2.1 Reagents and antibodies
GPS (used in animal experiment, purity>98%) was obtained from NanJing Dilger Medical Technology Co.,Ltd (Nanjing, China). GPS (used in vitro experiment, purity>99%) was obtained from Nanjing ZheWeiKang Biological Technology Co., Ltd (Nanjing, China). Metformin (Met, used in vitro experiment, purity>99%), methyl thiazolyl tetrazolium (MTT) and insulin were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Met (used in animal experiment) was obtained from American Shanghai Squibb Pharmaceuticals Ltd (Shanghai, China). Dulbecco's modified eagle (DMEM) medium, fetal bovine serum (FBS), trypsin and phosphate buffer solution (PBS) were purchased from Gibico (New York, USA). Palmitic acid (PA), dimethyl sulfoxide (DMSO) and oil red O were purchased from Sigma Aldrich (Missouri, USA). Bovine serum albumin (BSA) was purchased from solarbio (Beijing, China). The detection kits of glycated serum protein (GSP), hemoglobin A1c (HbA1c), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) were purchased from Jiancheng Bioengineering Institute (Nanjing, China).
Antibody against FGFR1 was obtained from Cell Signaling Technology (Massachusetts, USA). Antibodies against FRS2α, AKT, phosphorylated AKT (p-AKT), GCK and α-Tubulin were purchased from Proteintech Group (Wuhan, China). Antibodies against PI3K and phosphorylated PI3K (p-PI3K) were purchased from ABclonal Technology (Massachusetts, USA). Antibodies against FoxO1, phosphorylated FoxO1 (p-FoxO1), LDLR and Lamine B1 were purchased from Abcam (Massachusetts, USA). Antibody against G6Pase was obtained from Boster (Wuhan, China). Antibody against phosphorylated FRS2α (p-FRS2α) was obtained from Affinity Biosciences (Ohio, USA). GFP antibody was purchased from Beyotime Institute of Biotechnology (Shanghai, China).
2.2 Animal experiments
Six-week-old male db/db mice (Animal Quality Certificate Number: 201919681) obtained from Gem Pharmatech Co., Ltd (Nanjing, China) were selected in animal experiments. The mice were kept in specific pathogen free condition and given normal feed. All experiments were conducted in accordance with the China Animal Welfare Legislation and were approved by Ethics Committee on the Care and Use of Laboratory Animals of Sun Yat-sen University (Guangzhou, China).
After adaptation for one week, the db/db mice were divided randomly into five groups (n=8) including diabetic model group, low-dose group (50 mg/kg), medium-dose group (100 mg/kg), high-dose group (200 mg/kg) and Met group (195 mg/kg) based on fasting blood glucose (FBG) value. Wild db/m (n=8) were the control group. The same volume of normal saline was given to the mice in both control group and diabetic group. Body weight and FBG were measured weekly. After intragastric administration for 10 weeks, all mice were sacrificed to collect the serum. The livers were fixed in 4% paraformaldehyde or frozen at -80 ℃ for further experiments.
2.3 Liver tissue staining and immunohistochemistry
Hematoxylin eosin (HE) staining was conducted as reported method [20]. In short, liver tissues were fixed in 4% paraformaldehyde at room temperature for more than 24 h. Slices (5 µM) were made after being paraffin embedding and then dried at 60 ℃ for 24 h. After that, the procedures of deparaffinization, rehydration, hematoxylin and eosin staining were conducted in turn. Finally, the sections were dehydrated and covered with neutral balsam. Meanwhile, periodic acid schiff (PAS) staining and immunohistochemistry were conducted as reported method after paraffin sections were made [21, 22].
Fresh liver tissues were fixed in 4% paraformaldehyde at room temperature for more than 24 h, and then dehydrated by immersing in 15% and 30% sucrose solution. Then optimal cutting temperature compound-embedded slices (10 µM) were made and kept at -20 ℃. After that, oil red O staining was conducted as reported method [23].
2.4 Cell culture
HepG2 cells (American Type Culture Collection, Maryland, USA) were cultured with DMEM medium containing 25.5 mM glucose and 10% fetal serum at 37 ℃ in a humidified incubator with 5% CO2. After serum-deprivation for 12 h, the cells were divided into different groups. Next, HepG2 cells were treated with GPS and 0.25 mM PA (dissolved in 20% BSA) for 24 h. The cells were stimulated with 100 nM insulin for 10 min before collecting for western blotting. Met was the positive control in this study.
2.5 MTT assay
The cytotoxic of GPS on HepG2 cells was measured by MTT assay. Briefly, HepG2 cells (5×103 cells/mL, 100 µL) were inoculated into 96-well plate. After reaching about 50% confluence, the cells were cultured with FBS-free DMEM that contained different concentrations of GPS for 24 h. Then 10 µL of MTT (5 mg/mL) was added to each well. After 4 h, the supernatant was removed and 200 µL of DMSO was added into wells. Finally, the optical density was measured at 570 nm with microplate reader (Omega Bio-Tek, Georgia, USA).
2.6 Glucose consumption assay
Firstly, HepG2 cells were treated with various concentrations of GPS for 24 h. After that, the supernatants were replaced with DMEM without phenol red (New York, USA) containing 100 nM insulin for 4 h. Later, the supernatants were collected to examined glucose content with the method of glucose oxidase (Jiancheng, Nanjing, China). Finally, glucose content was computed by subtracting glucose content in DMEM without phenol red and standardized using the number of cells in each wells (detected by MTT method).
2.7 Cellular Oil Red O Staining
Oil red O powder was diluted in isopropyl alcohol with the concentration of 0.5%. Then above solution was mixed with distilled water by 3:2 and filtered to remove residual particles. After washing with PBS for 3 times, HepG2 cells were fixed with 4% paraformaldehyde for 10 min and then recolored with oil red O dye liquor for 30 min at room temperature. Finally, the cells were observed by cell imaging system (EVOS FL Auto, Thermo Fisher Scientific, Massachusetts, USA) after hematoxylin staining for 5 min.
2.8 Western blot
The liver tissues or HepG2 cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) mixed with protease and phosphatase inhibitor cocktail (Selleck Chemicals, Texas, USA). Nuclear and cytoplasmic proteins were extracted by special protein extraction kit (Active Motif, Carlsbad, USA). Later, the concentration of protein was measured by BCA Protein Assay Kit (Pierce, Illinois, USA). After that, equal amount of proteins boiled with loading buffer for 5 min were subjected to 8-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Mellipore, Massachusetts, USA). After sealing with 5% skim milk at room temperature for 1 h, PVDF membranes were incubated with primary antibodies and secondary antibodies labeled with HRP (Promega, Wisconsin, USA). The signals of antibodies were visualized with ECL detection kit (Pierce, Illinois, USA) and observed by Tanon 5200 Chemiluminescent Imaging System (Shanghai Tanon Technology Co., Ltd., Shanghai, China). The software of Image J was applied for quantitative analysis.
2.9 Immunofluorescence
HepG2 cells were cultured on glass coverslips placed in 24-well plate. After intervention, the cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 (dissolved in PBS) for 10 min successively. Next, the cells were blocked with 10% goat serum (Boster Biological Technology Co., Ltd., Wuhan, China) for 1 h at room temperature and incubated with primary antibody at 4℃ for 24 h. After that, HepG2 cells were incubated with secondary antibody (Thermo, Massachusetts, USA) and DAPI (Beyotime Institute of Biotechnology, Shanghai, China). Finally, the fluorescent images were captured by Zeiss LSM 510 laser confocal fluorescence microscope (Oberkochen, Germany).
2.10 Immunoprecipitation assay
The proteins were extracted by lysing HepG2 cells with immunoprecipitation (IP) buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitor (Selleck Chemicals, Texas, USA) on ice for 30 min. Next, 400 µg proteins were incubated with 20 µL protein agarose A/G beads (Thermo, Massachusetts, USA) to reduce the nonspecific combination, followed by collecting the supernatants with transient centrifugation. Later, the supernatants were incubated with antibodies (against FGFR1 or IgG) with shaking for 12 h at 4 ℃. Subsequently, protein agarose A/G beads (20 µL) were added to the supernatants for further incubation (4 ℃, 4 h). Collecting beads by transient centrifugation and washing beads with IP buffer for 3 times. Finally, 20 µL loading buffer (Fdbio, Hangzhou, China) was added and the mixtures were boiled for 5 min to denature proteins. The protein levels were detected according to the method of western blot.
2.11 Transfections of plasmids and small-interfering RNA
FGFR1 plasmid was proved by Shanghai Genechem Co.,Ltd. (Shanghai, China) and transfected according to instruction of LTX reagent and PLUS™ reagent (Life Technologies, New York, USA).
The FGFR1 siRNA oligonucleotides was purchased from GenePharma (Shanghai, China). The sequences are as follows:
sense: 5'-CGGUCAUCGUCUACAAGAUTT-3';
antisense: 5'-AUCUUGUAGACGAUGACCGTT-3'.
The transfection of siRNA was conducted according to the instruction of Lipofectamine RNAiMAX reagent (Life Technologies, New York, USA).
2.12 Statistical analysis
All experimental data were analyzed by the software of Graphpad Prism 5.0 and expressed as mean ± standard deviation (SD). The experiments in this study were performed at least three times with similar results. Unpaired Student's test was adopted to analyse the data between two groups. For multiple comparisons, one-way ANOVA with post hoc was performed. A value of P < 0.05 was considered as statistically significant.