Chlamydia Pneumonia Disturbs Cholesterol Metabolism In Human THP-1Macrophages Via NF-κB Signaling Pathway

Part I Effects of Chlamydia pneumoniae infection on the activation of NF-кB signaling pathway in THP-1-derived macrophagesObjective To investigate the effects of Chlamydia pneumoniae (C.pn) infection on the activation of NF-кB signaling pathway in THP-1-derived macrophages.Methods THP-1monocytes were induced differentiation into macrophages using160nmol/L phorbol myristate acetate (PMA) for48h, next they were randomly allocated into four groups:(1) negative control group:culture medium containing50μg/ml low density lipoprotein (LDL) for48h;(2) positive control group:culture medium containing50μg/ml oxidized-low density lipoprotein for48h;(3) different concentrations of C.pn infection group:50μg/ml LDL with1×105,4×lO5,5×105, and1×106IFU C.pn infection for48h;(4) different time of C.pn infection group:50μg/ml LDL with1×106IFU C.pn infection for24,48and72h. the activation of NF-кB signaling pathway was assessed by electrophoretic mobility shift assay.Results In the negative control group, the activation of NF-кB signaling pathway was not detected. However, the NF-кB activation was involved in the rest groups. In different concentrations of C.pn infection group, NF-кB activity increased along with its increasing concentration. In different time of C.pn infection group, NF-кB activity increased to highest level at24h after the same dosage of C.pn infection, and then gradually dropped down with time.Conclusion C.pn may dose-dependently increase the activation of NF-кB in THP-1-derived macrophages exposed to LDL, which reaches its maximal activity at24h. This will lay a theoretical foundation for our further study on the involvement of NF-кB signaling pathway in C.pn induced foam cell formation. Part Ⅱ The involvement of NF-кB signaling pathway in Chlamydia pneumoniae induced macrophage-derived foam cell formation.Objective To investigate the involvement of NF-кB signaling pathway in C.pn induced macrophage-derived foam cell by small interfering RNA (siRNA) technique.Methods1pairs of6-FAM-siRNA sequence,1pairs of negative control siRNA sequence, and3pairs of NF-кB P65siRNA sequences were synthesized and transfected into macrophage by lipofectamine2000according to the instructions. The transfection efficiency was observed and calculated under fluorescence microscope at6h after transfection using6-FAM-siRNA sequence. The silencing efficacy of the3pairs of NF-кB P65siRNA sequences were evaluated with Real Time-PCR (RT-PCR) for NF-кB P65mRNA expression and with Western-blot for protein expression. Subsequently, macrophages exposed to50μg/ml LDL were incubated with1×106IFU C.pn for48h, whereafter the negative control siRNA and2pairs of NF-кB P65siRNA with better silencing efficacy transfection for24h respectively. Oil Red O staining was used to observe the lipid droplets in cytoplasm and foam cell formation. Results The transfection efficiency was83.33%±2.52. Compared to negative control siRNA, the NF-кB P65siRNA sequences transfection inhibited NF-кB P65mRNA and protein expression at different degrees. Among the3pairs of NF-кB P65siRNA sequences, NF-кB P65siRNA1and NF-кB P65siRNA3, which were used in our further experiments, have the better silencing efficacy. Also, the contents of intracellular lipid droplets and foam cell formation in NF-кB P65siRNA1and NF-кB P65siRNA3group decreased markedly compared to negative control siRNA.Conclusion NF-кB P65siRNA1and NF-кB P65siRNA3can significantly inhibit NF-кB P65expression in macrophage. The NF-кB gene silencing alleviates the formation of macrophage-derived foam cells induced by C.pn infection. It clarifies the significance of NF-кB signaling pathway in regulation of C.pn induced foam cell formation, which will provide potential new target for the therapy of clinical therapy of atherosclerosis. Part III The role of NF-кB signaling pathway in Chlamydia pneumoniae induced imbalance of macrophage cholesterol metabolismObjective To investigate the role of NF-кB signaling pathway in the regulation of key genes involved in macrophage cholesterol metabolism induced by C.pn infection.Methods THP-1monocytes were induced differentiation into macrophages using160nmol/L PMA for32-48h, next they were randomly allocated into three groups:(1) negative control siRNA group:50μg/ml LDL with1×106IFU C.pn infection for32h after interfered by negative control siRNA for32h;(2) P65siRNAl group:50μg/ml LDL with 1×106IFU C.pn infection for32h after interfered by NF-кB p65siRNAl for32h;(3) P65siRNA3group:50μg/ml LDL with1×106IFU C.pn infection for32h after interfered by NF-кB P65siRNA3for32h. RT-PCR and western blot analysis were used to evaluate Acyl-CoA cholesterol acyltransferase-1(ACAT1), scavenger receptor A-1(SR-Al), and ATP-binding cassette transporter Al and G1(ABCAl/Gl) mRNA and protein expressions, respectively.Results Compared to negative control siRNA group, C.pn infection after siRNA-mediated silencing of NF-кB P65gene resulted in down-regulation of AC ATI、 ABCAl/G1mRNA and protein levels, however, the expression of SR-Al did not change significantly in general.Conclusion NF-кB signaling pathway takes part in C.pn induced macrophage-derived foam cells formation by regulating key genes involved in macrophage cholesterol metabolism to induce imbalance of cholesterol metabolism homeostasis. Part IV The interplay between NF-кB and PPAR α/γ signaling pathway in Chlamydia pneumoniae induced macrophage-derived foam cell formation.Objective To investigate interplay between NF-кB and PPAR α/γ signaling pathway in Chlamydia pneumoniae induced macrophage-derived foam cell formation.Methods THP-1monocytes were induced differentiation into macrophages using160nmol/L PMA for32-48h and were randomly allocated into groups, next the experiment was divided into two protocols:Protocol1to investigate the effect of siRNA-mediated Silencing of NF-кB p65gene on PPAR a/y expression:(1) negative control siRNA group:50μg/ml LDL with1×106IFU C.pn infection for32h after interfered by negative control siRNA for32h;(2) P65siRNA1group:50μg/ml LDL with1×106IFU C.pn infection for32h after interfered by NF-кB p65siRNAl for32h;(3) P65siRNA3group:50μg/ml LDL with1×106IFU C.pn infection for32h after interfered by NF-кB p65siRNA3for32h. RT-PCR and western blot analysis were used to evaluate PPAR a/y expressions, respectively. Protocol2to investigate the effect of PPAR a/y activation on NF-кB activity:(1) negative control group:50μg/ml LDL with1×106IFU C.pn infection for32h;(2) Fenofibrate group:50μg/ml LDL with1×160IFU C.pn infection for32h after20μM/L specific PPAR a agonist fenofibrate pretreatment for2h;(3) Rosiglitazone group:50μg/ml LDL with1×106IFU C.pn infection for32h after20μM/L specific PPAR y agonist rosiglitazone pretreatment for2h. NF-кB activity was assessed by electrophoretic mobility shift assay.Results In the first part, compared to negative control siRNA group, C.pn infection after siRNA-mediated silencing of NF-кB P65gene resulted in significant up-regulation of PPAR a/y mRNA and protein levels. In the second part, compared to negative control group, both fenofibrate and rosiglitazone pretreatment inhibited the activation of NF-кB induced by C.pn infection.Conclusion There exist interaction between NF-кB and PPAR a/y signaling pathways in C.pn induced macrophage-derived foam cells formation, which has a combined effect in regulating key genes involved in macrophage cholesterol metabolism to induce imbalance of cholesterol metabolism homeostasis. It is thereby provide theoretical basis for molecular mechanisms involved in C.pn infection induced atherosclerosis and its subsequent potential therapeutic target.