Study Of Effects Of Single Immunoglobin IL-1Receptor Related Protein On High Mobility Group Box1-Induced Inflammation In A549Cells

BackgroundALI(Acute Lung Injury) is a severe clinical syndrome with high mortality.It features the acute pulmonary dysfunction, associated with obvioushyoxemia and massive exudation. ALI is also one of the major post-surgerycomplications in the sphere of thoracic surgery. HMGB1(high mobility groupbox1) is a kind of DNA-binding protein, which mainly serves as the functionof stabilizing the structure of nucleosome and regulating the gene transcription.The proteins come out of the damaged cells which are experiencing apoptosisor necrosis, playing the role of the pro-inflammatory factors which may causeinflammation. HMGB1constantly stimulates the body system and provokesinflammation in the case of ALI featuring septic shock. Therefore, inhibitingthe immune activity of HMGB1might offer better time frame for treatingsystemic diseases such as ALI. Recent researches suggest the receptors ofHMGB1is TLR2(Toll-like receptor2), TLR4, and RAGE(the receptor for advanced glycation end products), in which TLR2and TLR4might be morerelevant than RAGE. SIGIRR(Single Immunoglobin IL-1Receptor Related protein)is one of the members of TIR(Toll-IL-1Receptor) superfamily, which maynegtively regulate many kinds of innate immune reaction mediated byTLRs(Toll-like Receptors). Studies have shown that SIGIRR dampens theTLR2signal cascades in kidney mononuclear cells induced by Pam3Cys andalleviates TLR4signal pathway induced by LPS. And HMGB1has beenconfirmed to cause the extracellular inflammation through TLR2and TLR4,and therefore it can be inferred SIGIRR might moderate the HMGB1-inducedinflammation.ObjectiveTo identify the effects of single immunoglobin IL-1receptor relatedprotein （SIGIRR） on high mobility group box1（HMGB1） inducedinflammation in human alveolar epithelial cells A549, make sure theanti-inflammatory effects of SIGIRR in the later phase of ALI, and offertheories for gene therapy of ALIMethodsEukaryotic expression vectors pCDNA3.1（+） constructed with SIGIRRcDNA were transiently transfected into the human type II alveolar epithelialcell derived A549cells，in which SIGIRR was forced to be over-expressed.Western blot and RT-PCR were applied to detect the expression levels ofSIGIRR after transfection. After the stimulation on A549cells by HMGB1，the transcriptional activity of NF-κB was detected by dual-luciferase reporterassay system，and the protein levels of inflammatory cytokine TNF-α and IL-1β were measured by ELISA.ResultsThe expression levels of SIGIRR increased significantly in A549cellstransfected with SIGIRR vectors. The transcriptional activity of NF-κB wasenhanced obviously after HMGB1treatment in A549cells by dual-luciferasereporter assay system，while the transfection of SIGIRR vectors decreased theactivity. The protein levels of TNF-α and IL-1β were down-regulated in A549cells over-expressing SIGIRR after HMGB1stimulation，compared with thenon-transfected cells.ConclusionsUp-regulated SIGIRR expression could inhibit HMGB1-inducedcytokine release in A549cell such as TNF-α and IL-1β. The transcriptionalactivity of NF-κB was dampened by SIGIRR transfection，implying that theanti-inflammatory effects of SIGIRR might be its involvement in theregulation of NF-κB.