The Influnce Of Liver X Recepter Alpha Promoter Activity Under Inflammatory Cytokines In HepG2and HMCs Cells

PART ONE: CONTRUCTION OF THE HUMANLIVER X RECEPTOR ALPHA PROMOTER LUCIFERASEREPORTER PLASMIDObjectives: This section is intended to construct the human liver Xreceptor alpha promoter luciferase reporter plasmid.Methods: Liver X receptor alpha promoter fragments was amplifiedby PCR, the product was digested by restrict enzymes and then combinedwith the luciferase report vector pGL3-Basic digested by the sameenzymes.Results: DNA sequencing showed pGL3-Basic-LXRα-P wasconstructed successfully.Conclusion: The LXRα promoter luciferase reporter plasmid wasconstructed successfully. PART TWO: THE INFLUENCE OF LXRα PROMOTERACTICITY UNDER INFLAMMATORY CYTOKINES INTHE HEPG2CELLSObjectives: To investigate whether inflammatory cytokines aggravatedcholesterol accumulation in liver by inhibiting LXRα signaling pathway inHepG2cells.Methods: pGL3-Basic-LXRα and pRL-TK were cotransfected intoHepG2cells and incubated with serum free medium or10μmol/LT0901317or20ng/ml inflammatory cytokines or100μg/ml LDL or20ng/ml TNF-α plus10μmol/L T0901317or20ng/ml TNF-α plus100μg/ml LDL or20ng/ml inflammatory cytokines or20ng/ml IL-6plus10μmol/L T0901317or20ng/ml IL-6plus100μg/ml LDL for24h to detectthe LXRα promoter activities. The gene and protein expression of LXRα，ABCA1and ABCG1were examined by RT-PCR and western blotting.Oil Red O staining and intracellular cholesterol assays were used toquantify cellular cholesterol levels.Results: Inflammatory cytokines decreased LXRα promoter activitiesin the absence or presence of LDL and T0901317. Inflammatory stressinhibited LXRα, ABCA1, and ABCG1mRNA and protein expression. Theresults of Oil Red O staining and quantitative intracellular cholesterolassays demonstrated that inflammatory stress increased cholesterol levels in HepG2cells.Conclusion: Inflammatory cytokines TNF-αexacerbates the hepaticcholesterol accumulation via inhibiting LXRα promoter activities and geneexpression. PART THREE: THE INFLUENCE OF LXRα PROMOTERACTICITY UNDER INFLAMMATORY CYTOKINES INTHE HMCS CELLSObjectives: To investigate whether inflammatory cytokines aggravatedcholesterol accumulation in kidney by inhibiting LXRα signaling pathwayin HMCs cells.Methods: pGL3-Basic-LXRα was transfected into HMCs cells andincubated with serum free medium or10μmol/L T0901317or20ng/mlinflammatory cytokines or100μg/ml LDLor20ng/ml TNF-α plus10μmol/L T0901317or20ng/ml TNF-α plus100μg/ml LDL or20ng/mlinflammatory cytokines or20ng/ml IL-6plus10μmol/L T0901317or20ng/ml IL-6plus100μg/ml LDL for24h to detect the LXRαpromoter activities. The gene and protein expression of LXRα，ABCA1wereexamined by RT-PCR and western blotting. Oil Red O staining andintracellular cholesterol assays were used to quantify cellular cholesterollevels.Results: Inflammatory cytokines decreased LXRα promoter activitiesin the absence or presence of LDL or T0901317. Inflammatory stressinhibited LXRα, ABCA1, and ABCG1mRNA and protein expression. Theresults of Oil Red O staining and quantitative intracellular cholesterolassays demonstrated that inflammatory stress increased cholesterol levelsin HMCs cells.Conclusion: TNF-αexacerbates the hepatic cholesterol accumulation viainhibiting LXRα promoter activities and gene expression.