Extracts Of Cistanche Deserticola Can Antagonize Immunosenescence And Extend Life Span in Senescence-Accelerated Mouse Prone 8 (SAM-P8) Mice

Ke Zhang, Xu Ma, Wenjun He, Haixia Li, Shuyan Han, Yong Jiang, Hunan Wu, Li Han, Tomohiro Ohno, Nobuo Uotsu, Kohji Yamaguchi, Zhizhong Ma, and Pengfei Tu

1 Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191, China

2 Medical and Healthy Analytical Center, Peking University, No. 38 Xueyuan Road, Beijing 100191, China

3 Fundamental Research Faculty, Fancl Research Institute, FANCL Corporation, 12-13 Kamishinano, Totsuka-Ku, Yokohama, Kanagawa 244-0806, Japan

4 Department of Integration of Traditional Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191, China

Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791

The senescence-accelerated mouse prone 8 substrains (SAM-P8), widely accepted as an animal model for studying aging and antiaging drugs, was used to examine the effects of dietary supplementation with extracts of Cistanche deserticola (ECD) which has been used extensively in traditional Chinese medicine because of its perceived ability to promote immune function in the elderly. Eight-month-old male SAM-P8 mice were treated with ECD by daily oral administrations for 4 weeks. The results showed that dietary supplementation of 150 mg/kg and 450 mg/kg of ECD could extend the life span measured by Kaplan-Meier survival analysis in a dose-dependent manner. Dietary supplementation of SAM-P8 mice for 4 weeks with 100, 500, and 2500 mg/kg of ECD was shown to result in significant increases in both naive T and natural killer cells in blood and spleen cell populations. In contrast, peripheral memory T cells and proinflammatory cytokine, IL-6 in serum, were substantially decreased in the mice that ingested 100 and 500 mg/kg of ECD daily. Additionally, Sca-1 positive cells, the recognized progenitors of peripheral naive T cells, were restored in parallel. Our results provide clear experimental support for long-standing clinical observational studies showing that Cistanche deserticola possesses significant effects in extending life span and suggest this is achieved by antagonizing immunosenescence.

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Introduction

Cistanche deserticola, one of the most popular traditional Chinese herbal medicines/health products, has been described in a number of historical Chinese herbal pharmacopeias as having antiaging properties. Consequently, it has been widely used in China for treating various age-related disorders including senile dementia, impotence, infertility, chronic infection, and hematopoietic disorders in the elderly [1]. Modern chemical approaches have allowed two principal types of compounds, phenylethanoid glycosides, and oligosaccharides, to be isolated as the main active ingredients of Cistanche deserticola [1]. In the last decade, Cistanche deserticola and its extracts have been studied intensively and shown to be capable of protecting neurons from injury induced by neurotoxins [2], inhibiting carbon tetrachloride-induced hepatotoxicity [2], and promoting the recovery of bone marrow cells from Co60 induced radiation damage [3]. It has also been shown to have anti-inflammatory, antioxidant, and antiaging effects [4]. However, whether Cistanche deserticola can increase life span and what are the underlying molecular mechanisms [3] associated with its antiaging properties have not been rigorously tested.

Immunosenescence, that is, alteration of the immune system with age forms the background against which increased susceptibility to infections, cancer, neurodegenerative diseases, and autoimmune diseases in the elderly has been noted [5]. Therapeutic interventions, such as caloric restriction [6] and vitamin E supplementation, have been reported to be effective at delaying the progression of immunosenescence and hence reducing the morbidity of some age-related diseases as well as prolonging the life span of both humans and rodents [7, 8]. However, studies in this area are complicated by the fact that aging is associated with the paradox of simultaneous immune deficiency and chronic inflammation [9]. This means that simple stimulation of lymphocyte proliferation or anti-inflammation does not represent ideal therapeutic interventions in dealing with aging and age-related conditions [10]. Consequently in searching for medical interventions capable of preventing or alleviating age-related conditions including infections, cancer, autoimmune diseases, atherosclerosis, and neurodegenerative diseases which are leading causes of death and disability, and repair of immune system defects must be accompanied by an inhibition of inflammatory responses.

The senescence-accelerated mouse [11] is an inbred mouse model, derived from the AKR/J strain, that is widely used in studies of aging. The P8 substrain (SAM-P8) of these mice has a markedly shortened life span when compared to the R1 substrain (SAM-R1), which also shows a slower aging process [12]. In parallel with their premature aging, SAM-P8 mice also exhibit increased neurological senescence, immunosenescence, and age-related hematopoietic deficits which closely mimic typical human aging characteristics [13, 14]. Analysis of the underlying mechanisms responsible for the accelerated aging process and age-related disorders indicates that mitochondrial dysfunction [15], oxidative stress, and increased somatic DNA mutation rate all appear to be involved [16, 17]. This mouse system, with its homogeneous genetic background, therefore, provides an excellent experimental model for studying aging and antiaging therapeutics [12]. This study has focused on investigating whether extracts of Cistanche deserticola are able to extend the life span of SAM-P8 mice and reverse their immunosenescence status.

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Results

3.1. Analysis of Potential Active Components in Extracts of Cistanche deserticola (ECD). As shown in Table 1, ECD is composed mainly of two types of compound, phenylethanoid glycosides, and oligosaccharides. In the phenylethanoid glycosides, the echinacoside, acteoside, and 8-epiloganic acid have been identified, whilst in the oligosaccharides, only galactitol was identified.

3.2.The Impact of Cistanche deserticola Extracts on the Average Life Span of SAM-P8 Mice. For this and subsequent studies, eight-month-old male SAM-P8 mice were divided into 4 groups. Among them, one group of mice were fed with a normal diet without ECD, the other 3 groups have separately ingested the diets which contain different proportions of ECD. Mice from the SAM-R1 substrain, which have a normal aging process and life span, were used as a control group in all experiments.

Compared to the control SAM-R1 group, the average life span of SAM-P8 mice was significantly shortened (Figure 1(a); 𝑃 < 0.001). Although supplementation of diet with the low dose (50 mg/kg) of ECD failed to produce a significant increase in a life span of SAM-P8 mice, at the medium (150 mg/kg) and high (450 mg/mL) supplementary doses there was a dose-dependent increase in life span (Figures 1(a) and 1(b); 𝑃 < 0.05–0.01) that was confirmed by Kaplan-Meier survival analysis, which included the use of both the Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests.

3.3. Reversal of Immunosenescence in SAM-P8 Mice by Extracts of Cistanche deserticola (ECD). A decrease of peripheral naive T lymphocytes and concomitant increase of peripheral memory T lymphocytes are prominent features of immunosenescence which are widely regarded as the main underlying reasons for age-related immunological abnormalities. This immunosenescence in SAM-P8 relative to SAM-R1 animals was clearly evident when FACS analysis was used to enumerate (CD3+ CD44low CD45RBhigh) lymphocytes as an indicator of naive T cells and (CD3+ CD44high CD45BRlow) lymphocytes as an indicator of memory T cells. Thus, in both peripheral blood (Figure 2) and spleen cell (Figure 3) populations reduced levels of naive T cells and increased levels of memory T cells were seen in SAM-P8 mice. Supplementation of the diet of SAM-P8 mice with ECD was found to be able to reverse these indicators of immunosenescence in a dose-dependent fashion in both peripheral blood (Figure 2) and spleen cell (Figure 3) populations. As an additional indicator of the reversal of immunosenescence by ECD supplementation of diet, the level of natural killer [18] cells, a major cellular marker of the innate immune system, was analyzed. This showed that ECD diet supplementation resulted in a dose-dependent increase in NK (CD3+ CD49+) cells in both peripheral blood (Figure 4) and spleen cell (Figure 5) lymphocyte populations.

3.4. Extracts of Cistanche deserticola Strengthen the Relative Fluorescence Intensity of Sca-1 Positive Cells in SAM-P8 Mice. Stem cell antigen-1 (Sca-1) is one of the most prominent biomarkers of hematopoietic stem cells (HSC) in bone marrow cell populations. Sca-1 positive cells also represent lymphocyte progenitors that have been newly exported from the bone marrow into peripheral blood where they undergo further differentiation into various types of mature lymphocytes. Consequently, these Sca-1 positive cells represent the main source of naive T lymphocytes in peripheral blood. Figure 6 shows that compared with SAM-R1 control mice, the relative fluorescence intensity of Sca-1 positive cells in SAM-P8 mice was substantially lower. Diet supplementation of SAM-P8 mice with three different doses of ECD was shown to significantly enhance the relative fluorescence intensity of Sca-1 positive cells (Figure 6).

3.5. Extracts of Cistanche deserticola (ECD) Promote Apoptosis and Inhibit Necrosis of Lymphocytes in SAM-P8 Mice. The levels of necrosis and apoptosis in blood lymphocyte populations were analyzed by double staining with Annexin V-FITC V/PI. This revealed that the proportion of necrotic lymphocytes in SAM-P8 mice was significantly higher than in SAM-R1 control animals, while the proportion of apoptotic lymphocytes was lower (Figure 7). Supplementation of the diet of SAM-P8 mice with three different doses of ECD was able to inhibit the levels of necrosis seen in peripheral lymphocyte populations, whilst only the two higher levels of ECD supplementation produced a significant change in the level of apoptotic lymphocytes (Figure 7).

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3.6. Extracts of Cistanche deserticola (ECD) Decrease the Proinflammatory Cytokine IL-6 in SAM-P8 Mice. As a further indicator of ECD’s capacity to influence age-related changes in the immune system, the effect of ECD diet supplementation on the level of a number of cytokines (IFN-𝛾, TNF- 𝛼, IL-2, IL-6, IL-10, GM-CSF, and IL-3) was analyzed using cytometric bead array analysis (CBA) which involves staining with fluorescent-dye-labeled antibodies coupled with capture cytometric beads. The results (Figure 8) showed that the inflammatory cytokine IL-6 was not increased in 8-monthold SAM-P8 mice compared with control SAM-R1 mice of the same age, but it was significantly increased when compared with 6-month-old SAM-R1 mice (results not shown). However, dietary supplementation with high and medium doses of ECD was able to produce a statistically significant decrease in plasma IL-6 levels (Figures 8(a) and 8(b), 𝑃 < 0.05). The plasma levels of the other cytokines examined (IFN-𝛾, TNF-𝛼, IL-2, IL-10, GM-CSF, and IL-3) showed no significant variations between SAM-P8, SAM-R1, and ECD treated groups of mice (data not shown).

4. Discussion

The immune defense against newly invasive microorganisms or endogenous tumor cells depends on the diversity of T cell repertoire, which in turn relies on the generation and maintenance of naive T cells [19]. During the aging process, the diversity of the T-cell repertoire has been shown to shrink dramatically due to the progressive depletion of naive T cells in the peripheral reserve pool. It is this shortage of responsive naive T cells that have been thought to be responsible for the susceptibility of elderly people to infection, cancer, and poor outcomes following vaccination [20]. Various interventions, including caloric restriction [7], exercise [21], and vitamin E supplementation [8], have all been used to successfully replenish naive T cells and thereby extend life span and reduce the onset of infection and cancer among the elderly. In this study, we have demonstrated that ECD is able to increase the level of naive T cells in the peripheral pool and there was a concomitant extension of life span and reduction in the frequency of tumor formation in senescence-accelerated mice (ECD therapeutic group versus SAM-P8, 0 versus 1/6). Therefore, it seems reasonable to conclude that ECD’s capacity to reverse the age-dependent depletion of peripheral naive T cells may contribute to its known effects in reducing age-related disease which derive from long-term clinical observations.

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