The Mechanism Underlying RIG-I-involved In Inflammation In Astrocytes Under Chemical Hypoxia

The retinoic acid-inducible gene I（RIG-I）is a crucial cytoplasmic pathogen recognition receptor and it plays a vital role in the inflammatory process.Cellular damage factors,such as ischemia,traumatic injury,poisons,and irradiations are recognized triggers of pattern recognition receptors.These drive inflammatory responses and contribute to inflammation stress.Recent studies suggest that RIG-I mediates its response through several pathways.The innate immune system is a pivotal component of inflammation.IPS-1 is a critical RIG-I downstream adaptor,and is essential for the innate immune response.IPS-1contains multiple TRAF-interacting motifs in the proline-rich region,which correlate with domain of TNF receptor-associated factor（TRAF）family.Specifically,TRAF6has been suggested to play a vital role in the expression of proinflammatory cytokines.TRAF6 signaling activates the canonical IKK complex,resulting in nuclear factor-κB（NF-κB）activation.Furthermore,RIG-I associated with apoptosis-associated speck-like protein（ASC）can trigger caspase-1-dependent inflammation and cytokine releases such as IL-1βand IL-18.They orchestrate the innate immune response to infection and regulate the expression of IL-1β,IL-6,IL-8,and TNF-α.Intracellular pattern recognition receptors are involved in the pathogenesis of a variety of inflammation-related diseases.Astrocytes,the largest of the glial cells,exert a important role in neuroinflammation.Aside from infection by viruses and other pathogens,hypoxia-related stimuli can also trigger inflammation in the nervous system.RIG-I is closely related to the inflammatory stress of brain glial cells and might participate in the astrocytic response to various stimuli,providing a potential mechanism underlying neuronal injury.And its role in regulating hypoxia-induced glial inflammation is unknown.To further characterize the mechanism underlying RIG-I-induced inflammation in astrocytes subjected to hypoxia,we established a model of chemical hypoxia in human astroglial cell lines in vitro.OBJECTIVES1.To confirme whether RIG-I is involved in the neuroglial inflammation under chemical hypoxia.To identify the role of NF-κB in RIG-I-induced neuroinflammation2.To study role of the RIG-I/IPS-1 pathway in chemical hypoxia-induced inflammation.To explore the signaling pathway regulated by Rig-I in neuroglial inflammation induced by chemical hypoxia.3.To characterize the mechanism underlying RIG-I-induced caspase-1 activation in astrocytes subjected to chemical hypoxia and Changes in intracellular osmotic pressure associated with glial cell edema.METHODSThe human astrocyte U87 cell line was exposed to CoCl₂ for 24 hours to establish a model of chemical hypoxia in human astroglial cell 87 in vitro.Real-time RT-PCR and Western Blot were respectively used to detected mRNA levels and protein levels.Subcellular isolation was used to study the nuclear translocation of NF-κB.1.we analyzed mRNA levels or protein levels of RIG-I extracted from U87 cells at different time-points following chemical hypoxia（0,1,3,6,12,and 24 h）.The position of RIG-I protein was observed by immunofluorescence staining.U87 cells were transfected with RIG-I plasmids of high expression,mRNA levels and protein levels of RIG-I or activity and cellular localization of NF-κB was investigated.An NF-κB inhibitor,ammonium pyrrolidinedithiocarbamate（PDTC）,was used to block the NF-κB pathway.Then,we detected mRNA levels of pro-inflammatory cytokines,such as IL-1β,IL-6,and TNF-α.2.NF-κB/P65 activity was first studied following CoCl₂ stimuli at differenttime-points（0,1,3,6,and 24 h）.U87 cells were transfected for 24 hours with siRNA against IPS-1 prior to CoCl₂ treatment.Activity of NF-κB was investigated following CoCl₂ treatment.Secondly,the protein level of IPS-1 were detected after 24 hours of CoCl₂treatment.U87 cells were transfected for 24 hours with siRNA against Rig-I prior to CoCl₂ treatment.The protein levels of IPS-1 and the mRNA levels of inflammatory factors,such as IL-1β,IL-6,and TNF-α,were measured following CoCl₂ treatment.Finally,we investigated the role of TRAF6 in RIG-I/IPS-1-induced NF-κB activation and inflammation in response to chemical hypoxia.Immunoprecipitation was used to detect the combination of IPS-1 and TRAF6.U87 cells were transfected for 24 hours with siRNA against TRAF6 before CoCl₂ treatment.Western blotting was performed to detect activity of NF-κB after CoCl₂ treatment.U87 cells were transfected for 24 hours with siRNA against TRAF6 or RNA fragment as control.The total RNA of U87 was extracted and transcribed into cDNA,and the levels of mRNA of the pro-inflammatory cytokines,such as IL-1β、IL-6 and TNF-α,were detected following CoCl₂ treatment.3.The distribution of caspase-1/P20 was detected by immunofluorescence,and the cell nucleus were marked with blue fluorescence,and the green fluorescence indicated the protein levels of caspase-1/P20.The protein of caspase-1/P20 was investigated following CoCl₂ stimuli for 24 hours.U87 cells were transfected for 24 hours with siRNA against Rig-I prior to CoCl₂ treatment,the protein of caspase-1/P20 was measured following Co Cl₂ stimuli for 24 hours.The protein of caspase-1/P20 was measured following U87 cells treated for 24 hours with CoCl₂ accompanied by Calcium Channel Blpckers Nifedipine or Carboplatin or Na-K-2Cl inhibitor Bumetanil or Free radical scavenger NAC.The changes of intracellular osmotic pressure related to edema induced by chemical hypoxia were detected by freezing point osmotic pressure instrument.U87cells were treated with CoCl₂ and Nifedipine or Carboplatin or NAC or Bumetanil,or transfected for 24 hours with siRNA against Rig-I before CoCl₂ stimuli.The cell and cytoplasm were extracted to detect the changes of cell osmotic pressure after CoCl₂treatment.RESULTS1.The data from real-time RT-PCR indicate that mRNA of RIG-I was up-regulated at 3,6,12,and 24 h following chemical hypoxia.,Western blots also demonstrate that protein levels of RIG-I were up-regulated compared to control（0h）（P<0.05）.Immunofluorescence staining showed that the protein levels of RIG-I significantly increased after CoCl₂ treatment.（P<0.05）.Both mRNA of RIG-I and protein levels of RIG-I were up-regulated,the phosphorylation of NF-κB/P65 at Ser536 was increased in response to RIG-I overexpression compared to control（empty plasmid transfection）（P<0.05）.But total protein levels remained unchanged in U87 cells（P>0.05）.Protein levels of NF-κB in the nucleus were increased and protein levels of cytoplasm were decreased following RIG-I activation（P<0.05）.Compared to control（empty plasmid transfection）,RIG-I activation could raises the mRNA levels of pro-inflammatory cytokines,such as IL-1β,IL-6,and TNF-α（P<0.05）,which was significantly attenuated by PDTC treatment in comparison with solvent groups（P<0.05）.2.The phosphorylation of NF-κB/P65 at Ser536 was increased at 3,6,12,and 24 h following chemical hypoxia compared with control（0h）（P<0.05）.U87 cells were transfected with siRNA against IPS-1 prior to CoCl₂ treatment.IPS-1 siRNA significantly attenuates NF-κB activity or mRNA of IPS-1 and protein levels of IPS-1under chemical hypoxia（P<0.05）.The protein levels of IPS-1 in astrocytes were significantly increased after chemical hypoxia,which could be reversed by siRNA-RIG-I compared with control（disorderly RNA fragment）（P<0.05）.The mRNA levels of IL-1β,IL-6,and TNF-αin astrocytes treated by IPS-1 siRNA were significantly lower than those elicited only by RIG-I overexpression（P<0.05）.Chemical anoxia can induce distinct increase in the combination of TRAF6 and IPS-1 in contrast with Con（si-Con+CoCl₂）（P<0.05）.si-RIG-I treatment results in the decrease of TRAF6 and IPS-1 binding（P<0.05）.The phosphorylation of NF-κB/P65 at Ser536 was decreased or mRNA levels of pro-inflammatory cytokines,such as IL-1β,IL-6,and TNF-α,were reduced through si-TRAF6 dispose（P<0.05）.3.Immunofluorescence shows that green fluorescent of U87 cells in Norm or control group turned fainter.Compared to control,green fluorescent of U87 treated with CoCl₂ for 24 hours brighten and protein levels of caspase-1/P20 rised significantly（P<0.05）.U87 cells were transfected for 24 hours with Rig-1 siRNA and the protein levels of caspase-1/P20 significantly decreased following the down-regulation of RIG-I compared to control RNA（si-con）（P<0.05）.U87 cells were treated with CoCl₂ accompanied by Nifedipine or Carboplatin or Na-K-2Cl inhibitor Bumetanil or Free radical scavenger NAC and the protein levels of caspase-1/P20were reduced compared with solvent group（P<0.05）.Intracellular osmotic pressure significantly increased after chemical hypoxia compared with solvent control（P<0.05）.Calcium antagonist can partially inhibit the osmotic pressure enhancement induced by chemical hypoxia in comparison with control group of chemical hypoxia（P<0.05）.However,NAC or Na-K-2Cl inhibitors or RIG-I inhibition did not cause significant changes in intracellular osmotic pressure.CONCLUSIONS1.Chemical hypoxia in astrocytes can lead to up-regulation of RIG-I genetranscription and protein translation and enhance its biological function,which result in the expression of the inflammatory factors depended on NF-κB.RIG-I is involved in neuroinflammation through NF-κB Pathway in human astrocytes response to hypoxia2.Inflammatory effect of astrocytes induced by chemical hypoxia and is closely related to the upregulation of RIG-I function.Activation of RIG-I lead to NF-κB activation and nuclear translocation by inducing binding activation of IPS-1 and TRAF6,which in turn promotes the expression of pro-inflammatory cytokines in chemical hypoxia.Blocking of the IPS-1/TRAF6 pathway relieves RIG-I-induced neuroinflammation in astrocytes.3.RIG-I is also involved in the regulation of chemical hypoxia-induced caspase-1activation of glial cells and is associated with the activation of calcium signaling-dependent inflammatory factors.However,upregulation of RIG-I function was not associated with chemical hypoxia-induced regulation of glial edema.The above studies revealed that RIG-I regulates the expression and activation of inflammatory cytokines through NF-κB and caspase-1 signals in chemical hypoxia models,and participates in the regulation of IPS-1/TRAF6 signaling pathway.Therefore,it may be developed as a potential drug target for clinical treatment of ischemic stroke and provide new ideas for improving ischemic brain damage in middle-aged and elderly patients.