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.

Keywords: Rehmannia glutinosa; anti‐aging; hematopoietic stem cells; quiescence.

Figure 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

Figure 2

Rehmannia glutinosa and Astragalus membranaceus affected the hematopoietic stem/progenitor cells number. Mice (aged 20 months) were fed diets supplemented with R. glutinosa or A. membranaceus (200 mg/d) for 10 months (n = 5/group); the control group was fed a standard diet. Freshly isolated bone marrow (BM) cells were stained with the indicated antibodies and analyzed by flow cytometry. A, Representative staining profiles of BM hematopoietic stem cell and progenitor cell populations. B, The percentage of Lin^–Sca1⁺c‐kit^– cells (LSKs) in BM cells. C‐I, Cell numbers of (C) long‐term (LT; Lin^–, Sca‐1⁺, c‐Kit⁺, CD34^–, Flt3^–); D, short‐term (ST; Lin^–, Sca‐1⁺, c‐Kit⁺, CD34⁺, Flt3^–); E, multipotent progenitor (MPP; Lin^–, Sca‐1⁺, c‐Kit⁺, Flt3⁺); F, common myeloid progenitor (CMP; Lin^–, Sca‐1^–, c‐Kit^–, CD34⁺, CD16/CD32^–); G, granulocyte‐macrophage progenitor (GMP; Lin^–, Sca‐1^–, c‐Kit^–, CD34⁺, CD16/CD32⁺); H, megakaryocyte‐erythroid progenitor (MEP; Lin^–, Sca‐1^–, c‐Kit^–, CD34^–, CD16/CD32^–); and I, common lymphoid progenitor (CLP; Lin^–, Sca‐1^low, c‐Kit^low, CD127⁺). Data represent the mean ± SD; n = 5 mice/group. *P < 0.05, **P < 0.01

Figure 3

Function of hematopoietic stem cells enhanced by dietary Rehmannia glutinosa supplementation. In vitro clonogenic potential of long‐term hepatic stellate cells from mice (n = 3)

Figure 4

Dietary supplements of Rehmannia glutinosa and Astragalus membranaceus maintained the quiescence of hematopoietic stem cells. A, The percentage of cells in each phase of the cell cycle. Flow cytometric analysis of 7‐aminoactinomycin D (7‐AAD) and Ki‐67 staining of Lin^–Sca1⁺c‐kit^– cells (LSKs). B, Real‐time polymerase chain reaction analysis of sorted LSK cell gene expression; GAPDH was used for normalization. Data represent the mean ± SD of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 5

Dietary Rehmannia glutinosa supplementation reduced the hepatic stellate cell (HSC) senescence. Mice (aged 20 months) were fed diets supplemented with Rehmannia glutinosa or Astragalus membranaceus (200 mg/d) for 10 months (n = 5/group); the control group was fed a standard diet. A, Flow cytometric analysis of SA‐β‐gal‐positive cells Lin^–Sca1⁺c‐kit^– (LSK) using a fluorescent β‐galactosidase substrate (C₁₂ FDG). B, The percentage of reactive oxygen species (ROS)‐positive cells in LSKs. Flow cytometric analysis of ROS‐positive LSKs. 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

Figure 6

Flow cytometric analysis of the percentage of mature immune cells in peripheral blood (PB), bone marrow (BM), spleen and thymus. Mice (aged 20 months) were fed diets supplemented with Rehmannia glutinosa or Astragalus membranaceus (200 mg/d) for 10 months (n = 5/group); the control group was fed a standard diet. A, Flow cytometric analysis of the percentage of B cells (B220⁺) in PB, BM and spleen. B, Flow cytometric analysis of the percentage of monocyte and granulocyte cells (CD11b⁺) in PB and BM. C and D, Flow cytometric analysis of the percentage of CD4⁺ and CD8⁺ cells in PB, spleen and thymus. Data represent the mean ± SD. *P < 0.05