LPS Up-regulates The Expression Of SR-PSOX And Lipid Accumulation In THP-1 Cells Via TLR4

OBJECTIVE:To study the relationship between TLR4 and SR-PSOX, and to investigate the possible mechanism in which LPS accelerate the progression of atherosclerosis.METHODS:THP-1 cells were incubated for 24 hours (h) with 160nmol/L Propylene Glycol Methyl Ether Acetate (PMA) before fully differentiated into adherent cells, and then different factors were used to treat cells as indicated bellow. Experiment 1: THP-1 cells were incubated for 24 h with 0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml LPS, respectively. TLR4 mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR). The optimal LPS concentration was adopted. THP-1 cells were incubated for 0 h, 3 h, 6 h, 12 h, 24 h with LPS at the optimal concentration, respectively. The optimally incubating time was adopted. Experiment 2: The experiment was divided into two parts according to the aim. (1) The expression of SR-PSOX was determined in THP-1 cells after being treated with different factors. The experiment had four groups: PBS group, LPS group, IgG+LPS group, TLR4 blocking antibody +LPS group. THP-1 cells were pretreated for 2 h with 10ug/ml antibody before incubated with LPS for 3 h. SR-PSOX mRNA and protein expression were determined by RT-PCR and Western blot, respectively. (2) Lipid content was detected in THP-1 cells after being treated with different factors. The experiment had four groups: oxLDL group, LPS+oxLDL group, IgG+LPS+oxLDL group, TLR4 blocking antibody+LPS+oxLDL group. After being treated for 2 h with 10ug/ml antibody, THP-1 cells were incubated with LPS for 3 h, after which 30ug/ml oxLDL was used to treat cells for another 24 h. High performance liquid chromatography (HPLC) and oil red O were conducted to determine the lipid content. Experiment 3: The experiment was also divided into two parts according to the aim. (1) The expression of TLR4 was determined in THP-1 cells after being treated with different factors. The experiment had four groups: PBS group, LPS group, IgG+LPS group, SR-PSOX blocking antibody +LPS group. THP-1 cells were pretreated for 2 h with 2ug/ml antibody before incubated with LPS for 3 h. TLR4 mRNA and protein expression were determined by RT-PCR and Western blot, respectively. (2) The lipid content was determined in THP-1 cells after being treated with different factors. The experiment had four groups: oxLDL group, LPS+oxLDL group, IgG +LPS+oxLDL group, SR-PSOX blocking antibody+LPS+oxLDL group. After being treated for 2 h with 2ug/ml antibody, THP-1 cells were incubated for 3 h with LPS, after which 30ug/ml oxLDL was used to treat cells for another 24 h. HPLC and oil red O were conducted to determine the lipid content.RESULTS:The expression of SR-PSOX mRNA was up-regulated in THP-1 cells incubated with LPS, and the optimal LPS concentration was 100 ng/ml, the optimally incubating time was 3 h. SR-PSOX mRNA and protein expression was significantly down-regulated in THP-1 cells incubated with TLR4 blocking antibody compared with LPS group(p<0.05). Oil red O indicated that the red lipid droplets were reduced. HPLC showed that the lipid content was decreased significantly, compared with LPS+oxLDL group(p<0.05). The expression of TLR4 mRNA and protein was but not significantly decreased in THP-1 cells incubated with SR-PSOX blocking antibody. Results from HPLC and oil red O suggested the lipid content was significantly reduced compared with LPS+oxLDL group(p<0.05).CONCLUSIONS:1. LPS up-regulated the expression of SR-PSOX in THP-1 cells which wasinhibited by TLR4 blocking antibody,this indicated that LPS up-regulated the expression of SR-PSOX via TLR4 in THP-1 cells.2.LPS promoted oxLDL-uptaking in THP-1 cells ,which was decreased by incubated with TLR4 blocking antibody,this suggested LPS up-regulated the SR-PSOX-mediated accumulation of lipid in THP-1 cells via TLR4.