All animals were barrier-housed according to the standards for laboratory animals established by the People's Republic of China (GB14925-2001), and the experimental protocol was approved by the Committee on the Ethics of Animal Experiments of Capital Medical University (Permit Number: SCXK-2012-0001). To minimize animal suffering, the lowest number of animals required for a statistically valid result was used. All experiments were performed under sodium pentobarbital anesthesia.
Homozygous ApoE-/- mice and wild type C57BL/6J mice (male, 10 weeks old, 22.85± 0.45g) were purchased from Beijing Vital River Laboratory Animal Technology Co Ltd.. All mice were adaptively barrier-housed for one week in an air-conditioned room without specific pathogens, with a light-dark cycle of 12 h. Then ApoE-/-mice were given a high-fat diet containing 21% (w/w) fat and 0.15% (w/w) cholesterol from 11-48 weeks of age, while C57BL/6J mice (groupA, n=10) were given a normal diet. Autoclaved food and water were provided ad libitum .
At 36 weeks of age, ApoE-/-mice (n=50) were randomly divided into the following 5 groups (n=10 per group): hyperlipidaemia model (group B), positive control (group C), low-dose β3-AR agonist (groupD), high-dose β3-AR agonist (group E)and β3-AR antagonist (group F). Mice in groupA and B received vehicle (0.9% saline, intraperitoneal injection, twice a week). Mice in group C received atorvastatin at a dose of 10 mg/kg by gavage once a day. Mice in groupD received the β3-AR agonist BRL37344 at a dose of 1.65 μg/kg by intraperitoneal injection, twice a week. Mice in groupE received the β3-AR agonist BRL37344 at a dose of 3.30 μg/kg by intraperitoneal injection, twice a week. Mice in groupF received the β3-AR antagonist SR59230A at a dose of 50 μg/kg by intraperitoneal injection, twice a week. All mice were treated for 12 weeks.
Reagents and drugs
The β3-AR agonist BRL37344 and the β3-AR antagonist SR59230A were obtained from Sigma Chemical Co. (St Louis, MO, USA). Atorvastatin was purchased from Pﬁzer Inc. (Dalian, China). Trizol reagent was produced by Invitrogen Co. (Carlsbad, CA, USA). Reverse transcription-polymerase chain reaction (RT-PCR) kit A3500 was obtained from Promega Co. (Madison, WI). Random primers were obtained from Invitrogen Co. (Shanghai, China). SYBR Green Real-Time PCR Master Mix kit was purchased from Katara Bio. (Shiga, Japan). Antibodies against α1A-AR, α1B-AR, α2A-AR, β1-AR, β2-AR and β3-AR, agiotensin II type 1 receptor (AT1R) and β-actin were produced by Abcam Co. (California, USA). Secondary antibody was obtained from Sigma Chemical Co. (St Louis, MO. USA).
Measurement of glucose and insulin
All mice were fasted for 10 hours at 48 weeks of age and anesthetized using 1% sodium pentobarbital by intraperitoneal injection, and then blood samples were collected by retro-orbital sinus puncture. nHDL-C, Glucose (Glu) and Insulin (Ins) were detected using a Beckman CX7 (Beckman Coulter, Fullerton, CA, USA).
Quantitative real-time PCR
Total RNA was extracted from renal tissues with TRIZOL Reagent, which was then reverse transcribed into double-stranded cDNA using RT-PCR kit based on the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) used a SYBR Green Real-Time PCR Master Mix kit (Katara Bio., Shiga, Japan). The reaction was performed as follows: 40 cycles of denaturation at 94℃ for 30s, annealing at 60℃ for 30s, and extension at 72℃ for 30s. Details of gene-specific primers were described in Table 1. The housekeeping gene β-actin was used as control. Results were normalized against β-actin and expressed as fold change for each gene using the 2–ΔΔCT method .
Table 1 Sequences of the primers used in this study. The sizes of the PCR products were given in base-pairs (bp).
Forward primers (5’-3’)
Reverse primers (5’-3’)
Protein expression of α1A-AR, α1B-AR, α2A-AR, β1-AR, β2-AR, β3-AR and AT1R in renal tissues was detected by western blot (WB). Total protein was extracted with RIPA buffer (50 mM Tris/HCl pH7.4, 150 mM NaCl, 2mM EDTA, 1% NP-40, 0.1% SDS). The protein concentration of the kidney was quantiﬁed using the Bradford protein assay. The samples were boiled for 5 min followed by loading on a 10% sodium dodecylsulfate polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene diﬂuoride membranes. After transfer, the membrane was blocked for 2 h at room temperature with 5% nonfat dry milk in Tris-buffered saline-Tween (TBST; 20 mM Tris, 500 mM NaCl, pH 7.5, 0.1% Tween 20). The membranes were then incubated with diluted primary antibodies against α1A-AR (rabbit polyclonal, 1:250), α1B-AR (rabbit polyclonal, 1:1000), α2A-AR (rabbit polyclonal, 1:500), β1-AR (rabbit polyclonal, 1:1000), β2-AR (rabbit polyclonal, 1:1000), β3-AR (rabbit polyclonal, 1:1000)，AT1R (rabbit polyclonal, 1:1000) in 5% nonfat dry milk in TBST for 2 h at room temperature. After washing three times with TBST, the membranes were incubated with a secondary antibody (goat anti-rabbit IgG-HRP, 1:5000) conjugated to horseradish peroxidase in 2.5% nonfat dry milk in TBST for 1 h at room temperature. The immunoreactive proteins were detected by enhanced chemiluminescence (ECL). The WB results were quantiﬁed by measuring the relative intensity compared with the control using Quantity one 4.6 (Bio-Rad, California, USA).
The data was analyzed with SPSS23.0, and GraphPad Prism 8.0 was used for plotting. All data was expressed as means ± SE.M. One-way ANOVA was used to determine the difference between groups. Post-hoc comparisons were performed using a Bonferroni t-test (equal variances) or Dunn's multiple comparison test (unequal variances). A value of P<0.05 was considered statistically significant.