LPS (Escherichia coli, O111:B4) was purchased from Sigma (St. Louis, MO, USA) and reconstituted in phosphate-buffered saline (PBS). PPARγ antibody (rabbit anti-mouse) and PPARγ antibody (goat anti-rabbit) were purchased from Sigma. Phosphor-nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α (IκBα; Ser32) was purchased from Youyizhonglian Bio-Corporation (Beijing, China). [H³] cholesterol and apolipoprotein AI (ApoAI) were purchased from Sigma. Ciglitazone was purchased from Sigma, and the final concentration of ciglitazone dissolved in dimethylsulfoxide (DMSO) was 3 μmol/ml. The sequences of the PPARγ antisense and missense oligonucleotides were 5′-CATGAGGCTTATTGTAGAGCTGA-3′ and 5′-GCCAGGTACCACTCACTCTGCAGT-3′, respectively. The procedure of synthesis, purification and subpackage of the sequence was operated by Shenggong Bio-Corporation (Shanghai, China).

Fifteen C57BL/6 mice (8–10 weeks old, males, weighing 20–26 g) were obtained from the Laboratory Animal Centre of Chongqing Medical University (Chongqing, China). These mice were housed in an animal room and fed a standard diet. All experimental protocols described in this study were approved by the Ethics Review Committee for Animal Experimentation of Chongqing Medical University.

The 15 mice were randomly divided into three groups. Proceeding from isolation and culture of peritoneal macrophages from the C57BL/6 mice, the cells were divided into three groups: The control group, the ciglitazone group and the PPARγ antisense oligonucleotide group. The expression of PPARγ and IκBα in each group was observed through the levels of protein and mRNA, and then the cholesterol efflux of each group was investigated. The intraperitoneal injection of LPS into mice is a widely used method of constructing inflammatory animal models (15). In addition, the same experiment was repeated subsequent to stimulation of each group with LPS.

Pre-cooled PBS (2 ml) was injected into the abdominal cavity of the mice, whilst the abdomen was kneaded softly for 2 min. The PBS was drawn out and collected, and then centrifuged for 10 min at 2,000 × g. The supernatant liquid was discarded and placed in RPMI-1640, which regulated the concentration of the cells at 3–5×10⁶cells/ml. The cells were cultivated in 24-well plates at 37°C for 2 h until they had adhered, then the cultivation holes were washed with pre-cooled PBS. The adherent cells were peritoneal macrophages. The peritoneal macrophages were cultivated for 24 h and then randomly divided into three groups: The control group (RMPI-1640+25 μl DMSO), the ciglitazone group (RMPI-1640+25 μl DMSO+ciglitazone; final concentration, 10 μmol/l), and the PPARγ antisense oligonucleotide group (RMPI-1640+25 μl DMSO+PPARγ antisense oligonucleotide; final concentration, 400 nmol/l). The final concentration of LPS was 80 ng/ml.

The peritoneal macrophages were cultured on a chamber slide, which was washed with PBS and air-dried. The slide was fixed with methanol for 30 min at −20°C and then stained with PPARγ antibody (rabbit anti-mouse; Abcam, Cambridge, MA, USA) and PPARγ antibody (goat anti-rabbit; Abcam) for 24 h at room temperature. The cells that were stained purple were considered positive.

Total RNA samples of the peritoneal macrophages were extracted using an RNA extraction kit Takara Bio Inc. (Shiga, Japan) according to the manufacturer’s instructions. Total RNA was quantified with the ratio of absorption values of RNA samples at 260 and 280 nm. Each total RNA sample was reversely transcribed to complementary DNA using an RT-PCR kit and stored at −70°C. All PCR products were electrophoresed on 2% agarose gels. The RT-PCR was performed using the sense and antisense primers for PPARγ or β-actin (Table I). The relative expression of mRNAs were assessed by taking the ratio of the intensity of the DNA bands of PPARγ to the β-actin band using the Bio-Image analysis system (Gel Doc 2000; Bio-Rad, Hercules, CA, USA) and expressed as arbitrary units.

Total protein of the peritoneal macrophages was extracted by homogenizing the macrophages in a cell lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), then by two cycles of centrifugation at 12,000 × g for 15 min. Protein concentration was determined using a Bradford assay kit (Beyotime Institute of Biotechnology). The total protein was separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were then incubated with rabbit anti-mouse PPARγ polyclonal antibody (diluted 1:1000, IMG-441; Sigma) and horseradish peroxidase-conjugated goat anti-rabbit IgG (diluted 1:2000; Zhongshan Jinqiao, Beijing, China). The immune complexes were developed with an Enhanced Chemiluminescence Detection kit (Pierce Biotechnology, Inc., Rockford, IL, USA) and the membranes were then immediately exposed to autoradiographic film (Kodak, Rochester, NY, USA). The relative amount of PPARγ protein was quantified from the optical density of the corresponding band by Bio-Image analysis system (Gel Doc 2000; Bio-Rad).

The concentration of macrophages was regulated at 3.0×10⁹ cells/l and the macrophages were transferred into RPMI-1640 containing fetal bovine serum, penicillin and streptomycin, and [³H] cholesterol. After 24 h, the cells were cultivated in new medium containing 50 μg/ml ApoAI for 12 h. The [³H]cholesterol in the culture solution and cells was detected by liquid scintillation counting. The effluxion of cholesterol was calculated using the following formula: [³H] (culture solution)/[³H] (culture solution and cells) × 100.

Data are reported as the mean ± standard deviation and were analyzed using one-way analysis of variance with Tukey’s multiple comparison test, and the statistical program SPSS, version 11.0 (SPSS, Inc., Chicago, IL, USA). P≤0.05 was considered to indicate a statistically significant difference.

Peritoneal macrophages from C57BL/6 mice were isolated and cultured (Fig. 1). Subsequently, PPARγ in peritoneal macrophages prior to and following stimulation by LPS was examined using immunocytochemistry (Figs. 2 and 3). It was found that the number of PPARγ-positive cells following stimulation by LPS was greater than that prior to stimulation by LPS. The PPARγ-positive cells were stained purple.

The results of the RT-PCR indicated that there was no significant difference in the average relative gray value between the control group and ciglitazone group. The average relative gray value of the PPARγ antisense oligonucleotide group was evidently lower than that of control group. Following stimulation with LPS, the expression levels of PPARγ mRNA in the ciglitazone group were higher than those in the control group, while the PPARγ mRNA expression levels of the PPARγ antisense oligonucleotide group were lower than those of the control group (Figs. 4 and 5).

The results of the western blotting suggested that there was no significant difference in the expression of PPARγ protein between the control group and ciglitazone group. The PPARγ protein expression levels of the PPARγ antisense oligonucleotide group were considerably lower than those of the control group. Subsequent to stimulation with LPS, the expression levels of PPARγ protein in the three groups were higher than those of each group prior to stimulation, and the IκBα protein expression levels of the three groups were lower than those of each group prior to stimulation (Figs. 6–9).

The cholesterol efflux of the ciglitazone group was suppressed following stimulation with LPS, and the suppression ratio was lower than that of the control group. However, the cholesterol efflux of the PPARγ antisense oligonucleotide group was greatly suppressed following stimulation with LPS, and the suppression ratio was higher than that of the control group (Table II, Fig. 10).