2.1 Chemicals and Reagents
Cinnamic acid (≥99.5% purity) was purchased from Shanghai YuanYe (Shanghai, China). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), and 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco (Carlsbad, CA, USA). OA was purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA) and carboxymethyl cellulose (CMC) were purchased from Solarbio Science & Technology (Beijing, China). CA was dissolved in 0.5% CMC buffer solution before being orally administered to the mice.
2.2 Cell Culture
HepG2 cells were purchased from KeyGEN Biotech (Nanjing, China). The cells were cultured in DMEM with 10% FBS and maintained in a humidified, 37℃, 5% CO2 environment, with the media changed every 24 hours. Based on previous studies, 0.5 mM OA was used to establish a high-fat model in vitro [27,28]. After reaching 80% confluence, the cells were placed in 6-well plates and stimulated with 0.5 mM OA media with or without CA of different concentrations. Cells cultured without OA were used as normal controls. The OA/BSA complex used for cell stimulation was prepared as follows: OA was dissolved in 0.1M NaOH and heated at 70℃ for 30 min to form a 100 mM OA solution. This was further mixed with 10% BSA in phosphate-buffered saline to acquire a 10 mM OA/BSA complex. This complex was diluted in DMEM with 10% FBS to a concentration of 0.5 mM for final use. All cell experiments were performed in at least three replicate wells.
2.3 Cell Viability Assay
The effect of CA on the viability of HepG2 cells was assessed by Cell Counting Kit (CCK)-8 assays. CCK-8 assays were conducted as previously described [22,29] with slight modification. Briefly, 200 µl HepG2 cells were seeded at a density of 1×104/well in a 96-well plate and cultured for 12 h. Thereafter, the cells were treated with CA (diluted to 12.5, 25, 50, 100, or 200 µM) for 24 h, followed by changing the media to 200 µL DMEM containing 10 µL CCK-8 solution (Dojindo Molecular Technologies, Kumamoto, Japan) and incubating for 1 h in 37℃. Absorbance at 450 nm was measured using a microplate reader (Promega BioSystems Sunnyvale, Sunnyvale, CA, USA). Cells cultured in DMEM with 10% FBS (without CA) were used as normal controls.
2.4 Oil Red O Staining and TG Assessment
After treatment for 24 h, HepG2 cells were stained with Oil Red O to determine the intracellular lipid accumulation. The staining was performed using a commercial kit (Solarbio Science & Technology) according to the manufacturer’s instructions. TG content was also determined using a TG measuring kit (Nanjing Jiancheng Bioengineering, Nanjing, China) following the manufacturer’s instructions. Cells cultured in DMEM with 10% FBS were used as normal controls.
2.5 Animal Experiments
The animal experiments conducted in this study were approved by the Animal Care and Ethics Committee of Beijing University of Traditional Chinese Medicine (approval no. BUCM-4-20190931002-1088). 14 5-week-old male C57BL/KsJ-db/db mice and 7 age-matched male C57 BL/KsJ-wt/wt mice were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China) and housed in specific-pathogen-free conditions under a 12/12-h light/dark cycle, with free access to water and food. The body weight of mice was measured every week. After 1 week of acclimatization, all mice were fasted overnight and blood glucose was measured using a portable glucometer (Glucocard 01-mini; Arkray, Kyoto, Japan) using blood collected from the tail vein.
The db/db mice were then randomly divided into two groups with fasting blood glucose and body weight of which at the same baseline: the CA and model groups. The CA mice were orally treated with 20 mg/kg body weight CA every day. The dosage was chosen based on previous studies. The model mice were treated with the same volume of the vehicle (0.5% CMC buffer). The wt/wt mice were used as the normal control group and treated the same as the model mice. After 4 weeks of treatment, the mice were sacrificed. Blood samples were collected and centrifuged to acquire serum. Additionally, liver and epididymal adipose tissues were removed and weighed. A small slice of each tissue was fixed in 4% paraformaldehyde solution (Solarbio Science & Technology) and the remining tissues were immediately frozen in liquid nitrogen.
2.6 Assessment of Serum Lipid Profile and IHTG
The serum was stored at -20 ℃ before the total cholesterol (TC), TG, high-density lipoprotein (HDL), low-density lipoprotein (LDL), free fatty acid (FFA), and glucose levels were tested using an automated chemistry analyzer (AU480; Beckman Coulter, Brea, CA, USA). IHTG levels (mmol/g protein) in the mice were measured using a commercial kit (Nanjing Jiancheng Bioengineering, Nanjing, China).
2.7 Histological Analysis of Liver and Adipose Tissues
Liver tissues and epidydimal adipose tissues fixed in 4% paraformaldehyde were embedded in paraffin, cut into 4-μm-thick slices using a microtome, and then stained with hematoxylin and eosin (H&E). Immunohistochemical (IHC) staining of liver tissues was used to examine protein expression. Primary antibodies against SREBP1 (ab191857; Abcam, Cambridge, UK) and CPT1A (ab234111; Abcam) were used, followed by incubation with horseradish peroxidase (HRP)-labeled anti-rabbit IgG secondary antibody for 20 min. The tissues were then stained with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. The IHC staining images were analyzed based on the mean optical density using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
2.8 Quantitative Real-Time PCR (RT-qPCR)
Total RNA was extracted from HepG2 cells and the liver tissues using a commercial kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. The RNA was then homogenized by RNase-free water to the same concentration of 0.5ng/tube and cDNA was synthesized using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara Bio, Kusatsu, Japan). Thereafter, the RT-qPCR analyses were conducted on Applied Biosystems 7500 Real time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) using a GoTaq® qPCR Master Mix Kit (Promega). The expression levels of target genes were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the results were calculated using the relative quantitative (2-DDCT) method. The primers used are listed in Table 1.
2.9 Western Blot Analysis
Total protein was extracted from the liver tissues as follows. The tissues were homogenized and lysed using radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor, phosphatase inhibitor, and phenylmethanesulfonyl fluoride (KeyGEN Biotech). The concentration of each extracted protein solution was then measured using a Bradford protein quantitative assay with Coomassie brilliant blue G-250 (Solarbio Science & Technology). Protein samples were prepared at a concentration of 1 µg/µl and denatured at 100°C for 5 min in loading buffer (Solarbio Science & Technology). The proteins were loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and separated according to their molecular weights. They were then transferred onto methanol-soaked 0.45-μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). These membranes were then blocked using Blocking-One solution (Nacalai Tesque, Kyoto, Japan) for 1 h and then incubated with primary antibodies against the following proteins: ATP-citrate lyase (ACLY; #13390; Cell Signaling Technology, USA), acetyl-CoA carboxylase (ACC; #3676; Cell Signaling Technology), fatty acid synthase (FAS; #3180; Cell Signaling Technology), stearoyl-CoA desaturase 1 (SCD1; #2794; Cell Signaling Technology), CPT1A (ab234111; Abcam), and β-actin (#4970; Cell Signaling Technology) at 4°C overnight. After washing with Tris-buffered saline with Tween 20 (TBST; Beijing Applygen Technologies, Beijing, China), the membranes were incubated with HRP-conjugated AffiniPure goat anti-rabbit IgG secondary antibody (Proteintech Group, Rosemont, IL, USA) for 1 h and washed with TBST again. Enhanced chemiluminescence (ECL) reagent (Solarbio Science & Technology) was reacted with the HRP to generate fluorescence. The gray values of the protein bands were calculated by normalization to the endogenous control protein β-actin.
2.10 Statistical Analysis
All data are presented as mean ± standard deviation (SD). SPSS 23.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis. Normally distributed data were compared between two groups using Student’s t-test. Normally distributed data were compared between three or more groups using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test for groups with equal variances or Dunnett’s test for groups with unequal variances. Non-normally distributed data were compared between two groups using the Mann–Whitney U test. Non-normally distributed data were compared between three or more groups using the nonparametric Kruskal–Wallis test. A statistically significant difference was defined as p < 0.05 or p < 0.01.