The process of aging is an inherent physiological phenomenon, which progressively impacts human cognitive function, metabolic processes and immune system. Given the imminent advent of an aging society, extensive research on anti-aging has assumed paramount significance. Bioactive compounds derived from plants and animals hold great promise as potential sources for development of pharmaceuticals targeting anti-aging conditions. Previous studies have demonstrated that SVHRSP has protective effects on dopamine neurons in 6-OHDA-induced PD mouse model mice by reducing neuroinflammation [27–29] and upregulating the expression of brain-derived neurotrophic factor (BDNF) [17]. In addition, it significantly enhance resistance to oxidative stress and heat stress through activation of the insulin/IGF-1 signaling pathway [15]. Cognitive learning and memory dysfunction are important behavioral changes in the aging process. Previous laboratory findings have demonstrated that SVHRSP in mitigating cognitive dysfunction and enhancing synaptic plasticity in SAMP mice. However, the underlying mechanism by which SVHRSP improves cognitive function in these mice remain unexplored. In this study, we have identified SVHRSP as a potential therapeutic agent for ameliorating aging-related phenotypes both in vivo and in vitro, particularly by conferring protecting against oxidative damage during aging through activation of the Nrf2 transcription factor. Moreover, our results indicate that SVHRSP reduces cell cycle arrest and senescent cells by regulating cell cycle distribution. We postulate that SVHRSP may exert a protective role against aging by activating the Sirt1-p53 signaling pathway to promote nuclear translocation of Nrf2 for reducing stress levels, as well as inhibiting MAPKs/NF-κB signaling pathway to decrease inflammatory factor release.
The SAMP8 mice serve as a valuable mouse model for accelerated aging and shortened lifespan, enabling the study of age-related cognitive decline. Previous research has indicated that these mice exhibit impaired learning and memory function as early as 4 months of age [30]. Following a 12-week treatment with SVHRSP in 4-month-old SAMP8 mice, a battery of behavioral tests was conducted to assess their cognitive abilities. Notably, administration of SVHRSP significantly improved short-term spatial memory performance in the Y-maze test. Moreover, the passive avoidance test, widely employed to evaluate memory and cognition-enhancing effects, demonstrated that SVHRSP effectively enhanced memory and cognitive performance compared to control conditions. In the novel object recognition test, SVHRSP administration resulted in a significant increase in both the discrimination index (DI) of SAMP8 mice and their exploration time towards the novel object. The Morris water maze task, a classic assessment of spatial memory, evaluates mice on their ability to escape from water based on environmental cues. On the fifth day of navigation trials, mice treated with SVHRSP exhibited reduced escape latencies and swimming distances along with increased preference for searching within the target quadrant. During probe trials, SVHRSP significantly decreased latency to reach the platform while promoting higher frequency of entries crossing it; additionally, it increased both the duration and distance of search within the target quadrant. Overall, these findings demonstrate that SVHRSP ameliorates cognitive performance deficits observed in SAMP8 mice across various behavioral paradigms.
Synaptic plasticity, a fundamental biological mechanism underlying learning, memory, and cognitive function [31, 32], refers to the ability of synapses to efficiently transmit nerve activity changes. It is well established that synaptic plasticity deficiency is implicated in cognitive impairment associated with neurodegenerative diseases [33]. The reduction of synaptic plasticity observed in the aging hippocampus is generally believed to be associated with cognitive decline [34]. Therefore, it is crucial to explore promising targets for enhancing synaptic plasticity in neurodegenerative disorders. Long-term potentiation (LTP), which represents the most dominant form of synaptic plasticity, has been strongly correlated with memory formation and retention. Its impairment closely correlates with cognitive deficits. In our study, we measured the slope and amplitude of fEPSP as reference values for LTP (fEPSP slope reflects the rate of excitatory synaptic response to stimuli while amplitude indicates the intensity of synaptic stimulation). Our results indicated that administration of SVHRSP can enhance synaptic transmission by improving LTP through an increase in both the amplitude and slope of fEPSP, as compared to the SAMP8 group. Morphological analysis revealed that SVHRSP treatment restored damaged presynaptic and postsynaptic components as well as increased content of synaptic vesicles. These findings demonstrate that SVHRSP could inhibit structural damage at synapses in the hippocampus of SAMP8 mice. SYN is widely used for studying synaptogenesis and synaptic function [35]. After phosphorylation, SYN, the most abundant presynaptic vesicle transmembrane protein, binds to synaptic vesicles to regulate their movement and release in nerve endings, thereby modulating neurotransmitter release [36]. PSD95 is widely expressed in excitatory glutamatergic synapses' postsynaptic membranes and its reduction indicates damage to the postsynaptic density. In this study, SVHRSP significantly increased SYN and PSD95 expression in SAMP8 mice, suggesting a protective effect and improvement of memory in aging mice.
The accumulation of senescent cells progressively increases with age in the body, and this accumulation to a certain extent leads to degeneration of nervous system function. According to literature reports, the proportion of senescent cells in the hippocampus of 6-month-old mice was 14.57 ± 2.74%, which doubled to 31.66 ± 14.12% at 18 months old, and reached 50.76 ± 14.41% in 24-month-old mice. The presence of a substantial population of senescent-positive cells in aged mice may have an impact on nervous system function to some extent. Several studies have demonstrated that transplantation of a small number of senescent cells into young mice can induce physical dysfunction, while elimination of these cells can alleviate physical dysfunction in aged mice and increase their remaining lifespan. Timely elimination of senescent cells may be considered as an important strategy for anti-aging [37]. The SA-β-gal staining assay has been widely employed as a biological marker for aging cells [37]. We utilized flow cytometry to assess the percentage of cell volume at different time points. Propidium iodide (PI) is a fluorescent dye used for DNA detection, and its fluorescence intensity is proportional to the DNA content. After intracellular DNA staining with PI, we analyzed cell numbers in each cell cycle phase based on their DNA content using flow cytometry. G1/G0 phase represents living cells, S phase indicates material synthesis, and G2/M phase reflects the number of cell divisions. In this study, SVHRSP not only reduced brain SA-β-gal activity but also alleviated G0/G1-phase cell cycle arrest in SAMP8 mice. Cellular senescence can manifest as cell cycle arrest without cell division, and p16 and p21 can be regarded as protein markers for cell cycle arrest, which are crucial indicators of cellular senescence. These findings suggest that SVHRSP may alleviate the state of cellular senescence in SH-SY5Y neuronal cells induced by H2O2 and in SAMP8 mice, indicating that SVHRSP could reduce the expression of aging markers. Sirtuins are a family of NAD+-dependent deacetylases that have been highly conserved throughout evolution [38].
Substantial evidence suggests that Sirt1 is involved in numerous biological processes, including the regulation of cell cycle, DNA repair, inflammation [39], aging [40], and protection against neurodegenerative diseases [41]. Previous studies have demonstrated that increased expression of the Sirt1 gene extends lifespan in Caenorhabditis elegans [8]. Similarly, overexpression of Sirt1 homolog in mice can promote lifespan extension [9]. Therefore, Sirt1 has garnered significant interest as a regulator of longevity. As a non-histone substrate of Sirt1, p53 activation has been implicated in apoptosis, cell cycle arrest, and aging [42, 43]. P21 is known to be a target of p53 and its expression regulates cell cycle arrest during cellular senescence. Acetylation of p53 by Sirt1 reduces the expression of p21[39] and inhibits DNA damage- and stress-mediated cellular senescence [44]. Pre-treatment with SVHRSP activates Sirt1 and inhibits p53 to attenuate cell injury; thus, it may serve as a potential therapeutic strategy for aging-related diseases.
The regulation of oxidative stress is influenced by multiple factors, among which Nrf2 plays a crucial role as a transcription factor in this process. Under normal physiological conditions, Nrf2 activity is suppressed and remains localized in the cytoplasm. However, upon occurrence oxidative stress, Nrf2 can initiate a cascade of antioxidant and detoxification genes to safeguard the organism against the deleterious effects of reactive oxygen species (ROS) [45, 46]. Additionally, Nrf2 has close associations with aging. Studies have reported that down-regulation of Nrf2 in the hippocampus of SAMP8 mice triggers oxidative stress and neuroinflammation, thereby resulting in cognitive dysfunction [45, 46]. Activation of the Nrf2 signaling pathway has been shown to ameliorate AD-related pathological characteristics in SAMP8 mice [45, 46]. Therefore, we investigated whether SVHRSP could induce nuclear translocation of the Nrf2 transcription factor in the brains of SAMP8 mice using karyoplasmic separation techniques. Our results demonstrated an increase in nuclear protein levels of Nrf2 in the SAMP8 + SVHRSP group while cytoplasmic protein levels remained unchanged. Consistent findings were observed through cellular immunofluorescence analysis for H₂O₂-induced cells regarding nuclear translocation of Nrf2. In conclusion, SVHRSP can mitigate age-related oxidative stress by promoting nuclear translocation of Nrf2. To further validate the role played by Nrf2 in SVHRSP's anti-aging effects, we assessed expression levels of downstream antioxidant proteins regulated by this transcription factor including SOD1, NQO1, and HO-1. The Western blot results clearly demonstrated that SVHRSP effectively enhanced the expression of SOD1 and HO-1 proteins in the brain of SAMP8 mice. Concurrently, the findings from in vitro cell experiments were consistent with those obtained in vivo, as SVHRSP significantly upregulated the expression of SOD1, NQO1, and HO-1 proteins in an H2O2-induced cellular model. These results provide evidence that SVHRSP facilitates Nrf2 activation, promotes downstream antioxidant protein expression, and mitigates oxidative damage during aging. The present findings suggest that the protective effect of SVHRSP in aging may be achieved through activation of the Sirt1-p53 signaling pathway. Both p53 and Nrf2 play crucial roles in the aging process. p53 can suppress the expression of antioxidant genes regulated by the Nrf2 transcription factor, while Sirt1 agonists can activate Nrf2 to exert antioxidant effects by inhibiting p53 [32, 33]. Furthermore, we hypothesized that SVHRSP activates the Sirt1-p53 signaling pathway and regulates the activation of Nrf2 transcription factor to provide protection against oxidative stress and delay the aging process.
Both oxidative stress and inflammatory responses are crucial components in the pathogenesis of neurodegenerative diseases. Accumulating evidence suggests that oxidative stress is closely associated with the inflammatory response. The NF-κB signaling pathway in the central nervous system plays a vital role in regulating various functions, including neuronal plasticity and growth [47]. Moreover, Sirt1 signaling negatively regulates NF-κB activity [48]. Therefore, our study focuses on exploring the potential of inhibiting NF-κB as a novel therapeutic target for age-related diseases [49]. Yeung et al. demonstrated that Sirt1 interacts with the RelA/p65 subunit of NF-κB. Since acetylation of p65 enhances transcriptional activity of the NF-κB complex, Sirt1-mediated deacetylation suppresses NF-κB signaling [50]. The SVHRSP treatment effectively enhances Sirt1 activity and significantly suppresses the phosphorylation of NF-κB p65, leading to a remarkable reduction in the expression of immune genes encoding cytokines such as IL-1β, TNF-α, and IL-6. Aging is known to induce the phosphorylation of NF-κB and members of MAPK family, such as JNK and p38-MAPK [51]. Therefore, targeted inhibition of NF-κB/MAPK phosphorylation plays a crucial role in preventing age-induced neural degradation. In this study, Western blot analysis revealed that aging models exhibited significant increases in the phosphorylation of p38, JNK, and p65 along with decreased protein levels of Sirt1; however, these changes were effectively inhibited by SVHRSP treatment. Henceforth, we infer that the Sirt1-NF-κB signaling pathway contributes to the anti-senescence effects of SVHRSP on neurodegenerative diseases associated with aging.
The hypothesis that glial cell initiation induces a brain inflammatory response is a well-established mechanism of aging. In this study, the immunohistochemical experiment demonstrated a significant increase in astrocyte and microglia activation in SAMP8 mice, which aligns with previous research [52, 53]. In aging animal models, glial activation leads to degenerative neuronal lesions. All these findings suggest that regulating neuroinflammation could be a potential therapeutic target. SVHRSP intervention effectively reduced astroglia and microglial activation in SAMP8 mice. Following priming, microglia can differentiate into two distinct phenotypes: cytotoxic M1 type and cytoprotective M2 type [54]. Transient priming of microglia is generally considered neuroprotective; however, prolonged exposure to inflammatory stimuli can result in the release of various cytotoxic molecules such as reactive oxygen species, chemokines, and proinflammatory factors. The levels of proinflammatory factors released by M1 microglia, such as TNF-α, IL-1β, and IL-6, were significantly elevated in SAMP8 mice. However, the intervention with SVHRSP resulted in a decrease in the expression of these proinflammatory factors. Astrocytes play a crucial role in either inhibiting or promoting neurodegeneration through their interaction with extracellularly released molecules and the shared microenvironment with neurons. Inflammatory factors released by microglia, including TNF-α, IL-1β, and IL-6, can directly activate astrocytes and induce the production of oxygen free radicals that contribute to neuronal damage [55]. Additionally, another study demonstrated that improved neuronal deactivation was accompanied by a reduction in astrocyte priming.
Neurons serve as the fundamental functional units within the intricate neural network of the brain, and excessive neuronal loss can detrimentally impact cognitive and other essential functions [56]. The Nissl body, a distinctive cellular structure reflecting neuronal function and aiding in protein synthesis, was found to exhibit damage in hippocampal and cortical neurons of SAMP8 mice. Additionally, disorganized arrangement of vertebral body cells and reduced number of normal cells were observed. Notably, these alterations were effectively reversed by SVHRSP treatment, suggesting a potential neuroprotective effect associated with its anti-aging properties in SAMP8 mice.
The effects of SVHRSP on age-related cognitive deficits, including the reduction of oxidative stress and neuroinflammatory responses through the Sirt 1 pathway, have been summarized in Figure. 5.