Plant material and extraction
The aerial parts of A. pilosa were purchased from the Gokseong Agricultural Association (Jeollanam-do, Korea) in 2011 as dried form and identified by the Classification and Identification Committee of the Korea Institute of Oriental Medicine (KIOM). A voucher specimen (KIOM109-122Aa) was kept in the herbarium of the Department of Herbal Resources Research of the KIOM. Dried A. pilosa was extracted twice with 10 volumes of water at 80 °C for 3 h. The extracts were filtered through No. 6 filter paper (Whatman, Maidstone, UK) and were concentrated under reduced pressure by a rotary evaporator (EYELA, Tokyo, Japan) at 40 °C. The water filtrates were frozen and lyophilised (Ilshin Lab). The final lyophilised extracts were stored at − 20 °C until use.
Animals and diets
Seven-week-old male Sprague-Dawley (SD) rats with an average weight of 271.9 ± 8.7 g were purchased from Orient Bio Co. Ltd. (Seongnam, Korea). All rats were housed individually in plexiglass cages and maintained in an air-conditioned room at 22 ± 2 °C under an automatic lighting schedule. To help with adaptation to the laboratory conditions, the rats were fed a standard pellet diet with free water for the first week prior to the experiment. After the acclimation period, the rats were randomly blocked into three groups (n = 10 per group) with similar mean body weights. The rats were assigned to the following groups: Normal-fat diet (NF); HFD (HF, 36% of energy as fat); HFD with 0.1% A. pilosa aqueous extract (HFA). These experimental diets were maintained for 16 weeks. The composition of the experimental diets is shown in Additional file 1. This experimental design was approved by the Institutional Animal Care and Use Committee (IACUC) of the National Academy of Agricultural Science (reference number: NAAS-1203).
Preparation of blood and tissue samples
On the last day of the experiment, after fasting for 16 h, all rats were anesthetized with CO2 and blood was collected via the heart. The collected blood was kept in cold water for approximately 30 min, and the serum was separated by centrifugation at 3000 rpm for 20 min at 4 °C. After cutting the abdomen open, small pieces of liver and fat tissues were rapidly excised and frozen immediately in liquid nitrogen for Real-time quantitative reverse transcription PCR. The liver and fat tissues were removed, rinsed with cold phosphate-buffered saline, and weighed. Hepatic sections for histological evaluation were stored in 10% buffered neutral formalin. The residue was frozen immediately in liquid nitrogen and stored –in a − 70 °C freezer.
Oral glucose tolerance test (OGTT)
Rats were subjected to an overnight fast for 16 h before the OGTT. The baseline glucose level was measured by OneTouch Ultra Glucose Monitoring System (Johnson & Johnson Medical, New Brunswick, NJ, USA). The 75% glucose solution (2 g/kg b.w.) was orally injected, and serum glucose in the blood sample from the tail vein was estimated by a glucometer at 0, 30, 60, and 120 min. Assays were carried out in triplicate (n = 10). The area under the curve (AUC) was also calculated.
Blood biochemical assays
Serum glucose was determined using commercial assay kits (Asan Pharmaceutical, Seoul, Korea) according to the manufacturer’s protocols. Insulin and adiponectin levels were analysed by commercial assay kits (Millipore Corp., Billerica, MA, USA). Serum TNF-α and IL-6 levels were also measured using commercial assay kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocols. Assays were carried out in duplicate (n = 10). The homeostasis model assessment of insulin resistance (HOMA-IR) was obtained by the calculation (HOMA-IR = glucose (mg/dL) × insulin (mU/L)/405).
Liver histology
Liver tissue was fixed with 10% neutral buffered formalin and embedded in paraffin. Sections were cut and stained with Oil red O staining. Images were captured using an Olympus AX 70 camera (Center Valley, PA, USA). The initial assessment was performed under low magnification (40 × to 200 ×), and confirmed under high magnification (400 ×). Hepatic steatosis was graded as 0 (fatty hepatocytes occupying < 5%), 1 (fatty hepatocytes occupying 5–33%), 2 (fatty hepatocytes occupying 34–66%), or 3 (fatty hepatocytes occupying > 66%) according to the percentage of hepatic lipid [16].
Real-time quantitative reverse transcription PCR
Total RNA was isolated from liver and fat tissues using the RNeasy Microarray Tissue Kit (Qiagen, Valencia, CA, USA), and RNA integrity (RIN > 9.0) was assessed using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). The Rat Fatty Liver PCR array and Insulin Resistance PCR array (SABiosciences, Frederick, MD, USA) were used to profile the genes differentially expressed in liver and fat tissue, respectively, according to the manufacturer’s instructions. The complete list of genes assayed on the array is provided on the manufacturer’s website (http://www.sabiosciences.com/Metabolic_Diseases.php). For each plate, 0.5 μg of RNA was converted to double-stranded cDNA using the RT2 first strand synthesis kit (Qiagen). After mixing this with the SABiosciences RT2 qPCR master mix, the cDNA was pipetted into the 96-well profile plate and amplified on CFX96TM Real-Time PCR Detection System Bio-Rad Laboratories (Hercules, CA, USA). Data were normalised using lactate dehydrogenase A as an endogenous control, and fold-changes in expression were calculated using SABiosciences online software (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php).
Statistical analysis
Data were expressed as mean ± standard error (S.E.). Statistical comparisons were performed by one-way ANOVA followed by Duncan’s multiple range test. Values were considered statistically significant when p < 0.05.