Multi-target regulatory mechanism of Yang Xin Tang - a traditional Chinese medicine against dementia

Abstract

Background: Yang Xin Tang (YXT) is a traditional Chinese herbal preparation which has been reported to improve cognitive function and memory in patients with dementia. As the underlying mechanism of action of YXT has not been elucidated, we examined the effects of YXT and its major herbal components in regulating gene transcription and molecular targets related to Alzheimer's disease (AD).

Methods: Aqueous and ethanol extracts of YXT and selected herbal components were prepared and validated by standard methods. A series of biochemical and cellular assays were employed to assess the ability of the herbal extracts to inhibit acetylcholinesterase, reduce β-amyloid aggregation, stimulate the differentiation of neural progenitor cells, suppress cyclooxygenase, and protect neurons against β-amyloid or N-methyl-D-aspartate-induced cytotoxicity. The effects of YXT on multiple molecular targets were further corroborated by a panel of nine reporter gene assays.

Results: Extracts of YXT and two of its constituent herbs, Poria cocos and Poria Sclerotium pararadicis, significantly inhibited β-amyloid aggregation and β-amyloid-induced cytotoxicity. A protective effect of the YXT extract was similarly observed against N-methyl-D-aspartate-induced cytotoxicity in primary neurons, and this activity was shared by extracts of Radix Astragali and Rhizoma Chuanxiong. Although the YXT extract was ineffective, extracts of Poria cocos, Poria Sclerotium pararadicis and Radix Polygalae inhibited acetylcholine esterase, with the latter also capable of upregulating choline acetyltransferase. YXT and its components significantly inhibited the activities of the pro-inflammatory cyclooxygenases. Additionally, extracts of YXT and several of its constituent herbs significantly stimulated the phosphorylation of extracellular signal-regulated kinases and cAMP-responsive element binding protein, two molecular targets involved in learning and memory, as well as in the regulation of neurogenesis.

Conclusions: Several constituents of YXT possess multiple regulatory effects on known therapeutic targets of AD that range from β-amyloid to acetylcholinesterase. The demonstrated neuroprotective and neurogenic actions of YXT lend credence to its use as an alternative medicine for treating AD.

Keywords: Alzheimer’s disease; Chinese medicine; Neural progenitor cell; Neurodegenerative disease; Neuroprotection; Yang Xin Tang; β amyloid.

Fig. 1

Effects of YXT and its selected herbal components on Aβ aggregation and cytotoxicity. A ThT assay was used to measure the aggregation of 5 µM Aβ42 in the absence (Con) or presence of (A) aqueous (Aq) or (B) ethanol (EtOH) extracts of YXT (left panel) and selected YXT herbal components (right panel). Data is illustrated in blue (YXT), dark red (PA), orange (PS), yellow (RA), green (RC), and beige (RP). A WST assay was used to assess Aβ42-induced cytotoxicity in primary cortical neurons cultured in the absence (Con) or presence of aqueous (left) or ethanol (right) extracts of YXT and its selected herbal components (C). Treatment was performed with extracts of RP at 10 μg/mL, and 100 μg/ mL for all the remaining herbs. 100% Aβ-induced cell death was defined as the loss of viable cells upon treatment with 5 µM Aβ42 for 48 h. *P < 0.05 (ANOVA); n = 6

Fig. 2

Effects of YXT and its selected herbal components on AChE activity and ChAT expression. A PC12 cell lysates were treated with extracts of YXT and its components at 1 mg/mL for 30 min, followed by an Ellman assay to determine the AChE activity (n = 6). B PC12 cell lysates were treated with ethanol extracts (EtOH) of PA, PS, and aqueous extract (Aq) of RP at various concentrations (100 µg/mL to 1 mg/mL) prior to the Ellman assay (n = 6). C Tacrine was used as a positive control for the Ellman assay, and the AChE activity measured in its absence was defined as 100% (Con). D RDIN cells were treated with the indicated herbal extracts for 7 days and analyzed by Western blotting to determine the amount of ChAT with β-actin as a loading control. The results were compared with basal group (Veh) in the absence of herbal treatment. *P < 0.05 (ANOVA); n = 6

Fig. 3

YXT and one of its herbal components promote the expression of neurofilaments. RDIN cells were treated with ethanol extracts of YXT and its selected components for 7 days. Cell lysates were subjected to Western blotting with the indicated antibodies. β-actin served as a loading control. Quantifications of immunoreactive band intensities were normalized against β actin and shown in the panels: A NF-L, B NF-M, C NF-H, D β-(III)tubulin, E GFAP; values represent fold-change as compared to basal (Veh) in the absence of herbal treatment. *P < 0.05 (ANOVA); n = 3

Fig. 4

Effects of YXT and its selected herbal components on NMDA-induced primary neuronal cell death. To monitor the level of cell death induced by 100 μM NMDA (48 h), a WST assay was used to measure cell viability of the NMDA-treated primary neuronal cultures in the absence or presence of aqueous (upper; Aq) and ethanol (lower; EtOH) extracts of YXT and its selected herbal components. NMDA-induced cell death observed in the absence of herbal treatment was defined as 100%. *P < 0.05 (ANOVA); n = 6

Fig. 5

Effects of YXT and its components on COX-1 and COX-2 activities. COX-1 and COX-2 activity assay kits were used to monitor the COX enzymatic activities. Ethanol extracts of YXT or its herbal components (100 µg/mL) were mixed with respective COX enzymes (COX-1 or COX-2), COX assay buffer, arachidonic acid in NaOH, and a fluorescence probe. Fluorometric reading of enzyme control (Con) was defined as 100% COX activity. COX-1 specific inhibitor SC560 (SC) and COX-2 specific inhibitor celecoxib (Cel) served as controls. *P < 0.05 (ANOVA); n = 7

Fig. 6

Effects of YXT and its selected herbal components on CREB and ERK phosphorylation. RDIN culture, SH-SY5Y and U-87 MG cell lines were treated with ethanol herbal extracts for 10 min. Western blotting analysis of (A) P-CREB and (B) P-ERK are shown. β-actin served as a loading control and the amount of phosphoprotein was normalized against total amount of the respective protein as a measure of the phosphorylation activity. Bar graphs illustrate the relative fold change of band intensities detected by anti-phosphoantibodies as compared to the basal group (Veh) detected in the absence of herbal treatment. *P < 0.05 (ANOVA); n = 3

Fig. 7

Schematic representation of the neuro-pharmacological effects of YXT on multiple molecular targets related to AD. The anti-AD effect of YXT on regulating (A) neurodegeneration, (B) neuroinflammation, (C) cholinergic dysfunction, and (D) neurogenesis. Activations are indicated by arrows, and blunt-end arrows represent inhibition. Potential activities of YXT as suggested by the actions of its component herbs are shown as dotted lines. The illustration was created with BioRender.com