Chemicals and reagents
Berberine chloride, lovastatin and 5α-cholestane were purchased from J&K Scientific Ltd (Beijing, China). Cholesterol and coprostanol were supplied by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and Shanghai Huicheng Biotechnology Co., Ltd. (Shanghai, China), respectively. The purity of all standards listed above was more than 98 %. A package of kits for CHO, TG, LDL-C, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyltransferase (GGT) were purchased from BioSino Bio-Technology & Science Inc. (Beijing, China). The total bile acid (TBA) assay kit was obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). High-performance liquid chromatography-grade n-hexane, ethyl acetate and methanol were obtained from J&K Scientific Ltd (Beijing, China). The distilled water was Wahaha purified water (Hangzhou, China).
A Shimadzu gas chromatograph mass spectrometer (GC–MS, Shimadzu Cooperation, Kyoto, Japan), EnSpire multimode microplate reader (PerkinElmer, Waltham, MA, USA), THZ-100-type incubation shaker (Shanghai, China), TG18G-II desktop universal high-speed centrifuge (Hunan Kaida Scientific Instruments Co., Ltd, Hunan, China), XW-80A miniature vortex mixers (Jintan Super Blue Instrument Manufacturing Co., Jintan, Ltd, China) and a MD200-2 nitrogen sweep blowing instrument (Hangzhou Dian Sheng Instrument Co., Ltd, Hangzhou, China) were used.
Animals and study design
Eight-week-old male Syrian Golden Hamsters weighing 110–140 g (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were housed in a controlled environment (21 ± 2 °C, 12-h light/dark cycle) at six per cage with free access to food and water during the acclimatization and study periods. The animal usage and experimental protocols were approved by the Laboratories’ Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College. The research was conducted in accordance with all guidelines and ethics of the Chinese Council on Animal Care.
One hundred and twenty-four hamsters were acclimatized for 7 days in their cages before the start of the study. All animals were then randomly separated into two groups according to their weights: the normal control group (thirty-four animals) and the hyperlipidemia model group (ninety animals). The animals that served as the normal control group were fed the standard diet ad libitum, whereas the hyperlipidemia model hamsters were fed a high-fat diet (HFD) daily for a period of 6 weeks to induce hyperlipidemia. The high-fat diet (Beijing HFK Bioscience Co., Ltd, China) was composed of 83.0 % standard diet, 15.0 % fat, 1.0 % cholesterol and 1.0 % sodium cholate .
At the end of 6 weeks, all animals were fasted for 12 h. Blood was collected, and the serum was obtained to measure the serum levels of TC, TG and LDL-C. The animals fed with a high fat diet showed significantly higher levels of TC, TG and LDL-C than the animals in the normal control group. According to their serum TC levels, the hamsters that were fed the normal diet (ND) were randomly allocated into two groups: 8 animals served as the ND control group, and 26 animals were treated with BBR (100 mg/kg/day). The hamsters that were fed the HFD were then randomly allocated into five groups: 8 animals served as the HFD control group, 26 animals were treated with BBR (50 mg/kg/day), 26 animals were treated with BBR (100 mg/kg/day), 15 animals were treated with lovastatin (10 mg/kg/day) and 15 animals were treated with lovastatin (30 mg/kg/day) .
All animals were orally administered the placebo, BBR or lovastatin, which was dissolved by saline into liquid suspension, in the morning for 10 days. After the animals were fasted for 12 h, the blood samples were obtained from posterior orbital venous plexus at 0, 3, 5, 7, and 10 days (n = 6 for each time point) by immediate centrifugation at 5,000 r/min for 5 min to obtain the serum samples. Feces were collected at the same time points. The hamsters were anesthetized for the collection of bile and liver samples. All samples were stored at −20 °C for the subsequent analyses.
Serum lipid, ALT, AST and GGT levels
The concentration of TC, TG, LDL-C, ALT, AST and ALT in serum was determined at the end of the treatment using enzymatic kits.
Cholesterol in bile, liver and feces
The liver samples obtained from the animals were washed with saline and dried. After weighing, they were homogenized with 2 volumes [v (mL)/w (g)] of saline. The bile and liver homogenates (50 μL) were extracted with 300 μL of ethyl acetate after the addition of 10 μL of an internal standard solution (100 μg/mL of 5α-cholestane in methanol) and 10 μL of methanol, respectively. To prepare the cholesterol standard curve, the bile and liver homogenates (infinitely diluted with saline, 50 μL) were extracted with 300 μL of ethyl acetate after the addition of 10 μL of an internal standard and 10 μL of a cholesterol standard working solution (prepared at a serial concentrations of 0.01, 0.1, 0.2, 1, 2, 10, 20, 100, and 200 μg/mL by dissolving in methanol), respectively. The samples were mixed using a vortex mixer for 20 min, followed by centrifuging at 5,000×g for 5 min. The supernatant was dried under N2 flow at 40 °C , and the residue was reconstituted with 200 μL of n-hexane and filtered through a 0.22-μm micropore membrane. An aliquot of 1 μL was injected into the GC–MS system.
The feces samples were triturated before weighing and dissolved with 10 volumes [v (mL)/w (g)] of methanol, followed by ultrasonic extraction for 30 min and centrifugation at 10,000 r/min for 10 min. The supernatant from the feces (200 μL) was extracted with 1 mL of ethyl acetate after the addition of 10 μL of an internal standard (100 μg/mL of 5α-cholestane), 10 μL of methanol and 500 μL of distilled water. To prepare the standard curve of cholesterol and coprostanol, the supernatants from the feces (infinitely diluted with methanol, 200 μL) were extracted with 1 mL of ethyl acetate after the addition of 10 μL of an internal standard and 10 μL of a cholesterol and coprostanol standard working solution (prepared at a serial concentrations of 0.5, 2.5, 5, 25, 50, 125, 250, and 500 μg/mL by dissolving in methanol), respectively. The samples were mixed using a vortex mixer for 20 min and centrifuged at 5,000×g for 5 min. The supernatant was dried under N2 flow at 40 °C, and the residue was reconstituted with 200 μL of n-hexane and filtered through a 0.22-μm micropore membrane. An aliquot of 1 μL was injected into the GC–MS system.
A GC–MS method was used to determine the cholesterol (as well as coprostanol) levels in bile, liver and feces. A capillary column (AT-1701, 30 m × 0.25 mm, and 0.25 μm phase thickness) was used, and high purity helium was used as a carrier gas at a constant flow rate of 1 mL/min. The temperature program started at 60 °C, was increased to 270 °C after 2 min at a rate of 20 °C/min, and was maintained at that temperature for 12.5 min. The sample was injected in a split-less mode. The injector temperature and transfer linear temperatures were set to 260 and 265 °C, respectively. The mass spectrometer was operated in the electron impact ionization mode with 70 eV, and the ion source temperature was set to 230 °C.
TBA in bile, liver and feces
The concentration of TBA in bile, liver and feces was determined at the end of the treatment using enzymatic kits.
The statistical analysis was performed using Graph Pad Prism 5 software (Graph Pad Software, San Diego, CA, USA). The data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test or two-way ANOVA plus Bonferroni’s post-test, as appropriate. The results are presented as the mean ± standard deviation (S.D.). P-values equivalent to a significance level of 0.05 were considered statistically significant.