The human hepatoblastoma cell line (HepG2) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). A total of 16 male 8-week-old ApoE^−/− (weight, 19.12±0.44 g) and 8 male 8-week-old C57BL/6 (weight, 20.08±0.31 g) mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Atorvastatin original powder was purchased from Abcam (Cambridge, UK), LXR agonist T0901317 was from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and a quantitative polymerase chain reaction (qPCR) kit (SYBR^® Premix Ex Taq^™ II) was obtained from Takara Bio, Inc. (Otsu, Japan). Antibodies against apoM (cat. no. ab122896) and LXRα (cat. no. ab41902) were purchased from Abcam, while a pre-β HDL ELISA kit (cat. no. ml001270) was obtained from Mlbio (Shanghai, China). Reverse transcription (RT)-qPCR primers were obtained from GENEWIZ (South Plainfield, NJ, USA).

Mice received humane care according to the Guidelines for the Care and Use of Research Animals established by Soochow University (Suzhou, China) and the experimental protocols were approved by the Ethics Committee of Soochow University. A total of 12 8-week-old apoE^−/− mice and 4 8-week-old C57BL/6 mice were acclimated to housing in standard polycarbonate cages in the Animal Facility of Soochow University under a 12 h light/dark cycle for 1 week prior to experimentation. Mice were housed with free access to food and water, and under the temperature of 23°C and air humidity of 55%. The in vivo study included four groups (16 mice total; 4 mice for each group): C57BL/6 mice and apoE^−/− mice fed with regular diet (control and apoE^−/− groups, respectively), apoE^−/− mice fed with high-fat diet treated with normal saline (vehicle control group) and apoE^−/− mice fed with high-fat diet and treated with atorvastatin (statin group). In the control and apoE^−/− groups, mice were fed with regular chow for 8 weeks and administered with 0.2 ml (0.9%) normal saline by lavage every day. For the vehicle control and atorvastatin groups, mice were provided with high-fat feed (Suzhou Shuangshi Animal Feed Technology Co., Ltd., Suzhou, China), comprising 4% cholesterol, 0.5% sodium cholate, 10% lard, 0.2% propylthiouracil and 85.3% normal chow diet, for 8 weeks to induce hyperlipidemia. Following the successful establishment of hyperlipidemia, mice were randomly divided into the vehicle control and statin groups. In the statin group, mice were administered with 10 mg/kg/day atorvastatin dissolved in 0.2 ml (0.9%) normal saline by oral gavage for 4 weeks. Mice in the vehicle control group were administered with 0.2 ml (0.9%) normal saline only.

In addition to the groups described above, a total of 4 male 8-week-old apoE-deficient mice (Model Animal Research Center of Nanjing University) were housed with free access to food and water in standard polycarbonate cages under a 12 h light/dark cycle, and under the temperature of 23°C and air humidity of 55%. The mice were used as the fifth experimental (agonist) group. Hyperlipidemia was induced in apoE-deficient mice as previously described. Thereafter, mice were administrated with 50 mg/kg/day of T0901317 + 10 mg/kg/day atorvastatin dissolved in 0.2 ml (0.9%) normal saline by lavage for 4 weeks. Following treatment, mice in all groups were anesthetized and sacrificed. Blood samples were collected through the tail vein and centrifuged at 4°C at 5,000 × g for 20 min to collect the serum, which was subsequently stored at −80°C. Liver samples were harvested and flash frozen for subsequent RT-qPCR and western blotting.

HepG2 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel), 1.0×10⁵ U/l penicillin and 1.0×10⁵ U/l streptomycin at 37°C. The medium was replaced every 2 days and cells were passaged. Cells were seeded in a 6-well plate at a density of ~5×10⁵ cells/well and grown to 50–70% confluence. Prior to experimentation, cells were washed twice with PBS and the medium was replaced. In vitro experiments involved four groups: Control, dimethylsulfoxide (DMSO), atorvastatin and agonist. Cells in the control and DMSO groups were treated with 200 µl PBS or 200 µl DMSO, respectively. In the statin group, cells were treated with 20 nmol atorvastatin dissolved in 200 µl DMSO. In the agonist group, cells were treated with 200 nmol T0901317 + 20 nmol atorvastatin dissolved in 200 µl DMSO. Cells were harvested following 24 h of treatment at 37°C for western blotting and RT-qPCR.

Mice were fasted for 12 h and sacrificed, following which blood samples were collected via the tail vein. Serum was separated by centrifugation at 600 × g under 4°C for 20 min, following which HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), total cholesterol (TC; all cat. no. ab65390), and triglyceride (TG; cat. no. ab65336) levels were measured (23) with assay kits (Abcam).

ApoM and LXRα protein expression in liver samples and HepG2 cells were detected using western blotting as previously described (24,25). Briefly, 40 mg tissue or approximately 2×10⁶ cells in each group were homogenized to obtain lysates from which protein was extracted using ProteoPrep^® Total Extraction Sample Kit (cat. no. PROTTOT-1KT; Sigma-Aldrich; Merck KGaA). The protein concentration was measured using a BCA protein assay kit according to the manufacturer's instructions. A total of 25 µg of each sample was separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Blocking was then performed by overnight incubation at 4°C in Tris-buffered saline/Tween 20 (TBST) containing 5% non-fat dried milk. Membranes were washed with TBST and incubated with anti-apoM (1:1,000), anti-LXRα (1:1,000), and anti-GAPDH (cat. no. AF0006; 1:1,000; Beyotime Institute of Biotechnology, Shanghai, China) primary antibodies for 1 h at room temperature. Subsequent to washing with TBST, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. A0181; 1:500; Beyotime Institute of Biotechnology) for 1 h at 37°C. A chemiluminescent substrate (cat. no. 34580; Thermo Fisher Scientific, Inc.) was used to detect the peroxidase-conjugated antibodies and membranes were exposed to X-ray film using BIO-RAD Gel Doc XR+ (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The intensity of bands was evaluated with ImageJ software (version 1.51; National Institutes of Health, Bethesda, MD, USA) and quantified relative to GAPDH bands from the same sample.

RT-qPCR was performed to determine the expression of LXRα and apoM mRNA in liver samples and HepG2 cells. Total RNA was extracted from liver tissues and HepG2 cells using TRIzol (Thermo Fisher Scientific, Inc.) and purified using a Qiagen RNeasy Mini kit (cat. no. 74104; Qiagen AG, Sollentuna, Sweden) as previously described (26). cDNA was synthesized from total RNA using the PrimeScript RT reagent kit (Takara Bio, Inc.). The temperature and duration for RT was 25°C for 10 min, 37°C for 60 min, 70°C for 10 min and 4°C on ∞ hold. A total of 2 µl cDNA was used for qPCR, which was performed using Power Syber Green and the StepOne-Plus real-time PCR system (Thermo Fisher Scientific, Inc.). The thermocycler protocol for qPCR was 95°C for 3 min, then 40 cycles of 94°C for 40 sec, 55°C for 30 sec, 72°C for 40 sec and 72°C for 5 min, then 4°C on ∞ hold. The endogenous housekeeping gene GAPDH was used to normalize expression levels. The sequences of primers used in qPCR are presented in Table I. The 2^−ΔΔCq method was used to evaluate changes in target gene expression relative to GAPDH (27).

Serum pre-β HLD levels were measured using a sandwich ELISA kit according to the manufacturer's protocol. Optical density (OD) values were measured at 450 nm (background reading at 620 nm) with an absorbance reader (BioTek Instruments, Inc., Winooski, VT, USA). The serum concentration of pre-β HLD (pg/ml) in each group was calculated using a standard curve.

Data are presented as the mean ± standard deviation. All data were analyzed using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA) using one-way analysis of variance with Bonferroni's correction for multiple group comparisons and Student's t-test for the comparisons between two groups. P<0.05 was considered to indicate a statistically significant difference.

Hyperlipidemia was successfully induced in apoE^−/− mice following 8 weeks of high-fat diet administration. As presented in Table II, TC, LDL-C and TG were significantly increased in apoE^−/− mice fed with a high-fat diet compared with C57BL/6 mice or apoE^−/− mice fed with regular chow for 8 weeks. Following 4 weeks of statin or vehicle treatment, the serum concentration of LDL-C was significantly decreased in the statin group compared with the vehicle control group, while HDL-C levels were significantly increased (Fig. 1A and B). Serum TC and TG levels were decreased by 2.99 and 6.38%, respectively, in the statin group compared with the vehicle control group (Fig. 1C and D). However, no statistically significant differences were observed between the statin group and the vehicle control group. Serum pre-β HLD levels were reduced in the apoE^−/− mice compared with the vehicle control group (Fig. 1E). However, reduced pre-β HLD levels were reversed in the statin group, resulting in significant pre-β HLD upregulation compared with the control group (Fig. 1E).

ApoM and LXRα expression was assessed in mouse liver samples using RT-qPCR and western blotting. No significant differences in apoM mRNA expression were observed between the control, apoE^−/− and vehicle control groups; however, apoM mRNA expression levels were significantly increased in the statin group compared with all other groups (Fig. 2A). LXRα mRNA expression was downregulated in apoE^−/− and vehicle control mice compared with the control group, however no statistical significance was recorded (Fig. 2B). Mice in the statin group exhibited a significant decrease in LXRα mRNA expression compared with the control, apoE^−/− and vehicle control groups (Fig. 2B). The expression of apoM was significantly increased in the statin group compared with the vehicle control group (Fig. 2C and D), consistent with the results of RT-qPCR. However, LXRα protein expression decreased in the statin group compared with the vehicle control group (Fig. 2C and E). These results suggest that atorvastatin is able to upregulate apoM expression in the liver of hyperlipidemic mice while simultaneously downregulating LXRα.

The in vivo study indicated that atorvastatin was able to downregulate serum lipid levels in hyperlipidemic mice by regulating liver apoM and LXRα expression at the mRNA and protein levels. In order to further study the mechanism underlying mechanism, an in vitro cell model was employed. RT-qPCR results suggested that apoM mRNA expression was significantly increased 1.6-fold compared with the DMSO group following statin treatment (Fig. 3A). Furthermore, LXRα was significantly downregulated at the mRNA level in response to statin treatment compared with the DMSO group (Fig. 3B). Western blotting results revealed that apoM protein was overexpressed following statin treatment compared with the DMSO group (Fig. 3C and D). Although LXRα mRNA expression levels were reduced following statin treatment, no significant differences were observed in LXRα protein expression between the DMSO and atorvastatin groups (Fig. 3C and E).

To investigate whether apoM upregulation may be mediated by the attenuation of LXRα, the LXR agonist T0901317 was used. T0901317 was administered in combination with atorvastatin to hyperlipidemic mice and HepG2 cells. The results revealed that apoM mRNA and protein expression was significantly decreased by combined treatment with the agonist compared with atorvastatin alone in vivo and in vitro (Fig. 4). These results suggest that T0901317 is able to inhibit atorvastatin-induced apoM upregulation.