Lycium barbarum увеличивает калорийность расходы и уменьшает окружность талии у здоровых полных мужчин и женщин: пилотное исследование

2.1. Preparation of LB Extract

Dried LB was purchased in raw powder form (batch no. 53101: DeDu Holdings, West Ryde, Australia). The preparation of an extract of LB was performed as previously described [29, 30] with modifications. Briefly, 250 mg of fine LB powder was extracted with 2.5 mL of methanol by sonication at room temperature for 15 min followed by centrifugation for 5 min. This step was repeated four times. The solvent was then evaporated under reduced pressure below 50°C, and the remaining solid was collected. The identification and quantification of taurine in the LB extract were undertaken by thin layer chromatography (TLC) analysis as previously described [29].

2. 
   Tissue Culture and Treatment
   

The human retinal epithelial cell line, ARPE-19, was provided by Dr. Weiyong Shen (Save Sight Institute, Sydney, Australia). Cells were cultured as previously described [31]. Briefly, the cells were cultured in a humidified incubator at 37°C in 5% CO₂ in 10% fetal bovine serum-defined minimal essential medium (FBS-DMEM)-F12 medium) containing 5.5 mM D-glucose, supplemented with 100 U/mL penicillin G and 100 μg/mL streptomycin. The culture medium was replaced with fresh medium every second day. Upon confluence, cultures were passaged by dissociation in 0.05% (w/v) trypsin (Gibco-Life Technologies, Roseville, MD, USA) in phosphate-buffered saline (PBS) pH 7.4. For high-glucose-induced functional studies, cells were maintained in fresh medium containing 1% FBS for 2 h prior to use in the experiments. Cells were then pretreated with samples, rosiglitazone (RG), 15-deoxy-delta (12, 14)-prostaglandin J₂ (PG), or vehicle (0.5% DMSO) for 6 h followed by further exposure to normal (5.5 mM) or high (33.3 mM) D-glucose for 48 h. Cells incubated in 27.5 mM mannitol (M) served as osmotic control [32].

2.3. Cell Viability by MTS Assay

Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA), as previously described with some modifications [33]. Briefly, ARPE-19 cells were cultured in 96-well plates (3 × 10⁴/mL). Upon confluence, cells were treated with vehicle (0.05% DMSO), RG, colchicine (Colch), or samples for 24 h, 48 h, and 72 h. For high-glucose cytotoxicity experiments, the medium was replaced with fresh medium containing 1% FBS for 2 h prior to use in the experiments. Cells were then preincubated with treatment samples, RG, PG, or vehicle (0.5% DMSO) for 6 h followed by exposure to normal (5.5 mM) or high (33.3 mM) D-glucose for a further 48 h. On the day of the proliferation assay, 20 μL of the MTS solution was added to each of the 96 wells and incubated at 37°C for 1 h in a humidified (5% CO₂) environment. The absorbance at 490 nm was read in a microplate reader (Bio-Rad Laboratories, Inc, CA). The percentage of cell proliferation was calculated as (OD of treated samples/OD of untreated control) × 100.

2.4. Detection of Apoptotic Cells

Cells undergoing apoptosis were determined by Hoechst 33342 staining and Annexin V flow cytometry analysis, as previously described with modifications [4]. Briefly, cells were fixed in 4% formaldehyde for 10 min and stained with Hoechst dye for 10 min. Cells were visualized under an inverted fluorescence microscope (excitation at 365 nm and emission at 480 nm, using a UV filter). A minimum of 300 cells in each cover slip, from six randomly selected fields, were counted, and apoptotic cells were expressed as percentage of total cells counts.

2.5. Quantification of Apoptotic Cells

Apoptotic cells were quantified by flow cytometry using FITC Annexin V apoptosis detection kit (BD Biosciences, San Jose, USA), as previously described [34]. Briefly, after appropriate treatments, 1 × 10⁵ cells/100 μL were collected, and 5 μL of FITC Annexin V and 5 μL of propidium iodide (PI) were added. Cells were incubated in the dark at 25°C. After 15 min, 400 μL of 1x binding buffer was added to the cells. The Annexin V-positive (+)/PI-negative (−) cells, indicating early apoptotic cells, and the Annexin V-positive (+)/PI-positive (+), indicating late apoptotic cells, were detected by FACSCalibur (BD Biosciences, San Jose, USA). The results were analysed using the WINDI 2.5 software. Annexin V-FITC conjugates were detected at the FL1 channel, and PI was read on the FL3 channel.

2.6. Caspase-3 Activity Assay

Caspase-3 activity was measured by the caspase-3 fluorometric assay system (BD Biosciences, San Jose, USA), as previously described [35] with modifications. Briefly, a total of 1 × 10⁶ cells were harvested and resuspended in cold cell lysis buffer (10 mM Tris-HCL, 10 mM NaH₂PO₄/NaHPO₄ (pH7.5), 130 mM NaCl, 1% Triton-X-100, and 10 mM sodium pyrophosphate (NaPPi)) for 30 min on ice. Cell lysate protein concentrations were determined by the BCA (bicinchoninic acid) assay (Thermo, USA) according to the manufacturer's instructions. Equal amounts of protein were added to each well in a 96-well plate containing 200 μL of HEPES buffer and 5 μL of the fluorogenic substrate, DEVD-AMC (7-amino-4-methylcoumarin), and incubated for 1 h at 37°C. Cleavage of the substrate by active caspase-3 resulted in an increase of fluorescence (excitation = 380 nm and emission = 460 nm), measured in a 96-well plate fluorometer (FLUOstar OPTIMA), and expressed as unit per milligram protein.

2.7. Protein Extraction and Semiquantitative Western Blotting Analysis

Immunoblots were conducted as described previously [36]. The proteins from the cells were prepared using the Ripa lysis buffer (25 mM Tris buffer (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS). The lysed cells were centrifuged at 12,000 rpm (Micromax RF centrifuge, Thermo IEC, MA, USA) for 10 min and supernatants resolved by SDS-PAGE, 4–12% (Invitrogen, Australia). Protein was transferred to cellulose membrane in transfer buffer (Tris base 25 mM, glycine 192 mM, pH 8.3) and blocked in 5% skim milk powder (Sigma-Aldrich, St. Louis, MO, USA) overnight. The primary antibodies (Santa Cruz Biotechnology, USA) were anticaspase-3 rabbit polyclonal primary antibodies (1 : 500 dilution). After incubation with the primary antibody for 1 hr at room temperature, the membrane was washed and further incubated with horseradish peroxidase-conjugated anti-mouse secondary antibodies (1 : 6000 dilution; Santa Cruz Biotechnology, USA). Bound antibodies were detected using enhanced chemiluminescence with Lumi-Light Western Blotting Substrate (Roche). The membranes were exposed to X-ray film (Kodak, USA) and developed using the SRX-101A X-ray developer (Konica, Taiwan). The resultant films were quantified by scanning densitometry using ImageJ (National Institutes of Health, Bethesda, MD). Protein expression was quantified by normalization to α-tubulin. The membranes were reprobed with anti-α-tubulin primary antibody (1 : 10,000 dilution; Santa Cruz Biotechnology, USA) after stripping and overnight blotting with 5% skim milk. The membranes were reincubated with horseradish peroxidase-conjugated anti-mouse secondary antibody and detected using the same procedure as described above. Cell lysate protein concentrations were determined by the BCA (bicinchoninic acid) assay (Thermo, USA) according to the manufacturer's instructions.

2.8. Chemicals

Rosiglitazone (RG) was purchased from Alexis Biochemicals (San Diego, CA, USA). Pure taurine compound, 15-deoxy-delta (12, 14)-prostaglandin J₂(PG), and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise indicated.

2.9. Statistical Analysis

All results are expressed as means ± SEM Data were analysed by 1-factor analysis of variance (ANOVA). If a statistically significant effect was found, the Newman-Keuls test was performed to isolate the difference between the groups. P values less than 0.05 (P < 0.05) were considered to indicate significance.