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

Abstract

Background: Danggui Buxue Tang (DBT) is a historical Chinese herbal decoction, and which has more than 800 years of applications. This herbal decoction solely contains two materials: Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) at a weight ratio of 5:1. Clinically, DBT aims to improve anemia syndrome. In complementary and alternative medicine theory, the cause of neurodegenerative disease is proposed to be related with anemia. In line to this notion, low levels of hemoglobin and red blood cell have been reported in patients suffering from Alzheimer's disease (AD), a chronic neurodegenerative disease caused by β-amyloid peptide (Aβ) accumulation. Therefore, we would like to probe the neuroprotective functions of this ancient herbal formula in vitro.

Method: The neuroprotective effects of DBT in the Aβ-induced cell death were detected in cultured cortical neurons by multiple techniques, i.e. confocal and western blot.

Results: In the cultures, application of DBT reduced Aβ-induced apoptosis rate in a dose-dependent manner. In Aβ-treated cortical neurons, the expression ratio of Bcl2 to Bax was altered by DBT. In parallel, application of DBT markedly suppressed the Aβ-induced expressions of apoptotic markers, i.e. cleaved-caspase 3/9 and PARP.

Conclusion: Taken these results, DBT shows promising protective effects against Aβ-induced stress or insult in cultured neurons.

Keywords: Alzhemier’s disease; Complementary and alternative medicine; Danggui Buxue Tang.

Fig. 1

Typical HPLC fingerprint of DBT herbal extract. Typical chromatograph of major chemical markers and 1 mg/mL of DBT were subjected to HPLC analysis, and then a typical chromatograph was presented at a wavelength 254 nm

Fig. 2

Neuroprotective functions of DBT against Aβ-induced cell death. a Cultured cortical neurons were treated with Aβ at different concentration for 48 h. b DBT extract (0–100 μg/mL) was applied 3 h before Aβ (20 μM) for another 48 h. c Crystal violet was employed for revealing cell viability after drug treatment for 48 h. Live cells were dyed and shown in purple color. In (c) and (d), Aβ (20 μM), DBT-L (12.5 μg/mL), DBT-H (100 μg/mL) were used here, and the treatment was similar as in (b). Values were expressed as the percentage of decrease or control reading in Mean ± SEM, where n = 3. Bar = 100 μm

Fig. 3

DBT inhibits Annexin V and PI rates. Representative images of Annexin V-FITC and PI fluorescence staining were shown for the cell apoptosis after drug treatment for 48 h (Aβ: 20 μM; DBT-L: 12.5 μg/mL; DBT-H: 100 μg/mL). Annexin V was visualized by purple signal, and PI was shown in blue color. Scale bar = 50 μM

Fig. 4

DBT regulates expressions of Bcl2 and Bax. Cultured cortical neurons were co-treated with various concentration of DBT (DBT-L: 12.5 μg/mL; DBT-H: 100 μg/mL) and Aβ (20 μM) for 48 h. The protein expression levels of Bcl2 (~ 27 kDa) and Bax (~ 21 kDa) were detected by immunoblot analysis using specific antibodies, and β-actin (~ 42 kDa) served as an internal control. Quantification of target protein expression was calculated by a densitometer. Values were expressed as the percentage of change as compared to control reading, in Mean ± SEM, where n = 3. Statistically significant changes were classified as more significant (**) where p < 0.01 and highly significant (***) where p < 0.001 as compared with control group. More significant (^^) where p < 0.01 and highly significant (^^^) where p < 0.001 are compared with Aβ group

Fig. 5

DBT attenuates the Aβ-induced apoptosis markers. Cultured cortical neurons were co-treated with different amounts of DBT (DBT-L: 12.5 μg/mL; DBT-H: 100 μg/mL) and Aβ (20 μM) or directly treated with Aβ or DBT alone for 2 days. The translational levels of cleaved-PARP (cl-PARP at ~ 85 kDa), cleaved-caspase 3 (cl-caspase 3 at ~ 17 kDa) and cleaved-caspase 9 (cl-caspase 9 at ~ 37 kDa) were detected by immunoblot analysis by specific antibodies. Beta-actin (~ 42 kDa) served as an internal control. Quantification of target protein expression was calculated by a densitometer. Values were expressed as the percentage of change as compared to control reading, in Mean ± SEM, where n = 3. 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. Significant (^) where p < 0.05 and more significant (^^) where p < 0.005 are compared with Aβ group

Fig. 6

The treatment of DBT mitigates Aβ-triggered caspase 3/9 levels. Cultured cortical neurons were co-treated with a series amount of DBT (DBT-L: 12.5 μg/mL; DBT-H: 100 μg/mL) and Aβ (20 μM) for 48 h. Then cells were labeled with caspase 3/9 detection kit and Hoechst for 30 min. The activities of caspase 3/9 were evaluated by measuring the fluorescence intensity. Micrographs were taken by a confocal microscope

Fig. 7

Synergistic properties of DBT extract. DBT (0.1 mg/mL), AR extract (0.1 mg/mL), ASR extract (0.1 mg/mL) were applied onto primary cultures for 2 days. The protein expressions of Bax (~ 21 kDa), Bcl2 (~ 27 kDa), cl-PARP (~ 85 kDa), cl-caspase 3 (~ 17 kDa) and cl-caspase 9 (~ 37 kDa) were detected by specific antibodies. Quantification of target protein expression was calculated by a densitometer. Values were expressed as the percentage of change as compared to control (untreated culture) reading, in Mean ± SEM, where n = 3. 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