Introduction
The traditional Chinese medicine strategy for body wellness and longevity focused on the balancing of Yin and Yang, or Xue (blood, nutrient) and Qi (energy). This anti-aging strategy applies to the prescription of Chinese medicine and generates varies classical prescriptions for treatment of aging-related disease and sustaining longevity. One of the most extensively used prescriptions for longevity is the Bazhen decoction. Bazhen decoction is composed of eight herbal medicine, including Panax ginseng, Radix rehmanniae praeparata, Radix paeoniae alba, Ligusticum wallichii, Poria cocos, Angelica sinensis, Rhizoma Atractylodis Macrocephalae, and Licorice. Bazhen decoction is the combined prescription of Sijunzi and Siwu decoction, and is famous for enhancing both Qi and Xue, and balancing Yin and Yang. Thus, it is the one of the extensively used prescription for the intervention of sub-health status, and the prevention of aging-related degenerative diseases [1-4].
It has been revealed that Bazhen decoction was effective in the treatment of 5-fluorouracil-induced anemia in mice. It could promote the proliferation and differentiation of bone marrow cells, elevate the transcription of EPO mRNA, increase the red blood cell count and the hemoglobin concentration [5]. The similar effect of Bazhen has been found in the bone marrow depression induced by cyclophosphamide in mice, by promoting the proliferation of hematopoietic progenitor cell and secretion of hematopoietic growth factor [6]. It has also been shown that Bazhen decoction combined with sequential treatment of chemotherapy on acute lymphoblastic leukemia patients with deficiency of Qi and Yin could protect the hematopoietic system from damages induced by chemotherapy, and decrease the incidence of nausea and vomiting, liver and kidney injury [7]. Together these data suggest the function of Bazhen decoction in promoting hematopoietic regeneration.
It has also been found that Bazhen decoction administration could decrease acetaminophen-induced liver injury marker, maintain the activity of anti-oxidative factors, and depress the expression of pro-inflammatory factors. These data suggest that Bazhen decoction protect against acetaminophen induced acute liver injury by inhibiting oxidative stress, inflammation and apoptosis in mice [8]. The modification of Bazhen decoction (Huangqi Bazhen decoction) has been used to interfere the chemotherapeutic intestinal mucositis induced by capecitabine in mice. The results showed that the intestinal injury (villus height shortening, crypt destruction, and apoptosis) was reversed by Bazhen decoction, and the weight loss and diarrhea during capecitabine treatment were reduced [9]. These data suggest that Bazhen decoction could prevent tissue damages induced by toxic chemicals, such as chemotherapy drugs. The Bazhen decoction has been also apply to the treatment of premature ovarian failure induced by D-galactose in rats, it could up-regulate the E2 and X-linked inhibitors of apoptosis protein (XIAP), and prevented the apoptosis of oocyte and granulosa cells [10].
Together these data reveal the promising application of Bazhen decoction in prevent tissue damage and aging-related tissue degeneration. However, due to the complicity of the decoction components, the full image of the pharmacological basis of Bazhen decoction is still unclear, which hinder the precise clinical application, especially for complicated aging related degenerative diseases. The further dissection of the molecular pathways regulated by Bazhen decoction might provide the molecular pathways for the evaluation of treatment effect, and provide readouts for more precise symptoms and timing for the clinical application of Bazhen decoction on aging related diseases.
The hallmarks of aging include the attenuation of stem cell capacity, abnormal intercellular communication, abnormal mitochondrial function, increased chromosomal instability, altered epigenetic modifications, decreased protein homeostasis, decreased telomerase activity, and abnormal telomere function [11].
Telomere is at the end of eukaryotic chromosomes and composed of repeat DNA sequence TTAGGG and shelterin protein complex. Telomere is essential in maintaining the integrity of chromosomal DNA, and preventing chromosome ends from being recognized as DNA damage [12, 13]. Telomere length is mainly maintained by telomerase, or by a class of mechanisms referred to as alternative lengthening of telomere (ALT) [14]. In the absence of telomere lengthening mechanism, telomere DNA will gradually shorten along with the cell division, the shortened telomere activates DNA damage responses, such as p53-regulated signaling and the inflammation response pathways, eventually induced cellular senescence, apoptosis, and organism progeroid diseases [15].
In addition to telomerase, DNA helicases are also required for proper replication and elongation of telomeric DNA. Telomeric DNA is rich in G and tends to forms G-quadruplex DNA (G4 structure), which requires helicases to unwind properly for further DNA replication [16]. DNA helicases are a large family of proteins that hydrolyze ATP to produce energy to unwind DNA double helix. The RecQ family of DNA helicases, such as Wrn, Blm, Recql4, are involved in DNA replication, recombination, DNA damage repair and telomere maintenance, and play an essential role in maintaining chromosome stability [17]. Thus, the function of DNA helicase improves the G4 DNA unwinding, facilitates the DNA replication and the telomere elongation.
It has been proved by omics and large sample population studies that telomere length gradually decreases with human aging, and interacts with other important aging markers (such as stem cell function, mitochondrial function, and immune function), leading to a gradual decline in tissue renewal ability and inflammation. Telomere length has become one of the gold standards for detecting the process of aging and its related diseases [18-20].
To explore the full image of the molecular pathways that Bazhen decoction regulates, in this study, we adopted the RNA sequencing technique to dissect the molecular pathways regulated by Bazhen decoction. RNA sequencing could reveal all the RNAs that expressed under certain circumstance, thus provide the full image of transcriptome, in this case, the cellular transcriptome induced by Bazhen decoction treatment. By further analysis of the transcriptome, we found the DNA replication and telomere maintenance function been improved by Bazhen decoction treatment. To verify these data, we utilize the mouse embryo fibroblasts (MEFs) generated from Werner syndrome mouse model. Werner syndrome (WS) is an autosomal recessive genetic disease caused by DNA helicase Wrn gene mutation and telomere dysfunction. WS is characterized by premature aging and shortened life span. The average lifespan of WS is only 46-48 years, the symptoms include premature atherosclerosis, osteoporosis, cataracts, reproductive deficiency, type 2 diabetes mellitus, and soft-tissue sarcoma. We revealed that the Bazhen decoction elevated the expression of DNA helicases, which promoted the G4 DNA resolving, facilitated the DNA replication and the telomere elongation, and facilitate the progeroid Werner syndrome cell proliferation.