Rehmannia glutinosa exhibits anti‐aging effect through maintaining the quiescence and decreasing the senescence of hematopoietic stem cells

Abstract Background The time‐related decline in regenerative capacity and organ homeostasis is a major feature of aging. Rehmannia glutinosa and Astragalus membranaceus have been used as traditional Chinese herbal medicines for enhanced immunity and prolonged life. However, the mechanism by which this herbal medicine slows aging is unknown. In this study, we investigated the mechanism of the herbal anti‐aging effect. Methods Mice were fed diets supplemented with R. glutinosa or A. membranaceus for 10 months; the control group was fed a standard diet. The phenotypes were evaluated using a grading score system and survival analysis. The percentages of the senescence phenotypes of hematopoietic stem cells (HSCs) were determined by fluorescence‐activated cell sorting analysis. The function and the mechanism of HSCs were analyzed by clonogenic assay and the real‐time polymerase chain reaction. Results The anti‐aging effect of R. glutinosa is due to the enhanced function of HSCs. Mice fed with R. glutinosa displayed characteristics of a slowed aging process, including decreased senescence and increased rate of survival. Flow cytometry analysis showed decreased numbers of Lin–Sca1+c‐kit– (LSK) cells, long‐term HSCs (LT‐HSCs) and short‐term HSCs (ST‐HSCs) in the R. glutinosa group. In vitro, clonogenic assays showed increased self‐renewal ability of LT‐HSCs from the R. glutinosa group as well as maintaining LSK quiescence through upregulated p18 expression. The R. glutinosa group also showed decreased reactive oxygen species levels and the percentage of β‐gal+ cells through downregulation of the cellular senescence‐associated protein p53 and p16. Conclusion Rehmannia glutinosa exerts anti‐aging effects by maintaining the quiescence and decreasing the senescence of HSCs.


| INTRODUCTION
Aging is defined as the time-dependent functional decline that affects most living organisms. 1 The longevity of organisms is mainly maintained by stem cells. 2 Tissue-specific stem cells have the capacity for self-renewal and differentiate into a variety of effector cells. 3 However, during the aging process, this selfrenewal capacity declines, eventually leading to the accumulation of unrepaired, damaged tissues in aged organisms. 4 Loss of the ability to maintain a balance between quiescence and differentiation in the stem/progenitor cell compartment may result in death or age-associated diseases. 2 Furthermore, recent studies suggest that stem cell rejuvenation may reverse the aging phenotype. 5,6 In the hematopoietic system, hematopoietic stem cells (HSCs) are responsible for blood cell production. In aged mice, the hematopoietic system shows T-and B-lymphoid cell impairment and the number of myeloid cells is increased. Aged HSCs showed reduced self-renewal activity and reduced hematopoiesis reconstructive ability. Notably, age-related changes in the HSC compartment are manifested in the aging host as anemia, increased propensity for myeloproliferative neoplasms, decreased immune function and increased cancer incidence. [7][8][9] However, the mechanism of HSC aging and the effects of herbal medicines on HSC aging are unknown. 10 People have been concerned with delaying the aging process and staying young from ancient times and anti-aging is a current focus of research. Traditional Chinese medicine has received increasing attention for the treatment of various aging-associated diseases.
Many herbs used in traditional Chinese medicine are known to provide positive effects against aging through different mechanisms.
Rehmannia glutinosa and Astragalus membranaceus have been widely used in this way for thousands of years.
Rehmannia glutinosa is used to treat various diabetic disorders, enhance the bone metabolism in osteoporosis, and inhibit liver inflammation and fibrosis. In addition, this herb has other effects including anti-fatigue, antidepressant and neuroprotective properties. In the past few years, pharmacological studies on R. glutinosa and its active components have focused mainly on its broad actions on the blood, endocrine, cardiovascular and nervous systems. [11][12][13][14] Thus, R. glutinosa was shown to possess strong immuno-enhancement activity, which has provided the theoretical basis for further studies.
Astragalus membranaceus possesses tonic, hepatoprotective, diuretic and expectorant properties 15 and has been shown to exhibit immunomodulatory, 16 anti-inflammatory and antioxidant effects. 17 The elucidation of the molecular mechanisms underlying the effects of traditional Chinese medicines in clinical practice is a key step toward their worldwide application, and this topic is currently a subject of intense research interest.
Thus, in this study, we fed mice diets supplemented with R. glutinosa and A. membranaceus for 10 months to explore the mechanism underlying the ability of R. glutinosa to increase longevity.

| Animal grouping and treatment
C57BL/6J female mice were maintained in a pathogen-free environment and fed with a standard diet.

| Evaluation of the degree of senescence
A grading score system was adopted to evaluate the degree of senescence according to criteria defined by Takeda et al. 18 Each category listed in the protocol was selected from the clinical signs associated with the aging process. Each mouse was scored at 18 months of age and the score in each category was summed to determine the overall grading score.

| Senescence-associated β-gal staining
Senescence-associated (SA)-β-gal activity was also assayed by flow cytometry using C 12 FDG as described by the manufacturer (Molecular Probes, Eugene, OR, USA). Briefly, stem cells were first stained for cell surface markers and then incubated with 100 nmol L −1 bafilomycin A1 for 1 hour at 37°C to induce lysosomal alkalinization.
After washing with PBS, cells were incubated with a 2 mmol L −1 C 12 FDG for 1-2 hours at 37°C and then analyzed using a FACS Aria I (Becton Dickinson).

| Clonogenic assays
Long-term (LT)-HSCs were sorted by fluorescence-activated cell sorting (FACS) and then cultured in 96-well cell plates using a methylcellulose-base medium (HSC007; R&D Systems, Minneapolis, MN, USA). Ten replicate wells were prepared for each sample. We used 96-well cell culture plate, three cells per well. Two weeks after plating, the number and sizes of colonies were counted under a microscope.

| Statistical analysis
Data were analyzed by the one-way ANOVA using Microsoft Excel A. membranaceus groups was slightly longer than that in the control group ( Figure 1C). These data confirmed the anti-aging effects of both R. glutinosa and A. membranaceus, although R. glutinosa was found to be more efficient in slowing the aging process.

| R. glutinosa reduced the number of hematopoietic stem cells
Stem cell exhaustion is thought to be the integrative consequence of multiple types of aging-associated damage and one of the major causes of tissue and organism aging. 1 Recent studies suggest that stem cell rejuvenation may reverse the aging phenotype at the organism level. 5 We hypothesized that the anti-aging effects of

| R. glutinosa enhanced the function and maintained the quiescence of HSCs
To evaluate the function of HSCs, the clonogenic potential of LT- We observed an increased number of LSK cells in the G0 phase in the R. glutinosa and A. membranaceus groups compared with that in the control group ( Figure 4A). These results indicated that R. glutinosa and A. membranaceus maintained the quiescence of HSCs.

Real-time PCR analysis of cell cycle regulators in LSK cells
revealed that p18, p19 and p57 played critical roles in the maintenance of HSC quiescence. [22][23][24] There were no obvious changes observed for p57 and p19 ( Figure 4C-D), and p18 was upregulated in the R. glutinosa group compared with that in the control group, and slightly upregulated in the A. membranaceus group ( Figure 4B).
F I G U R E 1 Rehmannia glutinosa and Astragalus membranaceus had anti-aging effects. Mice were fed diets supplemented with ground R. glutinosa or A. membranaceus (200 mg/d); the control group was fed a standard diet. A, Changes in bodyweight of mice measured every 2 months. B, Changes in the senescence grading score of mice with age. C, Survival curves. Data represent the mean ± SD; n = 20 mice/group. Data represent the mean ± SD; n = 5 mice/group. *P < 0.05, ***P < 0.001

| R. glutinosa delayed HSCs senescence
Long-term dietary supplementation with R. glutinosa can extend the lifespan of mice. Cellular senescence increases with age; therefore, we investigated HSC senescence in the aging mice by flow cytometric analysis of SA-β-gal stained HSCs using a fluorescent β-gal substrate (C 12 FDG). 25 The percentage of SA-β-gal-positive cells was decreased in the R. glutinosa group, indicating that the R. glutinosa delays LSK cell senescence ( Figure 5A).
Reactive oxygen species play a major role in HSC senescence, and loss of HSC quiescence is frequently correlated with increased cellular ROS. 26 Analysis of the intracellular ROS in LSKs showed that the levels were decreased in the R. glutinosa group compared with those in the control group ( Figure 5B). These data indicated that R. glutinosa may maintain HSC quiescence and enhance HSC function by decreasing ROS levels.
We then investigated the expression several genes involved in cellular senescence by RT-PCR analysis of LSK cells. Compared Rehmannia glutinosa, which has been used as traditional Chinese herbal medicine for thousands of years, can be used to treat hypoglycemia in various diabetic disorders. 27,28 It has also been reported that R. glutinosa extract enhances bone metabolism. 29 Furthermore, R. glutinosa inhibits inflammatory responses and syndromes, 30,31 and protects against cell damage by scavenging free radicals. 14,32 In this study, we found that mice dietary supplementation with R. glutinosa exerted anti-aging effects and enhanced B-cell immunity by increasing the proliferative capacity of LT-HSCs and decreasing the numbers of SA-β-gal-positive cells and ROS levels.
The degree of oxidative damage has been found to increase with age in a variety of cells and tissues. Oxidative stress is a critical determinant of HSC self-renewal. Loss of LT-HSC quiescence frequently correlates with increased cellular ROS, which is negatively associated with HSCs self-renewal. 26 Catalpol is an iridoid glucoside, which has been found in the root of R. glutinosa. Catalpol showed inhibiting oxidative stress, DNA damage and telomere shortening through PGC-1α/TERT pathway in a previous study. 33   Data represent the mean ± standard deviation (SD). *P < 0.05, ***P < 0.001. C, The expression pattern of cellular senescence-associated genes in LSKs; GAPDH was used for normalization. Data represent the mean ± SD of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 Astragalus membranaceus is also used in traditional Chinese medicine to promote immunity, reduce blood sugar and promote tumor cell apoptosis, and also has antioxidation and anti-aging properties.
In this study, we found that dietary supplementation with A. membranaceus for 10 months had no obvious effects compared with those observed in the control mice, while the weight of mice was reduced at 20 months. Therefore, A. membranaceus may be unsuitable for LT treatment to fed mice.
In conclusion, our results show that R. glutinosa can prolong the lifespan of mice by maintaining the quiescence and enhancing the function of HSCs. Furthermore, R. glutinosa can decrease the intercellular levels of ROS and the number of SA-β-gal-positive cells and enhance B-cell immunity.

ACKNOWLEDGMENTS
This study was supported partly by the National Science Foundation

CONF LICTS OF INTEREST
None.