D̲anggui B̲uxue T̲ang (DBT) is a classical Chinese herbal decoction containing only
two herbs, A̲stragali R̲adix (AR; roots of Astragalus membranaceus (Fisch.) Bunge var.
mongholicus (Bunge) P.K. Hsiao, and 2 qian A̲ngelicae S̲inensis R̲adix (ASR; roots of
Angelica sinensis (Oliv.) Diels. at the weight ratio of 5:1, which serves as a dietary
supplement for easing women's menopause syndromes. However, the currently
developed methods of molecular biology cannot reveal the mechanism of DBT due to
a complex mixture of herbal decoctions. Within a herbal mixture, the pharmacological
action is mediated by acting harmoniously via models of multi-components, multi-sites
and multi-functions. Although Chinese herbal decoction has been used clinically, the
underlying action mechanism and potential drug-drug interaction of complex
phytochemicals in the mixture, however, are not resolved.
Omics technology is characterized as the qualitative and quantitative studies of small
molecules in a biological system. In the cellular system, DNA, RNA, protein, and
metabolite are recognized as multi-functional molecules that can provide a regulatory
feedback to upstream processes. Omics employs the systems biology approach to supply a new paradigm in studying the cellular organization and regulatory issues: the
final goals of the analyses are to describe the system behavior emerging from
interactions of various functional units in biological systems. Hence, we aim to employ
omics technology in resolving the action mechanism of DBT.
The metabolic flux analysis was employed to evaluate the mitochondrial bioenergetics
in live cardiomyoblasts under the treatments of herbal extracts deriving from DBT, AR,
ASR, and AR+ASR (mix without boiling together). By comparing to other herbal
extracts, DBT showed the best effect on mitochondrial bioenergetics by increasing
basal respiration and ATP production and decreasing reactive oxygen species.
Meanwhile, the treatment of DBT was able to trigger mitochondrial biogenesis via
activation of transcriptional activities of genes responsible for DNA replication and
mitochondrial dynamics. The result illustrates the “Qi” stimulating effect of DBT, and
more critically, this effect has been achieved in a combination of AR and ASR in a
herbal formula.
The calycosin-depleted DBT (named as DBT
Δcal) was generated to understand the role
of the calycosin, the most abundant active flavonoid, in DBT. The osteogenic function
of DBT
Δcal was significantly decreased, i.e. osteoblastic proliferation and differentiation,
as compared to parental DBT. To fully understand the action mechanism of synergistic
effect in DBT from different aspects of systems biology, the experimental platforms of
integrated omics, e.g. transcriptomics, mass spectrometry (MS) based-proteomics and
lipidomics, and the metabolic flux analysis were established. A total of over one
thousand biomolecules was identified. Differentially expressed RNA, protein and
metabolite were identified and quantified by the omics analyses. The osteoblastic
function of DBT
Δcal was lost in relation to ossification, glycolysis, energy metabolism,
AMPK signaling, lipid metabolism, as well as immune response. In addition, the omics
data was confirmed by the experiments of cell biology both in models of in vitro or in
vivo, i.e. calcium signaling, mitochondrial morphology and micromass culture. Thus,
our omics findings strongly support calycosin could play an essential role in enhancing
osteoblastic function of DBT via multiple signaling pathways and targets.
The master herb in DBT is AR. AR is a common herb playing an essential role in
stimulating body temperature, and which is known as the best “Qi” stimulating herb in Chinese medicine. In our present research, we illustrated AR could serve as a hot-stimulating
herb in regulating the body temperature, and which was contrary to cold-stimulating
herbs. In yeast-induced inflammatory rats, the treatments of cold- or hot-herbal
medicine were used as the model system, and the serum was collected and
subjected to omics analyses. The MS-based multi-omics approach identified the
clustering of cold and hot herb-treated rat serum by using partial least squares
discriminant analysis (PLS-DA) model, and which subsequently identified that 8-hour
treatment was the metabolic perturbation point of herb-induced thermogenetic
regulation. Meanwhile, the levels of metabolites in the serum, i.e. lysoPE, lysoPC and
carnitine, showed positive relationship with the regulation of body temperature; while
the levels of amino acid, fatty acid and bile acid were negatively correlated with the
temperature change at 8-hour. In addition, the differential expressed metabolites were
utilized to pathway enrichment and network analyses in revealing the action
mechanism of herbal medicines relating to thermogenetic regulation. Our study
illustrates AR is acting an essential role in stimulating the “Qi” function of DBT via
thermogenesis.
The overall results here strongly support: (i) DBT enhances the mitochondria function
and energy metabolism, relating to “Qi” function as stated in Chinese medicine theory;
(ii) calycosin plays a key function in DBT, acting as a linker in orchestrating other
compounds from DBT to achieve the maximal functions via multiple pathways and
targets; and (iii) AR, the master herbs in DBT, triggers the thermogenesis regulation,
supporting the hot and “Qi” stimulation of DBT. Therefore, the novel omics approach
could serve as a crucial backdrop for future study that characterizes the impact of a
broad array of different phytochemicals of an herbal mixture. These methodologies
could be applied in the investigation of the action mechanism for multi-target drugs or
combinational therapeutics of herbal medicine.
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