The application of Bazhen decoction activated the telomere
maintenance pathways via up-regulating the DNA helicases and
telomere proteins
As we know, telomere function could be the sustaining mechanism for the
stem cell function, anti-inflammation, anti-DNA damage induced stress,
etc., however, due to the low abundance of the genes in telomere
maintenance pathway, it might not be able to stand out as top pathway in
the transcriptome. Thus, we further plotted the heatmap for telomere
function and DNA damage response related pathways and tried to dissect
the function of Bazhen decoction in telomere maintenance.
Interestingly, we found that the telomere end processing related
pathways, which is essential for telomere elongation and maintenance,
were up-regulated (Figure 2, A, GOBP TELOMERE CAPPING, REACTOME
POLYMERASE SWITCHING ON THE C STRAND OF THE TELOMERE, REACTOME TELOMERE
C STRAND LAGGING STRAND SYNTHESIS, REACTOME TELOMERE C STRAND SYNTHESIS
INITIATION, etc.). And the telomere DNA repair pathway was also
up-regulated (Figure 2, A, GOBP TELOMERE MAINTENANCE IN RESPONSE TO DNA
DAMAGE), which inhibited the telomere DNA damage induced cellular
senescence (Figure 2, A, REACTOME DNA DAMAGE TELOMERE STRESS INDUCED
SENESCENCE). We also found the up-regulation of DNA helicase pathways
induced by Bazhen decoction (Figure 2, A, GOBP REGULATION OF HELICASE
ACTIVITY, GOMF 3 5 DNA HELICASE ACTIVITY, GOMF DNA HELICASE ACTIVITY,
GOMF HELICASE ACTIVITY, GOMF SINGLE STRANDED DNA HELICASE ACTIVITY).
Interestingly, the telomerase pathway was not obviously regulated by
Bazhen decoction (Figure 2, A, REACTOME TELOMERE EXTENSION BY
TELOMERASE). We suspected that the telomere maintenance was through the
activation of telomere recombination (Figure 2, A, up-regulation of GOBP
TELOMERE MAINTENANCE VIA RECOMBINATION, and down-regulation of REACTOME
INHIBITION OF DNA RECOMBINATION AT TELOMERE). The score of BZT-L1 was
strangely high for some telomere and helicase pathways, but did not
impact the tendency of up-regulation of these pathways by Bazhen
decoction.
Consistent with this, the DNA damage repair pathways were also
up-regulated (Figure 2, A, GOMF DAMAGED DNA BINDING, GOBP DNA SYNTHESIS
INVOLVED IN DNA REPAIR, WP DNA REPAIR PATHWAYS FULL NETWORK, KAUFFMANN
DNA REPAIR GENES). While the p53 regulated cell cycle arrest pathways
were down-regulated by Bazhen decoction (Figure 2, A, REACTOME TP53
REGULATES TRANSCRIPTION OF GENES INVOLVED IN G1 CELL CYCLE ARREST,
REACTOME TP53 REGULATES TRANSCRIPTION OF GENES INVOLVED IN G2 CELL CYCLE
ARREST, REACTOME TP53 REGULATES TRANSCRIPTION OF CELL CYCLE GENES).
Together these data suggest that Bazhen decoction could promote DNA
damage repair and telomere maintenance, reduce DNA replication stress,
thus promote cell cycle progression, and prevent cellular senescence.
To verify these data and explore the role of Bazhen decoction in DNA
replication and telomere maintenance, we utilized the progeroid MEF
cells from Werner syndrome mouse (G3DKO cells, both DNA helicase Wrn and
telomerase Terc genes were knocked out), with WT MEF cells as control.
From the RNA-seq data, we realized that the low dose (10μg/mL) of Bazhen
decoction showed better effect in sustaining cell growth, thus we
treated the cells with 10μg/mL, and 20μg/mL Bazhen decoction for further
experiments. The Western blot data showed that Bazhen decoction
treatment up-regulated the protein level of DNA helicase Blm, Mcm7,
Parp1 in both WT (Figure 2, B) and G3DKO cells (Figure 2, C), which are
known to unwind DNA G4 structure, and promote DNA replication fork
progression, especially in telomere DNA. The telomere protein Terf1,
which is known to involve in telomere packaging and c-strand (lagging
strand) synthesis, was also up-regulated. While the Pot1, which is known
to involve in the negative regulation of telomere length, was
down-regulated (Figure 2, B, C). Together these data suggest that the
Bazhen decoction might regulate the telomere DNA replication and
telomere DNA damage response, and thus regulate telomere lengthening.
At the same time, we also observed that the Bazhen decoction did not
obviously affect the tumor suppressor p53 protein level, while the p53
down-stream effector p21 was up-regulated in both WT and G3DKO cells
(Figure 2, C, D). Interestingly, another important tumor suppressor Rb
family member Rb and p130, which known to be the cell cycle inhibitors,
were up-regulated by Bazhen decoction. However, Rb pathway down-stream
E2F1 was also up-regulated, which supposes to be the cell cycle
promoter. These data suggest a double-side regulation of cell cycle
progression by Bazhen decoction (Figure 2, C, D).
Together the above data suggest that Bazhen decoction could up-regulate
the expression of DNA helicase, promote the unwinding of G4 structure,
thus promote DNA replication, and regulate cell cycle progression.