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.