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.