Objective: IL-10 is a typical anti-inflammatory cytokine, it not only inhibits inflammatory response, but also has the neuroprotective function, with the underlying mechanisms not fully understood. Whether the regulation of tryptophan-kynurenine pathway is the mechanism of IL-10 is need to be further studied. To address this question, this study aimed to examine whether IL-10 could function as a potential regulatory mediator on IDO-tryptophan pathway in CNS.Methods:(1) Remove the cerebral cortex of new born 1-3 days of c57 mouse, astrocytes be acquired by tissue digestion method, then the cells are used for morphological observation and purity identification.(2) The primary cultured cells were divided into four treatment groups: a) control, b) IFN-γ treated, c) IFN-γ + IL-10 treated, d) IL-10 treated. Immunofluorescence(IF), real-time quantitative PCR(RT-PCR) and western blot were used to detect changes in the expression of IDO.(3) Western blot was adopted to detect the phosphorylation of signal transduction and activator of transcription-3(STAT-3) and downstream cytokine signaling inhibitor(SOCS3) expression and the interaction between its signal pathway and IDO expression. For further reaseach, STAT-3 inhibitor WP1066 was adopt to explore the molecular mechanisms of IL-10.Results: The results showed that(1) The purity of primary cultured astrocytes by mechanical shaking method was more than 95%, the branch and body of cells were obvious and stretch.(2) The results of immunofluorescence staining, RT-PCR and western blot showed, the expression of IDO in IFN-γ + IL-10 group was significantly lower than IFN-γ group(P <0.05).(3) Immunofluorescence staining and western blot showed that IL-10 greatly increased the expression of p-STAT-3 and SOCS3 and decreased the expression of tyrosine kinase 1(JAK1). STAT-3 inhibitor WP1066 can reverse the role of IL-10, which revealed the molecular mechanisms of the inhibition of IL-10 on IDO.Conclusions:(1) IFN-γ significantly induced the expression of IDO in astrocytes, and IL-10 has a significant inhibitory effect on the expression of IFN-γ induced IDO.(2) This effect is achieved by inhibiting the activity of JAK/STAT-1.(3) The expression and activity of STAT-3, SOCS3 and JAK is the inside signaling pathway of inhibited IL-10 on IDO expression.(4) Due to the important role of IDO in CNS diseases, therefore in the CNS, the role of IL-10 on IDO expression and activity, not only provide a new explain for the protective effect IL-10 in the CNS, but also discover a new therapeutic target for CNS disorders. |
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