1.1 Animals and regents
Male C57BL/6J mice (n=7, 8 weeks old) and male ApoE-/- mice (n=35, 8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Berberine was purchased from Sigma-Aldrich (St. Louis, MO, USA). The human hepatoma cell line, HepG2, obtained from Cell Resource Center, IBMS, CAMS/PUMC (Beijing, China), U0126 (ERK1/2 inhibitor) was purchased from Cell Signaling Technology (Beverly, MA). Anti-PCSK9, Anti-LDLR and GAPDH antibodies were obtained from Abcam (Cambridge, UK). Antibodies against phospho-ERK1/2, total ERK1/2, were purchased from Cell Signaling Technology (Beverly, MA).
1.2 Animal treatment
All animals were maintained in an air-conditioned environment with a controlled temperature at 22 ± 2 °C and 50–60% relative humidity under a 12:12 h light/dark cycle. After an adaptation period of a week, all mice were divided into the following groups: group 1 (wild typeC57BL/6J mice, normal diet), group 2 (ApoE-/- mice, normal diet), group 3 (ApoE-/- mice, high fat diet), group 4 (ApoE-/- mice, high fat diet, and treatment with low dose berberine of 50mg/kg/d ),and group 5 (ApoE-/- mice, high fat diet, and treatment with low dose berberine of 100 mg/kg/d ), group 6 (ApoE-/- mice, high fat diet , and treatment with atorvastatin of 20mg/k/d).After 16-week treatment,mice were euthanized using 1% sodium pentobarbital(50 mg/kg) after a 4-hour fast. The eyeballs were removedand blood samples were collected.The subsequent serumwas used to determine blood lipid parameters.The left ventricle was perfused with 4% neutral paraformaldehyde for 1 hour, then the abdominal cavity was opened and the liver, spleen and kidney were removed in turn, and the chest cavity was opened, the sternum was removed, the heart was exposed and the heart and aorta were removed.
1.3 Serum lipids analysis
According to the manufacturer's instructions, serum was prepared from each blood sample by centrifugation at 3500 rpm for 10 min. Serum TC, blood glucose, triglyceride (TG), LDL-C and high density lipoprotein cholesterol (HDL-C) were examined by the automatic biochemistry analyser (Hitachi 917, Tokyo, Japan).
1.4 Hematoxylin–eosin staining
Mouse liver specimens were processed according to a standard HE staining technique [17]. Briefly, liver tissues were fixed by 10% neutral formalin, dehydrated in ethanol, and then embedded. Subsequently, liver sections (4 μm) were stained with HE for pathological changes under an optical microscope.
1.5 Oil red O staining
Mouse liver tissues were immediately snap-frozen in liquid nitrogen and placed in OCT cryostat embedding compound (Tissue-Tek, Torrance, CA, USA). Frozen liver sections (8 μm) were stained with oil red O according to previous report [18], and the intracellularlipid droplets were observed andassessed by bright-field microscopy(Leica, Wetzlar, Germany).
1.6 Real time quantitative PCR (qRT-PCR) assay
SYBR green quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect mRNA levels of PCSK9, LDLR, ABCA1, ABCG1, SR-B1. The Trizol method was used to extract total RNA from mouse liver tissue. RNA yield and purity was confirmed by measuring the ratio of the absorbance at 260 nm and 280 nm. cDNA was synthesized using the SuperScript III First-Strand Synthesis System. The qRT-PCR reaction, containing target genes and SYBR Green PCR master mix, was performed on a Bio-Rad CFX connect real-time system (Bio-Rad, USA). The qRT-PCR, containing target genes and SYBR Green PCR master mix, was carried out on a Bio-Rad CFX connect real-time system (Bio Rad, USA) at95°C for 3min, cycled at 95°C for 10s,56°C for 30s and 72 °C for 30 s for 42 cycles. Melt curves were performed from 56.0 °C to 95.0 °C with intervals at 0.5 °C for 5 s. Relative RNA levels were determined by analyzing the changes in SYBR Green fluorescence by the 2−ΔΔCT method according to the manufacturer's instructions. GAPDH was amplified in parallel and the results were used for normalization. The PCR product was confirmed by gel electrophoresis on a 2% agarose gel stained with ethidium bromide. Purity of amplified PCR products was determined by melting point analysis using ICycler software. Experiments were performed in triplicate.
1.7 Western blot
Total proteins were extracted from liver (100 mg) derived from ApoE-/-mice. Liver were homogenized in 1 ml of RIPA lysis buffer containing protease inhibitors and vortexed on ice for 30 min. After centrifugation at 12,000×g for 20 min, the supernatant, containing total protein extract, was collected, and protein concentrations were determined by the bicinchoninic acid (BCA) method. Cytosolic proteins were extracted using cytosol protein kits. Equal amounts of protein (20–40 μg) were electrophoresed on 8–12% SDS-PAGE gels for 2 h at 90 V and electrotransferred onto PVDF membranes at 125 mA for 1–2 h. The membranes were blocked with 5% non-fat dry milk in 20 mM Tris–HCl, pH 7.4, 0.15 M NaCl, 0.05% Tween-20 (Tris-Buffered Saline and Tween 20, TBST) for 1 h at room temperature, then membranes were incubated overnight at 4 °C with one of the following primary antibodies: anti-proprotein convertase subtilisin/kexin type 9 (PCSK9), anti-LDLrecepyor (LDLR), anti-ABCA1, anti-ABCG1, anti-SR-BI. After washing with TBST, membranes were incubated with secondary antibodies derived from goat for 1.5 h at room temperature. The results of Western blots were analyzed by the Image J program. The expression of each protein was normalized to the corresponding GAPDH.
1.8 Statistical analysis
GraphPad Prism 7 software was used for data analysis. Results are expressed as mean ± SEM. p<0.05 was considered statistically significant, and p<0.01 was considered extremely significant.