The cellular transcriptome profile induced by application of
Bazhen decoction revealed its systematic function in anti-aging
To get the full image of the molecular pathways regulated by Bazhen
decoction, we applied the RNA sequencing (RNA-seq) technique to the
cells with or without Bazhen decoction treatment. Due to the fact that
many cell lines carry multiple gene mutations during the immortalization
process, which might alter the response of affected molecular pathways
to the Bazhen decoction, we utilized the primary cultured wild type
mouse embryonic fibroblasts (WT MEF) to obtain the cellular
transcriptome induced by Bazhen decoction.
After treatment of low dose (10μg/mL, BZT-L,1-3) and high dose
(100μg/mL, BZT-H,1-3) of Bazhen decoction, the WT MEFs were applied for
RNA sequencing to get the information of all the genes’ mRNA count
(expression level). The RNA-seq data was normalized and analyzed by
single sample gene set enrichment analysis (ssGSEA) to get the cellular
pathways activated (up-regulated) or inhibited (down-regulated) by
Bazhen decoction.
The heatmap of top 50 pathways regulated by Bazhen decoction were
plotted (Figure 1). Among the top up-regulated pathways, we found the
stem cell pathway (Figure 1, BOQUEST STEM CELL), and Wnt-B-Catenin
pathway which known to be important in stem cell regulation (Figure 1,
WP REGULATION OF WNTBCATENIN SIGNALING BY SMALL MOLECULE COMPOUNDS). We
also found the nicotinamide metabolism pathway (Figure 1, KEGG
NICOTINATE AND NICOTINAMIDE METABOLISM), which is known to be essential
for Sirtuins protein function and mitochondria function [21], was
activated by Bazhen decoction (Figure 1).
Interestingly, our data revealed multiple pathways related to the
protein glycosylation protein quality control and ER function were
up-regulated by Bazhen decoction, such as glycosylphosphatidyl inositol
(GPI) anchored protein synthesis (Figure 1, REACTOME POST TRANSLATIONAL
MODIFICATION SYNTHESIS OF GPI ANCHORED PROTEINS), protein glycosylation
(Figure 1, KEGG N GLYCAN BIOSYNTHESIS, REACTOME BASIGIN INTERACTIONS),
the ER associated protein degradation pathway (Figure 1, BIOCARTA ERAD
PATHWAY), the chaperon for unfolded proteins (Figure 1, REACTOME
CALNEXIN CALRETICULIN CYCLE). These pathways are essential for
maintaining protein homeostasis, reducing ER stress and cellular
apoptosis. The down-regulation of protein homeostasis has become one of
the aging hallmarks [11].
It is very interesting that we found the neuronal function related
pathways were up-regulated by Bazhen decoction, including the process of
synaptic vesicle endocytosis (Figure 1, BIOCARTA NDKDYNAMIN PATHWAY),
the sensation function (Figure 1, KEGG OLFACTORY TRANSDUCTION, REACTOME
VISUAL PHOTOTRANSDUCTION). The down-regulation of these pathways are
known to be the symptoms for neuron degenerative diseases.
Consistent with one of the original function (Nourishing hematopoietic
system) for Bazhen decoction, we found the VEGFR3 signaling in lymphatic
endothelium pathway (Figure 1, PID LYMPH ANGIOGENESIS PATHWAY) was
activated, and the hemoglobin degradation pathway (Figure 1, REACTOME
HEME DEGRADATION) was down-regulated. Other than this, the angiotensin
converting and aldosterone synthesis inhibitor pathway (Figure 1, WP ACE
INHIBITOR PATHWAY) was also up-regulated, which might help enhancing the
wellness of cardiovascular system. To add on this finding, we also found
that the transcriptional response to SARS-COV-2 (Figure 1, BLANCO MELO
COVID19 BRONCHIAL EPITHELIAL CELLS SARS COV 2 INFECTION) has been
down-regulated. Since the ACE2 is found to be the receptor for
SARS-COV-2, these data suggest that the Bazhen decoction could block the
SARS-COV-2 by affecting ACE proteins.
Among those down-regulated pathways, multiple inflammatory response
pathways were found, including BIOCARTA TNFR2 PATHWAY, WP
CORTICOTROPINRELEASING HORMONE SIGNALING PATHWAY, WP RESISTIN AS A
REGULATOR OF INFLAMMATION, WP IL4 SIGNALING PATHWAY, PID L2 PATHWAY,
BIOCARTA IL3 PATHWAY, REACTOME REGULATION OF TNFR1 SIGNALING, etc
(Figure 1). Interestingly, the MAPK related signaling pathways were also
down-regulated, including PID FCER1 PATHWAY, BIOCARTA TPO PATHWAY, WP
GALANIN RECEPTOR PATHWAY, REACTOME FCERI MEDIATED MAPK ACTIVATION, PID
FCER1 PATHWAY, WP HOSTPATHOGEN INTERACTION OF HUMAN CORONA VIRUSES MAPK
SIGNALING etc. (Figure 1). MAPK related pathways are known to induce
inflammatory response to stress, such as DNA damages, hypoxia, oxidative
stress, virus infection, etc. The activation of MAPK pathways is the
kind of double edged sword event. Consistent with this, the oxidative
stress pathways were also down-regulated (Figure 1, WP OXIDATIVE STRESS,
WEIGEL OXIDATIVE STRESS RESPONSE).
Although we observed the p53 regulated cell cycle arrest and caspase
pathways (Figure 1, PID CASPASE PATHWAY, REACTOME TP53 REGULATES
TRANSCRIPTION OF CASPASE ACTIVATORS AND CASPASES, REACTOME REGULATION OF
TP53 ACTIVITY THROUGH ASSOCIATION WITH COFACTORS, REACTOME TP53
REGULATES TRANSCRIPTION OF GENES INVOLVED IN G1 CELL CYCLE ARREST, PID
P73PATHWAY) were down-regulated by low dose of Bazhen decoction, we also
observed the down-regulated of several tumorigenesis pathways, such as
KEGG CHRONIC MYELOID LEUKEMIA, WP ENDOMETRIAL CANCER, WP PANCREATIC
ADENOCARCINOMA PATHWAY, etc. (Figure 1).
Surprisingly, the target of rapamycin (TOR) signaling was also
down-regulated (Figure 1, WP TARGET OF RAPAMYCIN TOR SIGNALING, PID TCR
RAS PATHWAY), the inhibition of which is widely applied in anti-aging
and anti-tumor drug screening.
Together these data revealed that the anti-aging function of Bazhen
decoction might be achieved through systematic regulation of multiple
anti-aging pathways, including stem cell regulation, maintaining protein
homeostasis, promoting cardiovascular function, improving neuronal
function, anti-inflammation, anti-DNA damage induced stress, etc.