Il - 10 For The Readjustment Of Ido Expression In Astrocytes

Indoleamine2,3-dioxygenase (IDO) is the first and rate-limiting enzyme incatalyzing tryptophan via the kynurenine pathway in mammals. In the condition ofinflammation, infection or injury, IDO can be induced to be over expression in centralnervous system(CNS), which lead to reduction of tryptophan level and production ofseries of toxic metabolites. These toxic metabolites make neurons dyfunction and death.It has been found that IDO increased expression and activity can be detected in manyneuro degenerative diseases, which revealed kynurenine pathway has a closerelationship with many neurodegenerative diseases.Neuroimmunology studies reveal that many neurodegenerative diseases are relatedto inflammation, because of pro-inflammatory and anti-inflammatory cytokines allexpress in CNS, and the imbalance between the two cytokines can determine the diseasecondition. Pro-inflammatory substance lipopolysaccharide(LPS), Interferon-γ(IFN-γ),interleukin-1β(IL-1β) may exacebrate the disease, correspondingly, anti-inflammatorycytokine IL-4, IL-10, transforming growth factor-β(TGF-β) may alleviate theinflammatory response in CNS. IL-10as a typical anti-inflammatory cytokine not onlyinhibit inflammatory response, has the function of neuron protection. Whether thefunction of IL-10is through regulation of tryptophan-the kynurenine pathway is still tobe further studied.This study used primary astrocyte culture as model, to discuss the effects of IL-10on IDO expression and activity, futher probe the signaling pathway involving in theregulation. We isolated the cortical glias from C57BL/6J mice which had born for oneor two days, then cultures in DMEM high glucose medium, using immunofluorescenceto inditify the astrocytes purity. Using1μg/ml LPS to stimulate astrocyte, adoptingReal-Time PCR to detect IDO mRNA, immunofluorescence and western blot to find outprotein expression, using colorimetric method to assay IDO activity. Add10ng/mlrecombinant murine IL-10to find out changes of IDO expression and avtivity whenLPS exits or not, at last use western blot to find out the effect of key transcription factor NF-κB.The results showed that the astrocytes purity is over95%.1μg/ml LPS caninduced IDO mRNA significantly, compared to control(P <0.05), after48h, there is aincreasing of IDO positive cells,and western blot revealed that there is a high proteinlevel(P <0.05),96h avtivity also increased(P <0.05). IL-10alone can enhance IDOgene, protein, and avtivity level, but it is not significant. But IL-10can inhibit IDOexpression induced by LPS, which displayed at gene(P <0.05), protein(P <0.05) andavtivity level. And IL-10can inhibit I-κBα phosphorylation, that inhibit NF-κB activity.In this study, we found that LPS can highly induce astrocyte IDO expression, andIL-10can downregulate IDO expression and activity induced by LPS, and it is supposedthat IL-10inhibit IDO expression partly through inhibition of NF-κB.IDO plays animportant role in CNS disease, found that the regulation of IDO, which provide the newtheoretical explanation for the neuron protection function of IL-10and new target forneurodegenerative diseases.