Danggui Buxue Tang, an ancient Chinese herbal decoction, protects β-amyloid-induced cell death in cultured cortical neurons

The herbal materials of A. memebranaceus var. mongholicus (AR) and A. sinensis (ASR) were obtained and morphologically identified by Dr. Tina TX Dong (one of the author) in 2013. The voucher specimens of AR (# 02–10-04) and ASR (# 02–09-01) were recorded and displayed in Centre for Chinese Medicine of HKUST. To generate DBT formula, 5 parts of AR and 1 part of ASR were mixed well and boiled in water for twice, according to previous reports [16, 17]. The water extract of DBT was dried by lyophilization and re-suspended in water at 100 mg/mL final concentration. Before application, DBT was diluted to the required concentration in the cultured medium. The treatment of DBT in the Aβ-treated cultures was about 48 h, unless otherwise specified.

An Agilent 1200 series system (Agilent, Santa Clara, CA) was utilized here to determine the chemical compositions of DBT decoction. The Agilent 1200 series system was equipped with degasser, binary pump, auto-sampler, and thermo-stated column compartment. Agilent, Eclipse Plus, C₁₈ column (4.6 × 250 mm, 5 μm) with acetonitrile (as Solvent A) and 0.01% formic acid (as Solvent B). The detailed HLPC condition was described before [10].

The experimental procedures were approved by The Animal Experimentation Ethics Committee of the Hong Kong University of Science and Technology (No. 17–283 for Animal Ethics Approval) and under the guidelines of “Principles of Laboratory Animal Care” (NIH publication No. DH/HA&P/8/2/3). Primary cultured cortical neurons were cultured as described previously with slightly modifications [18, 22]. Cortex was dissected by trypsin from embryonic day 18 rats by 100 mg/kg of Ketamine and 10 mg/kg of Xylazine treatment. The dissociated cortical neurons were cultured in neural basal medium with B27 and 0.5 mM GlutaMax at 37 °C incubator supplying with 5% CO₂. Cells were cultured for 14 days before treatments. Reagents for cell cultures were purchased from Invitrogen Technologies (Carlsbad, CA).

MTT assay was used for analysis cell viability (Sigma-Aldrich, St. Louis, MO). In brief, cells were seeded in 96-well plate and then performed drug treatment for 48 h. After that, MTT solution was added into the cultures and incubated for 2 h, the purple crystal production was dissolved by DMSO solvent and detected at 570 nm absorbance. In crystal violet assay, cells were seeded and cultured in 6-well plate. Colonies were fixed with an ice-cold methanol-acetic acid solution (3:1) for 30 min at − 20 °C and stained with 1% crystal violet for 30 min at room temperature and visualized.

The protein expressions of Bcl2, Bax, cleaved-PARP, cleaved-caspase 3/9 and β-actin were revealed by western blot. Cultures were seeded onto 6-well plate. After drug treatment, the cultures were harvested in high salt lysis buffer (1 M NaCl, 10 mM HEPES, pH 7.5, 1 mM EDTA, 0.5% Triton X-100). Before conducting SDS-PAGE analysis, equal amount of protein level were adjusted with 2X lysis buffer (0.125 M HCl, pH 6.8, 4% SDS, 20% glycerol, 2% 2-meracptoethanol and 0.02% bromophenol blue). The membranes were incubated with anti-Bcl2, Bax, cleaved- PARP and cleaved-caspase 3/9 (CST, Danvers, MA) at 1: 1000 dilutions, respectively, and anti-β-actin (Abcam, Cambridge, MA) at 1: 10,000 dilutions were kept for over 12 h at cold room. Horseradish peroxidase (HRP)-conjugated secondary antibodies were employed at 1: 5000 dilutions for 3 h at room temperature, the immune-complexes were visualized by the enhanced ECL method (Amersham Biosciences, Marlborough, MA).

All processes were instructed following the protocol of caspase-3/9 assay kit (Biovision, Milpitas, CA). Olympus Fluoview FV1000 laser scanning confocal system (Olympus America Inc., Center Valley, PA) at 400 nm excitation and 505 nm emission was employed here for photosensitive reaction measurement. The fold of changes was calculated relative to the untreated cells.

Cells were plated and cultured in 24-well plates. After drug treatment for 48 h and labeled with Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ) as followed manufacturer’s instructions. In brief, 100 μL of binding buffer containing annexin-V/FITC and propidium iodide (PI) were used for covering cells for 15 min at room temperature in the dark condition and then detected by the Olympus Fluoview FV1000 laser scanning confocal system.

Protein levels were measured by Bradford’s method (Herculues, CA). Statistical tests have been done by using one-way analysis of variance. Data were expressed as Mean ± SEM, where n = 3–4. Statistically significant changes were classified as significant (*) where p < 0.05, more significant (**) where p < 0.01 and highly significant (***) where p < 0.001 as compared with control group. Statistically significant changes were classified as significant (^) where p < 0.05, more significant (^^) where p < 0.01 and highly significant (^^^) where p < 0.001 as compared with Aβ group.

Prior to the biochemical analysis of any herbal extract, it is indispensable to perform chemical composition analysis. The HPLC chromatogram of DBT water extract was shown here (Fig. 1). The accurate amounts of each major chemicals were calibrated by a calibration curve, which was obtained from a series of different concentration of the chemical markers. The calibration curve of ferulic acid was y = 21.134x + 19.607; calycosin was y = 10.189x-10.129; formononetin was y = 13.602x + 12.705. After calibrated by the standard curve, 1 g qualified DBT water extract contained 809.44 μg of ferulic acid, 693.19 μg of calycosin and 164.58 μg of formononetin. The cyto-protective functions of DBT against the Aβ-induced cell death in primary cultured neurons were analyzed. Application of Aβ in cultured cortical neurons provoked cell death obviously in a dose-dependent manner (Fig. 2a). From the results, the death rate was ~ 60% under the challenging of 20 μM of Aβ (Fig. 2a). The treatment of DBT reduced the Aβ-induced cell death in a concentration-dependent manner (Fig. 2b). Higher dose of DBT significantly protected cells from the injury: the maximum protection was able to recover cell number up to over 80% at 0.1 mg/mL (Fig. 2b). We also utilized crystal violet to determine cell viability, directly. The death cells lose their adherence, and finally resulted in reducing amount of crystal violet staining in cultures [23]. The DBT-treated cells remained healthy even after the challenge of Aβ (Fig. 2c). The sensitive apoptotic markers, Annexin V- and PI, were measured by fluorescent microscope. The Aβ-treated cells had a higher apoptotic rate of PI and Annexin V, as compared to the blank (Fig. 3). The ratio of Annexin V(+)/PI(+) of blank was 19.5%, positive treatment was 43.7%, lower concentration of DBT application (DBT-L: 12.5 μg/mL) was 19.6%, higher concentration of DBT challenging (DBT-H: 100 μg/mL) was 11.4%, the cotreatment of herbal decoction and Aβ were 41.6 and 21.8%, respectively (Fig. 3). The treatment of DBT suppressed the Aβ-induced cell apoptosis, dramatically (Fig. 3).

The aforementioned results showed that DBT could protect cell death against Aβ challenge.

Apoptosis is a programmed cell death, and inhibitors of apoptosis, e.g. Bcl2, and inducers of apoptosis, e.g. Bax, have been reported and identified [24]. The protein ratio of Bcl2 to Bax gives an index to the cell survival or death, after an apoptotic stimulus [25]. Here, we utilized western blot to reveal the protein expressions of two apoptotic markers in cultured cortical neurons, i.e. Bcl2 (~ 27 kDa) and Bax (~ 21 kDa) (Fig. 4). The application of Aβ significantly up-regulated the expression of Bax by over 3-fold and down-regulated Bcl2 by over 50%, as compared to the negative control (Fig. 4). Thus, Aβ markedly increased the ratio of Bax/Bcl2. Application of DBT in the cultures was capable to reverse the expressions of Aβ-induced cell apoptotic markers in a concentration-dependent manner (Fig. 4). More importantly, DBT decreased the ratio of Bax/Bcl2 obviously, as compared to the Aβ-treated group (Fig. 4), which suggested that this herbal decoction possessed neuroprotective effects in AD cell model.

Caspases are a group of protease enzymes acting as important roles in manipulating cell apoptosis [26]. For example, the activation of caspases triggers degradation of cellular components in a programmed manner, which results in cell death [26]. In cultured cortical neurons, the amounts of cleaved-caspases 3, 9 and PARP were much higher in the Aβ-treated group, as compared to the negative control (Fig. 5). Challenging of DBT, on the other hand, suppressed the expressions of these cleaved-caspases in concentration-dependent manners (Fig. 5). The presence of high dose of DBT (0.1 mg/mL) in cultured cortical neurons decreased the levels of cleaved-PARP, caspase 3/9 at ~ 25%, ~ 50% and ~ 40% respectively, indicating the neuroprotective functions of DBT decoction (Fig. 5). Fluorescence signal, directly reflecting the amount of caspase activity in the cells, was also utilized and measured here [27]. In cultured cortical neurons, we found that the activities of caspases 3 and 9 were strongly activated in Aβ-treated group, as compared to others (Fig. 6). However, lower fluorescent signals were observed in DBT-treated group, and these results were fully in line with the western blotting results.

To reveal that synergistic effect of AR and ASR in the pharmacological properties of DBT, the specific apoptosis markers were determined here. One hundred μg of ASR significantly inhibited the expressions of Bax, cl-PARP and cl-caspase 9 in cultured neurons, as compared to the control. On the other hand, 100 μg of AR was capable to suppress the expressions of cl-PARP, cl-caspase 3/9 activities (Fig. 7). However, the application of DBT dramatically declined Bax/Bcl2 ratio and other apoptosis marker expression levels, and the rate of decline was much stronger than the sum of single application of ASR and AR (Fig. 7).