Discussion
The classical TCM prescriptions, such as Bazhen decoction, have been
applied clinically and showing effective in treatment of aging-related
degenerative diseases or for longevity sustaining function. However, due
to the limit in the pharmacological study of such complicated TCM
system, it is hard to evaluate the pharmacological effect and follow up
the clinical outcome, especially for aging related degenerative
diseases.
Here we try to apply a systematic pharmacological methodology to study
classical TCM prescriptions. Through RNA sequencing and ssGSEA analysis,
we obtained the transcriptome profile regulated by Bazhen decoction in a
wild type genetic background. This way we could avoid the alteration of
signaling pathways caused by gene mutations occurred during the cell
line establishment process. The transcriptome profile of wild type cells
regulated Bazhen decoction revealed a systematic regulation of multiple
anti-aging pathways, including stem cell regulation, protein
homeostasis, cardiovascular function, neuronal function,
anti-inflammation, anti-DNA damage induced stress, etc. by Bazhen
decoction (Figure 1). This transcriptome profile and detailed signaling
pathways will be very useful for further study of Bazhen decoction’s
interfering function in various organ degenerative diseases.
For the above possible anti-aging properties, telomere maintenance could
be a common molecular basis for sustaining the stem cell function,
preventing DNA damage, improving organ function, anti-inflammation, etc.
Consistent with this, our further analysis of the transcriptome revealed
that Bazhen decoction treatment could up-regulate the pathways for DNA
helicase activity and telomere lengthening (Figure 2, A).
It is well known that the smooth progression of DNA replication is
essential for telomere maintenance and cell cycle progression. However,
the DNA secondary structure, such as telomere G-quadruplex (G4)
structure, slows down the replication fork progression, cause
replication stress, and become the chromosome fragile sites [22].
The function of DNA helicase is to fire replication origins and unwind
the G4 structures, ensure the accurate formation of replication forks
and the smooth progression of DNA replication. The defect of helicase
function result in DNA replication stress, activates DNA damage response
pathways, and results in aging or tumorigenesis [16, 23]. The
genetic defect of helicase causes progeroid syndromes, such as Werner
syndrome (Wrn gene mutation) [24], Bloom syndrome (Blm gene
mutation) [25], Rothmund-Thomson syndrome (Recql4 gene mutation)
[26], etc.
To further understand the regulation of Bazhen decoction on DNA
helicases and telomere, with the progeroid cells derived from Werner
syndrome mice, we verified that DNA helicases Blm, Recql4, Parp1, Mcm7
and telomere regulating protein Terf1 were up-regulated by Bazhen
decoction, and Pot1 were down-regulated (Figure 2, B-E). Studies have
shown that helicase Blm has a strong activity for unwinding G4
structures and is important for the synthesis of telomeric DNA leading
strand [27]. Helicase Fancm, Brca1, and Blm regulate DNA end
processing and homologous recombination [28]. Other than this, the
Blm protein could resolve other secondary DNA structure, such as Holiday
junction, thus is important for the regulation of DNA replication
stress. The Recql4 protein is involved in the repair of DNA double
strand breaks, as well as the telomere DNA maintenance [29]. Thus
the up-regulated DNA helicases by Bazhen decoction promote the resolving
of G4 structure, and endowed the cellular resistance to DNA damages
induced by G4 stabilizer PDS, promoted the quality control of cell cycle
and telomere elongation (Figure 3-5).
Together these data suggest that Bazhen decoction facilitate G4
resolving and telomere maintenance, which might contribute to the
longevity sustaining properties revealed by transcriptome profile. Thus
the Bazhen decoction application could be a strategy of dealing with DNA
replication stress, and the prevention of the progress of aging, as well
as tumorigenesis.
Our data also suggest a possibility of Bazhen decoction in treating
genetic progeroid syndromes, especially those with telomere dysfunction.
Indeed, some physician have used Bazhen decoction to treat aplastic
anemia [30, 31]. The hereditary aplastic anemia is due to abnormal
telomerase (e.g., dyskeratosis congenica syndrome) or DNA helicase
(e.g., Fanconi’s anemia) function [32, 33]. While the cause of
sporadic aplastic anemia is mainly the DNA damage of hematopoietic stem
cells. Further study on Werner syndrome mouse model will be performed to
evaluate the function of Bazhen decoction on organ aging and related
diseases.
Surprisingly, from the transcriptome profile, we also revealed that
Bazhen decoction could be applied in prevention and treatment of
COVID-19 (Figure 1). Interestingly, the COVID-19 has known to damage the
digestion function and cardiovascular function, which is the two targets
for Bazhen decoction (The Sijunzi decoction protect the digestion
function, and the Siwu decoction protect the cardiovascular function).
The anti-inflammation profile might also contribute to the COVID-19
treatment.
Together, our data provide the full image of possible pathways regulated
by Bazhen decoction. The signature pathways revealed by transcriptome
analysis are more accurate and stable than individual gene variation,
could applied as biomarkers for TCM system. With further clinical
studies, these key signaling pathways of TCM prescription could be
applied in quantification evaluation of clinical pharmacological effect
to a certain extent, and explain the role and molecular mechanism of
classic longevity formulae in the prevention and treatment of
aging-related diseases, so as to guide the clinical precision treatment
of traditional Chinese medicine.