Telomeres are located at the ends of linear chromosomes capped by nucleoprotein structures that consist of tandem repeats of hexameric sequences (TTAGGG in vertebrates) bound by a dedicated set of proteins. Telomeres shorten with every mitotic event. Telomerase is a ribonucleoprotein complex that lengthen telomeres and fundamentally consists of catalytic reverse transcriptase enzymes (TERT) and a telomerase RNA component (TERC). A key function of telomeres is to protect chromosomal ends from fusion and degradation by capping chromosomal ends. Cells recognize critically short telomeres as DNA damage; therefore, the shortest telomere, rather than average telomere length, is critical for cell viability and chromosomal stability (5). CAG activates telomerase, lengthens telomeres, and exerts a variety of pharmacological effects as introduced below (Fig. 2).

Several studies have shown that CAG activates telomerase both in vitro and in vivo. CAG activates telomerase and lengthens telomeres in a telomerase-dependent way in vitro; therefore, CAG decreases the percentage of critically short telomeres and DNA damage in the cell. CAG could activate telomerase in various cell types, including hematopoietic progenitors and mouse embryonic fibroblast haplo-insufficient for the TERC (MEF Terc^+/−) cells. In vivo, dietary supplementation of CAG increases TERT expression in tissues such as bone marrow, lungs, heart, brain, and liver. Further, CAG rescued short telomeres in 1- and 2-year old female mice. However, data have shown that dietary supplementation with CAG does not impact the mean or maximum longevity of female mice (6,7). Therefore, a necessary relation between telomerase activity and longevity does not likely exist.

The above study led to another question: How does CAG activate telomerase? Telomerase activity in human embryonic kidney HEK293 fibroblasts increases upon treatment with CAG and is not meditated via common secondary messenger pathways, including Ca²⁺, inositol trisphosphate (IP3), cyclic adenosine monophosphate (cAMP), and protein kinase B (Akt). However, CAG induced the phosphorylation of extracellular signal-regulated kinase (ERK) in many cell lines, including HEK293 and HEK-neo keratinocytes, as well as cells from lung, brain, mammary, endothelial, and hematopoietic origins. Further, proto-oncogene tyrosine-protein kinase (c-Src), ERK kinase (MEK), and epidermal growth factor receptors are involved in CAG-induced ERK phosphorylation. CAG may activate telomerase and other cellular effects by activating the Src/MEK/ERK pathway (8). Our previous research showed that CAG induces expression of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5b (STAT5b) and enhances phosphorylation of STAT5b following an increase in TERT expression. Further, expression of molecules downstream from the JAK/STAT signaling pathway was also increased, suggesting that CAG activates telomerase through the JAK/STAT signaling pathway (9). In addition, CAG-mediated activation of telomerase is associated with the cAMP response element-binding protein (CREB) (3). The above preliminary studies of the mechanisms associated with CAG demonstrate that it activates telomerase and is related to the CREB, MAPK, and JAK/STAT pathways; however, further investigation is needed to comprehensively understand the mechanisms.

A prevailing view is that the immune system gradually ages during the process of senescence. Weak immune systems lead to an increased susceptibility to infections, which accelerates the process of senescence. The immune system consists of immune organs, immune cells, and active immune substances and is associated with senescence (10). Immune cells include T lymphocytes, B lymphocytes, and phagocytes, in which the number of lymphocytes is related to immune function. CAG enhanced both CD8(+) T lymphocytes and CD4(+) T lymphocytes, telomerase activity, and proliferative capacity in vitro (11,12). CD8(+) T lymphocytes from human immunodeficiency virus (HIV)-infected human donors treated with CAG showed an increase in telomerase activity, modest retardation of telomere attrition, increased proliferative potential of CD8(+) T lymphocytes, and enhanced cytokine/chemokine production, thus enhancing the antiviral potential. CAG-induced increases in telomerase activity are blocked by MAKP and ERK inhibitors; therefore, it is likely that CAG-associated enhanced antiviral functions are mediated through the ERK/MAPK pathway (13). In addition to T lymphocyte proliferation in HIV-infected human donors, CAG enhances normal human T lymphocyte proliferation.

An Astragalus membranaceus root extract (HTA; not a single purified compound) has also been shown to be a telomerase activator. However, CAG is a more effective telomerase activator, increasing telomerase activity and the proliferative potential of human CD4 and CD8 T cells more than that of HTA. Further, CAG has been shown to enhance the anti-viral ability of cells and has the potential to ameliorate acquired immunodeficiency syndrome (AIDS); however, the anti-viral properties of CAG have not yet been proven in animal models.

Within the past decade, numerous diseases or maladies have been associated with mutations in telomerase and/or are known to accelerate telomere loss, including AIDS, congenital dyskeratosis, aplastic anemia, and idiopathic pulmonary fibrosis (IPF). CAG, a telomerase activator, has the possibility to treat these diseases or maladies; to this end, several studies have been conducted. CAG dose-dependently hindered bleomycin-induced fibrosis in mTERT heterozygous mice (TRET+/−mice). However, CAG did not alter the inflammatory response to bleomycin-induced fibrosis, suggesting that the underlying mechanisms are not related to the anti-inflammatory properties of CAG. Prevention against fibrosis and suppression of senescence in cells are dependent on telomerase activation. Further, data suggest that the mechanisms associated with the protective effects of CAG against bleomycin-induced lung fibrosis involve specific types of lung cells rather than all cell types in the lung (7). The mechanisms by which CAG works in specific cell types are likely related to the basic cellular proliferative ability; however, this issue has yet to be resolved.

Telomere length is proportional to cell proliferation at the cellular level; that is, telomerase activation and elongation of telomeres can increase the proliferative ability of cells. The present study indicates that this hypothesis is correct. CAG can improve skin fitness, wound healing, and fur recovery in elderly mice and is a more remarkable agent for wound healing in both in vitro and in vivo assays than that of the other three main saponins from Astragalus species, including AG, cyclocephaloside I (CCI), and cyclocanthoside E (CCE). Of these compounds, CAG is the most effective at inducing human keratinocyte proliferation and migration at a concentration of 1 ng/ml. Further, a 5% preparation of CAG is the most effective for in vivo wound healing (14). CAG stimulates telomerase activity in human keratinocytes, which may contribute to the wound healing effects associated with CAG.

In addition to its wound healing properties, CAG promotes recovery from brain injuries, as shown in both cell and animal experiments. Neural stem cells are important for recovery from brain injuries such as hypoxic-ischemic brain injury. CAG increases proliferation and improves survival in neural stem cells and counters the effects of oxygen-glucose deprivation injury in vitro (15). Further, CAG reduces neuron apoptosis, improves nerve functional scoring, and reduces cerebral infarction volume in a cerebral ischemia reperfusion injury rat model in vivo. These functions are related to increasing expression of TERT (16,17). Furthermore, CAG promotes the renewal capacity of flight feathers in captive zebra finches, which are nonmammalian animals (18). In addition, CAG attenuates depression-like behavior in experimental mice. Telomere elongation and activation of telomerase activity are proportional to the tissue telomere-dependent recovery of cells and are potential treatment targets and indicators of tissue recovery.

Lipids store energy; however, when lipids accumulate too much, it is harmful for health. CAG could ameliorate various biomarkers associated with lipid metabolism. CAG has been shown to reduce cytoplasmic lipid droplets in 3T3-L1 adipocytes at a low dose. At high doses, CAG prevents differentiation of 3T3-L1 preadipocytes. Further, it was observed that CAG dose-dependently stimulates calcium influx in 3T3-L1 preadipocytes. Since elevated intracellular calcium plays a vital role in suppressing adipocyte differentiation, CAG may mediate the balance in lipid metabolism by activating calcium influx in 3T3-L1 preadipocytes (19).

Farnesoid X receptor (FXR) is a potential drug target for the treatment of non-alcoholic fatty liver disease (NAFLD), and CAG is a direct activator of FXR. Animal studies have shown that CAG reduces high-fat diet-induced lipid accumulation in the liver, accompanied by lowered blood glucose levels, serum triglyceride levels, and hepatic bile acids. CAG also ameliorates hepatic steatosis in methionine- and choline-deficient l-amino acid diet (MCD)-induced non-alcoholic steatohepatitis (NASH) mice. These results indicate that CAG ameliorates NAFLD via enhancement of the FXR signaling pathway (20). In addition, CAG increases TERT, c-Myc, and c-Jun expression in the liver and ameliorates liver lipid accumulation as well as glucose tolerance in 2-year old mice. CAG-ameliorated lipid metabolism may occur via multiple mechanisms, and CAG may be a multi-target monomer that binds with various receptors and exerts a variety of functions.

The oxidative stress hypothesis is a generally accepted mechanism of senescence. Reactive oxygen species (ROS), including superoxide (O²⁻), hydrogen peroxide (H₂O2), and the hydroxyl radical (-OH) damage cell functions and are a primary cause of disease and cell senescence. In a d-galactose-induced senescence mouse model, CAG treatment up-regulated total antioxidant capacity (T-AOC) and superoxide dismutase (T-SOD) activity, increased hydroxyproline (HYP) stores, and down-regulated malondialdehyde (MDA) in the skin, heart, and liver. Further, CAG enhanced the antioxidant capacity in a non-dose-dependent manner (21). The anti-oxidant effects of CAG may be related to the hydroxyl found in the chemical structure of CAG. Since oxidative stress is a main reason for telomere attrition (22), the telomere-protective effects of CAG may be related to both its antioxidant and telomerase activation properties.

Inflammation is a very common and important basic pathological process. Body surface trauma, infections, common organ diseases, and frequently-occurring diseases (such as pneumonia, hepatitis, and nephritis) are all related to inflammation. ROS generation was suppressed in human umbilical vein EA.hy926 cells exposed to CAG in the setting of endoplasmic reticulum (ER) stress. Further, CAG attenuates phosphorylation of inositol-requiring enzyme 1 (IRE1)-α, suppresses thioredoxin-interacting protein/NLR family pyrin domain containing 3 (TXNIP/NLRP3) inflammasome activation, inhibits mitochondrial cell death, and reduces apoptosis in endothelial cells. Further, CAG enhances 5′AMP-activated protein kinase (AMPK) phosphorylation, an effect that is diminished by AMPK inhibitors, indicating a potential role for AMPK in the anti-inflammatory properties of CAG (23).

We discussed the role of CAG in the normal immune system above; however, CAG also plays a role in the activated immune system. The effects of CAG in a concanavalin (Con) A pan-activated lymphocyte model were reported by Sun et al (24). CD69 (an early-activated marker) and CD25 (a middle-activated marker), both of which are expressed on the surface of lymphocytes, are inhibited in lymphocytes pretreated with CAG and are activated via Con A treatment in vitro. Further, pretreatment with CAG blocks Con A-induced lymphocyte mitogenesis, induces cell-cycle arrest in the G0/G1-phase, and downregulates cytokine expression. In addition, Ca²⁺ release from intracellular ER storage and Ca²⁺ influx from the extracellular environment are inhibited in Con A-induced activated T-cells treated with CAG; these changes may be related to the anti-inflammatory properties of CAG (24). CAG could activate the immune system and inhibit activated immunity similar to that of feedback inhibition. It is possible that the structure of CAG resembles substances found in our body, and it exerts its functions in a similar way.

The Patton protocol-1 provided 37 subjects with a comprehensive dietary supplement pack containing CAG for 12 months. This clinical trial concluded that CAG lengthens critically short telomeres and remodels the relative proportion of circulating leukocytes in cytomegalovirus-positive [CMV(+)] subjects toward a more ‘youthful’ profile, as seen in CMV(−) subjects (25). Furthermore, the supplement pack improved biomarkers of metabolic, bone, and cardiovascular health, such as fasting glucose, insulin, cholesterol, blood pressure, and bone mineral density (BMD). These effects are mostly attributable to CAG (26). A study reported by Harley et al (26), reported an increase in BMD in naturally aging rats; clinical trials have further verified this function. It is noteworthy that the Patton protocol-1 did not establish a control before initiating the study, which may have affected the trial results.

A random clinical trial conducted in 117 relatively healthy CMV (+) subjects 53–87 years old verified that TA-65 can significantly lengthen telomeres (4). Other TCM extracts, such as Cynomorium songaricum polysaccharides and Epimedium brevicornu flavonoids, can also activate telomerase or protect telomeres; however, CAG is the only compound that has been shown to activate telomerase in humans to date. Further, a randomized placebo-controlled study showed that CAG significantly improves the macular function of treatment subjects (27).

CAG activates human telomerase and has various positive functions. It is still unknown whether these different functions are related to each other and to CAG dosages and whether CAG functions differently in different groups of people.