Preparation of FGF21
The recombinant P-SUMO vector containing FGF21 gene which was constructed in our laboratory was transformed into host bacterium, Rosetta (DE3). Single colonies of the Rosetta (containing the plasmid of P-SUMO-FGF21) grew in LB media containing ampicillin (100ug/ml). When the OD600 reach 0.4 to 0.6, FGF21 protein expressed at 25℃ for 6 hours (IPTG final concentration: 0.25mmol/L). The FGF21 was purified by AKTA Purifier (GE Healthcare) [22]. The activity assay of FGF21 in vitro performed by HepG2 cells cultured as a materials of glucose uptake in a 96-well plate and incubated at 37℃ in a 5% CO2 humidified atmosphere. When the number of the cells reached 70% to 80%, the cells were starved for 12h in a serum-free medium followed by stimulation without or with various concentrations of FGF21 for another 24h. We used a glucose assay kit (Beijing Kingkawk pharmaceutical CO, LTD) to measured glucose uptake.
Assessment of SH-SY5Y cells
Cell viability was measured by a modified CCK8 assay (BestBio, Shanghai). SH-SY5Y cells were cultured on 96-well plates and incubated with LPS (100μg/ml, sigma, USA) and in the presence of FGF21 (0, 1, 5, 10μmol/L) for 48h, then 10% CCK8 was added in wells and incubated at 37℃ for 4h. Absorbance at 450nm were measured using ELISA. The cells experiment repeated in 6-well plates for testing the levels of protein by Western blot.
Animals and treatment regimens
Male C57BL/ksj-db/db mice (7-8 weeks, blood glucose concentration﹥20mmol/L, Shanghai Silaike Experimental Animals LTD) and the C57BL/6 mice (7-8weeks) fed and maintained under constant SPF conditions and temperature 23±2℃, humidity 50±5%, with 12h light and 12h dark cycles. In the mouse experiment, diabetic mice were divided into 5 groups : (1) wild type group for db/db mice (normal control, n=10), (2) diabetic mice treated with PBS (model control, n=10), (3) diabetic mice treated with FGF21 (0.5mg/kg, n=10), (4) diabetic mice treated with FGF21 (1.0mg/kg, n=10), (5) diabetic mice treated with FGF21 (2.0mg/kg, n=10). PBS or FGF21 injected into the mice once daily for four months. Blood samples were taken for serum T-AOC, CAT and T- SOD measurements. T-AOC (A015-1-2), CAT (A007-1-1) and SOD (A001-3-2) Assay Kits were purchased from Nanjing Jiancheng Bioengineering Institute, China.
In aging mice model, male C57BL/6 mice were divided into 4 groups: (1) 4 month C57BL/6 mice were fed before the experiment ending (n=10), (2) 14 month mice were treated by PBS (n=10), (3) 14 month mice were treated by FGF21 (1.0mg/kg, n=10), (4) 14 month mice were treated by metformin (20mg/kg, Sigma, USA, n=10). PBS, FGF21 or metformin were administrated in the mice once daily for 6 months. The relative situation of neurodegeneration were assessed by Water maze, Western blot and others.
Morris Water Maze
Before the experiment ending, the aging mice performed water maze test. Briefly, each mice was trained to find a hidden platform for six consecutive days, four trials per day with 120s rest period. The swimming pathway and escape latency were recorded in each trial. On the seven day, the swimming pathway, escape latency and number of platform quadrant crosses were examined in a 120s free swim period without platform.
H&E Staining
The brains of experimental animals were removed one lobe in 4% formaldehyde solution and embedded in paraffin. Sections were cut and stained with hematoxylin and eosin. The others were flash frozen in liquid nitrogen and stored at -80℃ for Real Time-PCR and Western blot.
Immunohistochemisty (IHC) Analysis
After dehydration, sections were subjected to antigen retrieval in 0.01 mol/L citrate buffer (pH=6.0) by microwaving, and then placed in 3% hydrogen peroxide for 30min at room temperature. After blocking with 5% BSA, the sections were incubated with anti-IL6 antibody (1:100, DF6087, Affinity, USA) and anti-IL8 antibody (1:100, DF6998, Affinity, USA) overnight at 4℃, followed by the anti-Rabbit secondary antibody (1:200, R&D, USA). The reaction was visualized with DAB solution. After counterstaining with hematoxylin, the sections were enveloped and views under a light microscope.
Immunocytochemistry (ICC) Analysis
The cells were immobilized on glass slides, then the follow-up operation is basically the same as IHC. The primary antibodys were Tau (1:100, Abcom, ab32057, USA) and β-Amyloid1-42 (1:100, bs-0107R, Bioss, China). The secondary antibody was anti-Rabbit antibody (1:200, R&D, USA)
Reverse Transcription and Real Time-PCR
Total RNA was isolated from tissues (100mg) using TRIZOL (Invitrogen, Carlsbad, CA) to perform Reverse transcription and Real Time-PCR. Real Time-PCR was carried out using the Eppendorf Realplex 4 instrument (Eppendorf, Hamburg, Germany). The β-actin was detected as an internal control. The primer sequences used were shown as Table 1.
Western Blotting
Tissues were lysed for total protein extraction in RIPA Lysis Buffer (Nanjing Jiancheng Bioengineering Institute, China) together with a protease inhibitor PMSF (Sigma-Aldrich Corporation, Saint Louis, MO, USA). The protein concentration was measured using the Pierce BCA protein Assay Kit (Thermo, USA). The membranes were block with the 5% skim milk in phosphate buffer saline (PBS) and probed with the following primary antibody overnight at 4C: AMPKα (1:1000, Cell Signaling Technology, D63G4, USA), P-AMPKα (Thr172) (1:1000, Cell Signaling Technology, 40H9, USA), AKT (pan) (1:1000, Cell Signaling Technology, C67E7, USA), P-AKT (Thr308) (1:1000, Cell Signaling Technology, D25E6, USA), Gsk-3β (1:1000, Cell Signaling Technology, D5C5Z, USA), P-GSK-3β (Ser9) (1:1000, Cell Signaling technology, D85E12, USA), Tau (1:1000, Abcom, ab32057, USA), P-Tau-s396 (1:1000, Abcom, ab151559, USA), BDNF (1:1000, Abcom, ab108319, USA), PPARγ (1:500, bs-0530R, Bioss, China), SIRT1 (1:500, bs-0921R, Bioss, China), β-Amyloid1-42 (1:500, bs-0107R, Bioss, China), NF-κB (1:500, AF5006, Affinity, USA), IL6 (1:500, DF6087, Affinity, USA), IL8 (1:500, DF6998, Affinity, USA). We used Rabbit or Mouse (1:7500, HAF008/HAF007, R&D, USA) IgG HRP-conjugated antibody as secondary antibody. Blots were developed using an ECL kit (Amersham Biosciences, Piscataway, NJ).
Enzyme-linked immunosorbent assays (ELISA)
The protein levels of Tau and β-Amyloid1-42 in the supernatants of the brains tissue or cells homogenate were measured using ELISA, according to the double antibody sandwich method. Briefly, Tau antibody (Abcom, ab32057, USA) and β-Amyloid1-42 (bsm-0107M, Bioss, China), was bonded to a solid state carrier, then incubated the supernatants of the brains tissue or cells homogenate, Tau antibody (bioss, bs-0157R, China) or β-Amyloid1-42 (bs-0107R, Bioss, China), and anti-Rabbit secondary antibody (HAF008, R&D, USA) in turn. Color lipuid was added in wells and incubated at 37℃ for 0.5h. Finally stop buffer was added. Absorbance at 450nm were measured.
Statistical analysis
Statistical analysis performed by SPSS 19.0, and date showed as mean ± SD. All data performed using one-way analysis of variance (ANOVA), followed by Student two-tailed-t-text. Statistical significance was defined as P﹤0.05.