Raw materials and preparation of DBT formula
Three-year-old Astragalus membranaceus var. mongholicus (AR) and two-year-old Angelica sinensis roots (ASR) were collected. The raw materials were qualified according to analysis listed in China Pharmacopeia, and the microscope identifications were carried by Dr. Tina Dong. In order to produce DBT formula, the amounts of ASR and AR were preciously weighed at 1:5. The herbs were mixed well and then boiled twice in water [1, 2]. Before performing biological assay, the water extract was lyophilized and re-suspended in water at final concentration at 100 mg/mL. All the samples were kept at −80 °C.
Fingerprint chromatograms of DBT
An Agilent 1200 series system (Agilent, Santa Clara, CA), supplied with auto-sampler, binary pump, degasser and thermo-stated column compartment, was involved here for chemical fingerprint analysis. Chromatographic conditions were performed on an Agilent, Eclipse Plus, C18 column (4.6 × 250 mm, 5 µm). Here, acetonitrile (as Solvent A) and 0. 1% formic acid (as Solvent B) were utilized as mobile phase, the flow rate was kept as 1.0 mL/min at room temperature. In brief, the chromatographic condition of DBT was shown here: 0–10 min, 15% of solvent A; 10–45 min, 15–50% solvent A; 45–50 min, 50% of solvent A; 50–70 min, 50–80% solvent A. All samples were able to pass through 0.45 µm Millipore syringe filter before injecting for analysis, and 10 µL was injected for HPLC. An ELSD detector and a DAD detector at an absorbance of 254 nm were used [2, 16].
SH-SY5Y cells, a human neuroblastoma line, were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). In brief, cells were supplied with Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/mL penicillin and streptomycin in 37 °C incubator. DMEM, FBS and penicillin and streptomycin were obtained from Invitrogen Technologies (Carlsbad, CA, USA).
Four promoter constructs were purchased from Addgene (Suite, MA), namely pBDNF-Luc, pGDNF-Luc, pNGF-Luc and pCRE-Luc carrying BDNF, GDNF, NGF, and CRE promoter sequences, respectively. Two hundred nanogram of each plasmid was transfected by Lipofectamine 3000 reagent (Invitrogen) in cultured SH-SY5Y cells. Cultured cells were seeded in 24-well plates at 6 × 104 cell/mL, and then added various concentrations of drugs for 2 days. After drug treatment, the medium was aspirated, and PBS was utilized twice for washing cells. Luciferase lysis buffer was stored at 4 °C, containing 0.2% Triton X-100, 1 mM dithiothreitol (DTT) and 100 mM potassium phosphate, was employed here to lyse cell. Centrifugation at 13,200 rpm for 10 min, and then the supernatant was harvested and used to carry out luciferase assay (Tropix Inc., Bedford, MA, USA). Forskolin (FSK) served as a positive control.
The phosphorylations of Erk1/2 and CREB were analyzed here by western blot with using specific antibodies. Serum-starved were at least 3 h of cultured before drug applications. The cultures were collected immediately in 2× lysis buffer (125 mM Tris–HCl, 2% SDS, 10% glycerol, 200 mM 2-mercaptoethanol, pH 6.8) after drug/inhibitor (U0126, 10 µM)/activator (TPA, 100 nM) applications, and the samples were prepared for SDS-PAGE. After transferring, the membranes were incubated with 1: 5,000 dilutions of anti-phospho-Erk1/2 (Upstate, Lake Placid, NY, USA), 1:5000 dilutions of anti-phospho-CREB (Cell Signaling, Danvers, MA, USA) overnight and incubated at cold room. Before adding secondary antibody, TBST should be employed here for washing membranes 4 times, and each time at 10 min. Lastly, 1:5000 dilutions of horseradish peroxidase (HRP)-conjugated anti-rabbit secondary were incubated at 3 h at room temperature, the immune-complexes were observed by the enhanced chemiluminescence (ECL) method (Amersham Biosciences, Piscataway, NJ, USA). The band intensities were compared on an image analyze tool.
The expression levels of NGF, BDNF and GDNF were analyzed by western blot. In brief, cells were seeded onto 6-well plate, and after 2 days of drug/activators (FSK, 10 µM)/blockers (U0126, 10 µM) treatments, the cultures were washed by PBS twice and harvested in high salt lysis buffer (1 M NaCl, 10 mM HEPES, pH 7.5, 1 mM EDTA, 0.5% Triton X-100). After 10 min of centrifugation at 16,100 rpm, supernatant was kept for further step. Equal amount of sample protein was added by 2× lysis buffer and heated at 95 °C before subjecting to SDS-PAGE. The specific antibodies, i.e. anti-GDNF, BDNF and NGF antibodies (Cell Signaling) were incubated with membranes after transferring at 1:1000 dilutions at cold room for 12 h.
Cell viability assay
MTT was employed for revealing cell viability. In brief, cells were seeded in 96-well plate. Drug treatments for 2 days, the final concentration of 0.5 mg/mL of MTT solution was applied into after 2 h durations, the production of purple crystal was dissolved in DMSO. The optimized absorbance was set at 570 nm.
Statistical analysis and other assays
One-way analysis of variance was utilized for statistical tests. Data were expressed as Mean ± SEM, where n = 4–5. The highly significant was labeled as (***) where p < 0.001, more significant (**) where p < 0.01 and significant (*) where p < 0.05 compared with corresponding control group without U0126; (^^^) where p < 0.001, more significant (^^) where p < 0.01 compared with corresponding FSK or DBT group without U0126, respectively. The Minimum Standards of Reporting Checklist (Additional file 1) contains details of the experimental design, and statistics, and resources used in this study.