Porcine CYP7A1 ELISA kit, porcine 3-hydroxymethyl glutaratemonoacyl CoA(HMGCoA) reductase ELISA kit and porcine lipoprotein lipase (LPL) ELISA kit was purchased from Wuhan Mershack biotechnology Co., LTD. PDK4 antibody (bs-0682r), PPAR-δ antibody (BSM-33263m), KLF10 antibody (bs-1838r), slc22a5 antibody (bs-8149r) and GAPDH antibody (BSM-33033m) were purchased from Beijing Bosenbiotechnology Co., LTD.The other reagents are all domestic analytical pure level.
Collection of muscle tissues and blood samples
A total of 24 muscle tissues and corresponding blood samples were collected from pigs at Hunan New Wellful Co., LTD. (Liuyang, Hunan, China). All tissues and blood samples were collected according to protocols approved by the Research ethics committee of the School of Veterinary Medicine, Hunan Agricultural University(433320027). All experiments were performed according to the institutional guidelines for the care and use of laboratory animals(200705041)
Animals were randomly divided into four subgroups (n = 15) after feeding with the regular diet for eight weeks: Group 1 was continuously fed with the regular diet for seven weeks (Control group); Group 2 was fed with G. elegans alkaloids diet for seven weeks (Low-dose group); Group 3 was fed with G. elegans alkaloids diet for seven weeks (Middle-dose group); Group 4 was fed with G. elegans alkaloids diet for seven weeks (High-dose group). After the treatment mentioned above, pigs were fasted for 12 h before being sacrificed. Bodyweight and liver weight were recorded. The serum was separated from the blood collected and the blood biochemical indexes were determined（Table 1）.
RNA extraction and qRT-PCR
Target RNA was extracted using TRIzol reagent according to the manufacturer's instructions. Primers for ANGPTL4、PPARD、PDK4、SLC22A5, GAPDH and KLF10, were obtained from Invitrogen Bioengineering Corporation (Shanghai, China). The sequences of the primers being used for PCR reactions were listed in Table 2 RT-PCR was performed for 40 cycles at the following conditions: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 58.3 °C for 30 s, extended at 72 °C for 30 s. In addition, the expression of target genes was normalized to that of GAPDH, and the relative quantification of mRNA levels was performed using the 2-ΔΔCt method.
Detection of LPL, CYP7A1 and HMG-CoA cytokines in tissue
Dissect the muscle tissue with clean tools, on ice preferably and as quickly as possible to prevent degradation by proteases. Place the tissue in round bottom microfuge tubes immersed in liquid nitrogen, and then keep on ice for immediate homogenization. Add 400 µL complete extraction buffer to the tube and homogenize with an electric homogenizer. Rinse the blade twice using 300 µL complete extraction buffer for each rinse, then maintain constant agitation for two hours at 4 °C. Centrifuge for 20 min at 13,000 rpm at 4 °C. Place on ice, aliquot supernatant to a fresh, chilled tube and store samples at -80 °C. ELISA test is based on the kits according to the manufacturer’s instructions.
Histological and immunofluorescence analysis
The muscle tissues were assessed by hematoxylin-eosin(H&E) staining as described in Liu et al. Frozen muscle sections (5 μm thick) were treated with 0.1% Triton X-100 in PBS and were incubated for 45 min at room temperature with the primary antibody mix and diluted in 0.05% Tween20 in PBS. After three washing steps with PBS, sections were incubated for 45 min at room temperature with the appropriate fluorescent-labelled secondary antibodies.
Total protein lysates of muscle tissues were obtained by incubation on ice with RIPA containing 1% PMSF, cocktail and phosphatase inhibitors. The supernatant was collected after centrifugation at 12,000×g for 15 min at 4 °C. Protein quantification was performed with a BCA kit. Aliquots containing 30 μg of protein were separated by SDS-PAGE (5% concentrated gel and 10% separated gel) and transferred to PVDF membranes. The membrane was incubated with 5% nonfat dried milk at room temperature for two hours, followed by specific primary antibodies at 4 ° C overnight and the corresponding secondary antibodies at room temperature for one hour. Immunoreactive bands were visualized by ECL and quantified by software Image J (NIH, USA).
All data are shown as mean± S.E.M. All statistical analyses, including one-way ANOVA, t-test and Pearson’s correlation, were executed using SPSS 20.0. All experiments were independently repeated at least replicates, where the alpha level is set to 0.05 for significance.