Effects Of ZAG On NF-κB Inflammatory Signaling Pathway In LPS-Induced HepG2 Cells And Its Mechanisms

Objective:To construct pIRES2-ZsGreen1(-)-hZAG recombinant mammalian expressionvector, which were transient transfected into HepG2cell and observe its effects onthe inflammation factor TNF-α after over-expression of ZAG in LPS-inducedHepG2cells.Methods:1. The total RNA from HepG2cells was extracted. The reverse-transcript (RT)-PCRmethod used to amplify the complete domain sequence of human ZAG. Theconfirmed PCR products were inserted into expression plasmid by DNA ligation.After identification by RT-PCR and DNA sequencing, human ZAG expressionplasmids pIRES2-ZsGreen1(-)-hZAG containing human ZAG cDNA werecloned and transient transfected into HepG2cells by lipofection2000for24hours.The transient expression and transfection efficiency were observed by invertedfluorescence microscope.The expression of ZAG mRNA were detected byRT-PCR.2. HepG2cells were cultured well. The pIRES2-ZsGreen1(-)-hZAG containinghuman ZAG cDNA were transient transfected into HepG2cells by lipofection2000for24hours. Then these cells were treated with LPS(1μg/mL). The mRNAexpression of TNF-α were detected by fluorescent quantitative PCR.Results:1. Human ZAG coding sequence was amplified successfully and ligated with vectorfragments by DNA ligation. The construction of pIRES2-ZsGreen1(-)-hZAGplasmid containing human ZAG coding sequence when digested by SacII and XhoI. pIRES2-ZsGreen1(-)-hZAG was transfected into the HepG2cell, theexpression of ZAG mRNA were up-regulated in the transfected HepG2cell.2. LPS promote the expression of the inflammation factor TNF-α in HepG2cell,ZAG can reduce the expression of the inflammation factor TNF-α in HepG2cell.Conclusions：1. The recombinant eukaryotic vector of pIRES2-ZsGreen1(-)-hZAG wasconstructed successfully.2. ZAG can reduce the expression of the inflammation factor TNF-α induced byLPS in HepG2cell. Objective:To explore the mechanism of NF-κB signaling pathway mediate the effectsof ZAG on inflammation induced by LPS in HepG2cell.Methods:Human ZAG expression plasmids (pIRES2-ZsGreen1(-)-hZAG)were transient transfected into HepG2cells by lipofection2000for24hours, thenthe cells were treated with lipopolysaccharide and NF-κB inhibitor BAY11-7082.The protein expression of ZAG and p50, p65and IKKβ were detected by WesternBlot. The ranslocation of Phosphorylated P65were observed byimmunofluorescence.Results:1. LPS promoted the expression of relevant protein of NF-κB. ZAG reduced theexpression of relevant protein of NF-κB, NF-κB inhibitor BAY11-7082promotedthe effects of reduction of ZAG the expression of relevant protein of NF-κB.2. The results indicated that the ranslocation of P65induced by LPS could beobviously inhibited by overexpression of ZAG.Conclusions:ZAG had an inhibition effect on relevant factors of inflammatory reaction inHpG2cells. NF-κB signal pathway can mediate this effect.