Ginsenoside Rg1 and Astaxanthin Act on the Hypothalamus to Protect Female Mice Against Reproductive Aging
Female infertility due to reproductive aging has emerged as a significant concern in the population. Oxidative stress is widely recognized as one of the primary mechanisms underlying reproductive aging. While the hypothalamic-pituitary-ovarian (HPO) axis undergoes gradual changes during female reproductive aging, the hypothalamus plays a particularly crucial role. Previous studies have demonstrated that estrogen-induced oxidative stress leads to the senescence of hypothalamic astrocytes, which precedes reproductive dysfunction. Antioxidants have been shown to delay reproductive aging by reducing oxidative damage to the ovaries. However, the effects of antioxidants on the hypothalamus remain unexplored. This study investigates the protective effects of two natural antioxidants, ginsenoside Rg1 and astaxanthin (AST), on the hypothalamus and ovaries in female mice, offering new insights into the mechanisms of reproductive aging.
Background and Significance
Reproductive aging in females is characterized by a decline in fertility, irregular estrous cycles, and eventual cessation of reproductive function. The HPO axis, which regulates reproductive function, is particularly vulnerable to aging-related changes. The hypothalamus, a key component of this axis, is responsible for releasing gonadotropin-releasing hormone (GnRH), which stimulates the pituitary gland to secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These hormones, in turn, regulate ovarian function and the estrous cycle.
Oxidative stress, a hallmark of aging, contributes significantly to the decline in reproductive function. Reactive oxygen species (ROS) accumulate in tissues over time, leading to cellular damage and senescence. In the hypothalamus, oxidative stress has been shown to induce the senescence of astrocytes, a type of glial cell that supports neuronal function. This astrocyte senescence precedes reproductive dysfunction, highlighting the importance of the hypothalamus in reproductive aging.
Ginsenoside Rg1, a steroidal glycoside found in ginseng, and astaxanthin, a xanthophyll carotenoid found in marine animals, are potent antioxidants. Both compounds can cross the blood-brain barrier, making them suitable candidates for targeting the hypothalamus. This study explores their effects on the hypothalamus and ovaries in naturally aging female mice, aiming to elucidate their potential in delaying reproductive aging.
Experimental Design and Methods
The study utilized intact naturally aging female mice to investigate the mechanisms of reproductive decline. Young mice (3–4 months old) and middle-aged mice (9–10 months old) were included. Vaginal smears were used to monitor the estrous cycle daily, which was divided into diestrus, proestrus, estrus, and metestrus based on the cell types present. The average estrous cycle of young mice was 4–5 days, while middle-aged mice exhibited irregular cycles, averaging more than 6 days.
To assess age-related changes in the HPO axis, senescence-associated β-galactosidase (SA-β-Gal) staining combined with glial fibrillary acidic protein (GFAP) immunohistochemistry was performed. This revealed higher SA-β-Gal activity in hypothalamic astrocytes of middle-aged mice compared to young mice. Increased peroxidase activity was also observed in the hypothalamus of middle-aged mice, indicating elevated oxidative stress.
Ovarian morphology was examined using paraffin sections and hematoxylin/eosin staining. While follicles and corpus luteum at various developmental stages were observed in both young and middle-aged mice, the number of primary, secondary, and antral follicles was reduced in the latter group. LH levels, measured via enzyme-linked immunosorbent assay (ELISA), showed no significant difference between the two age groups, suggesting that pituitary function remains relatively stable during this period.
Effects of Rg1 and AST on Reproductive Function
To evaluate the effects of Rg1 and AST on reproductive function, 80 young mice with regular estrous cycles were divided into four groups: a saline control group, an Rg1 group, an olive oil control group, and an AST group. Drug intervention began at 3 months of age and continued until 9–10 months. Estrous cycles were measured at 6, 10, and 12 months of age.
With advancing age, the percentage of mice with regular estrous cycles decreased in the saline control group. However, Rg1 treatment significantly increased the percentage of mice with regular cycles compared to the saline group. Similarly, AST treatment maintained a higher percentage of regular cycles than the olive oil control group at 6 and 10 months. By 12 months, no significant differences were observed among the groups, suggesting that the protective effects of Rg1 and AST diminish with extreme aging.
Mechanisms of Action in the Hypothalamus
To understand how Rg1 and AST delay reproductive aging, their effects on hypothalamic senescence were examined. Senescence-associated secretory phenotype (SASP) is characterized by the upregulation of p16 and p21, positive SA-β-Gal staining, and the secretion of proinflammatory cytokines. Rg1 treatment significantly reduced the expression of p16 and p21 in the hypothalamus of middle-aged mice. Similarly, AST treatment decreased the expression of these senescence markers compared to the olive oil control group.
Immunohistochemistry revealed that both Rg1 and AST treatments reduced peroxidase activity and the number of SA-β-Gal-positive astrocytes in the hypothalamus. Additionally, the expression levels of proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor-α (TNF-α), were significantly decreased in the hypothalamus of Rg1-treated mice compared to the saline control group. AST treatment also reduced the levels of these cytokines in comparison to the olive oil control group.
The redox status of the hypothalamus was further evaluated by measuring total superoxide dismutase (T-SOD) activity and malondialdehyde (MDA) content. T-SOD is a critical antioxidant enzyme that mitigates oxidative stress, while MDA is a biomarker of oxidative damage. Rg1 treatment significantly increased T-SOD activity and reduced MDA levels in the hypothalamus. Similarly, AST treatment enhanced T-SOD activity and decreased MDA content. These findings suggest that Rg1 and AST alleviate hypothalamic senescence through their antioxidant effects.
Effects on Ovarian Function
The study also examined the effects of Rg1 and AST on ovarian function. Neither compound significantly affected the number of growing or mature follicles compared to the control groups. Additionally, there were no significant changes in uterine or ovarian weight following Rg1 or AST treatment.
However, Rg1 and AST treatments reduced the expression of p16, p21, and proinflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) in the ovaries. T-SOD activity was significantly increased, and MDA levels were reduced in the ovaries of treated mice. These results indicate that Rg1 and AST downregulate ovarian SASP through antioxidant mechanisms, although their effects on ovarian morphology and weight are minimal.
Conclusion
This study demonstrates that chronic administration of ginsenoside Rg1 and astaxanthin effectively delays reproductive aging in female mice by targeting the hypothalamus and ovaries. Both compounds reduce oxidative stress and senescence in the hypothalamus, leading to improved estrous cycle regularity. While their effects on ovarian morphology are limited, they significantly enhance antioxidant activity and reduce oxidative damage in ovarian tissues. These findings highlight the potential of Rg1 and AST as therapeutic agents for mitigating reproductive aging, particularly through their actions on the hypothalamus. Further research is needed to identify specific molecular targets and mechanisms, paving the way for more effective treatments for age-related infertility.
doi.org/10.1097/CM9.0000000000001542
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