SiRNA Targeting Of PPARα Enhance Thp-1-derived Foam Cell Formation Induced By Chlamydia Pneumoniae

Background With the deeper understanding of peroxisome proliferator-activated receptors (PPARs),Several studies have indicated that PPARs are important nuclear receptors that regulate macrophage cholesterol metabolism. And PPARαplay a important role in controlling macrophage cholesterol circle.Objective We use the RNA interfering technology, inhibit the expression of PPARαin THP-1 cells which expressed high level of PPARα. We observe the changes of these cells to screening siRNA sequences that have the best interfere effect and establish fundament for next experiment.Methods 3 pairs of short hairpin RNAs(shRNA) targeting PPARαand 1 pairs of short hairpin RNAs(shRNA) non-targeting PPARαwere designed and synthesized. Two single-strand DNA sequences were annealed and then inserted the vector which has been digested and lineared to form the recombinant plasmid.The same methods were used to construct the negative control vector with general negative control. Plasmid was transformed into DH5a E.coli for amplification.Plasmid Was extracted using plasmid extraction kit for enzyme digestion appreciation and sequencing.THP-1 cells were cultured with conventional method.Transfected was done with lipofectamine 2000 according to the process of instructions.Transfection efficiency was observed under fluorescence microscope at 24h after transfection..Interfere efficiency wasanalysed by Western blotting.Result four recombinant plasmids (pGenesil-PPARα-1,pGenesil-PPARα-2,pGenesil-PPARα- 3,pGenesil-control) were constructed. Efficiency of transfection detected at 24h after transfection were 69.21%±0.21 , 65.57%±0.35 , 70.56%±0.69 , 62.58%±0.26, respectively.The silence efficiency of the special targeting sequence detected at 48h after transfection were 52.90%±4.35,45.31%±6.15,80.14%±5.30,3.51%±1.12, respectively. conclusion recombinant plasmidpGenesil-PPARα-3 can significantly inhibited PPARαexpression in THP-1 cell.Part II The influence of PPARαgene silencing in the the formation of THP-1-derived foam cells induced by C.pn infectionBackground peroxisome proliferator-activated receptors (PPARs) are key nuclear receptors which regulate macrophage cholesterol metabolism. The imbalance of cholesterol metabolism homeostasis in macrophage contributes a lot to the cell formation. One of the typical pathological hallmarks of atherosclerosis is the excessive accumulation of cholesteryl esters in macrophages and their conversion to foam cells. Chlamydia pneumoniae (C.pn) infection may disturb the homeostasis of intracellular cholesterol metabolism which can lead to foam cell formation. It suggest the key roles of PPARαin Chlamydia pneumoniae-induced foam cell formation.Objective This research was to to investigate the effects of PPARαin C.pn-induced foam cell formation.Methods THP-1 monocytes were induced into macrophages by 160 nmol /L PMA for 48 h after that they were randomly allocated into five groups:①negative control group, culture medium with 50μg / ml low density lipoprotein (LDL) for 48 h;②positive control group, culture medium with 50μg / ml oxidized low density lipoprotein (ox-LDL) for 48 h;③C.pn infection group, 50μg / ml LDL with 1×106 IFU C.pn for 48 h;④i nterference control group, RPMI 1640 medium containing 10﹪FBS and 50μmol /L LDL for 48 h after interfered by PPARα;⑤interference infection group, 50μg / ml LDL plus 1×106 IFU C.pn infection for 48 h after interfered by PPARα; The mRNA and protein expressions of SR-A1,CD36,ACAT1,ABCA1 and ABCG1 were tested by Realtime-PCR and Western-blot, The contents of intracellular total cholesterol (TC) and cholesteryl esters (CE) were tested by enzymic chromatometry.Result there were significantly increases in the accumulation of he ratio of CE to TC in positive control,interference control group ,infection group and interference infection group,Compared with negative control. Not only the gradually up-regulation of SR-A1 and ACAT1 but also the gradually down-regulation of CD36, ABCA1 and ABCG1 at mRNA and protein levels were detected(p < 0.05). Compared with positive control, interference control group and infection group, there were significantly increases of SR-A1 and ACAT1 and down-regulation of ABCA1 and ABCG1 at mRNA and protein levels. However, there were no significantly changes of CD36.Conclusion The PPARαgene silencing aggravate the formation of THP-1-derived foam cells induced by C.pn infection. It illustrate the antagonism role of PPARαin the fomation of C.pn induded foam cells , which provide a target point for the clinical therapy of atherosclerosis.