NOVOS Core Reduces Oxytosis/Ferroptosis and Inflammation, Processes Associated with Cognitive Decline & Cancer

The study, run by California’s prestigious Salk Institute for Biological Studies, explored the potential of a new formulation by NOVOS in combating oxytosis/ferroptosis and inflammation, pathways intricately linked to aging, neurodegenerative diseases, and cancer. Understanding this study necessitates a grasp of several foundational concepts.

Foundational Concepts

Ferroptosis is a recently identified form of regulated cell death characterized by iron-dependent lipid peroxidation. It is distinct from apoptosis, necrosis, and other forms of cell death, involving mechanisms such as glutathione depletion, reactive oxygen species (ROS) accumulation, and lipid peroxidation. Ferroptosis has been implicated in various diseases including cancer, neurodegenerative disorders, and ischemia-reperfusion injury, making it a focus of research for potential therapeutic interventions (Li et al., 2020; Xie et al., 2016; Stockwell et al., 2017).

Oxytosis, on the other hand, is a form of cell death first described over 30 years ago, primarily in nerve cells, as a non-excitotoxic pathway for glutamate-induced cell death. This process includes key steps such as glutathione depletion, activation of lipoxygenase, accumulation of ROS, and calcium influx. Oxytosis and ferroptosis share several pathophysiological changes observed in various neurodegenerative diseases and the aging brain. The discovery of ferroptosis, which shares characteristics with oxytosis, including the dependency on iron and involvement in glutathione depletion and lipid peroxidation, has led to discussions about their similarities and differences (Maher et al., 2020).

Oxytosis and ferroptosis are distinct but related cell death pathways, both characterized by their involvement in neurodegenerative diseases and linked to oxidative stress mechanisms. While ferroptosis is iron-dependent and associated with lipid peroxidation, oxytosis involves glutamate-induced excitotoxicity leading to cell death through depletion of glutathione and increased reactive oxygen species. Despite these differences, there is overlap in their mechanisms, such as the depletion of glutathione, suggesting a potential interconnection between these pathways in neuronal cell death processes. References like Maher et al. (2020) and Dixon et al. (2012) provide insights into their distinct yet potentially interconnected roles in neurodegeneration.

Required to Understand Table 1

In vitro assays for protection against glutamate, erastin, and RSL3, particularly in the context of ferroptosis/oxytosis, serve several key purposes in understanding and potentially treating various diseases, including cancer and neurodegenerative conditions.

Glutamate-induced Oxytosis/Ferroptosis: Glutamate can induce oxytosis in neural cells, a form of cell death characterized by the depletion of glutathione, accumulation of reactive oxygen species (ROS), and an influx of calcium. This pathway shares similarities with ferroptosis, particularly regarding the involvement of glutathione depletion and lipid peroxidation. Studying protection against glutamate-induced cell death provides insights into mechanisms that could be targeted to prevent neurodegenerative diseases associated with glutamate toxicity, such as Alzheimer’s disease (Maher et al., 2020).

Erastin-induced Ferroptosis: Erastin targets the cystine/glutamate antiporter leading to cystine deprivation, glutathione depletion, and subsequent lipid peroxidation-driven ferroptosis. Investigating compounds that can protect against erastin-induced ferroptosis is crucial for identifying potential therapeutic agents that could sensitize cancer cells to ferroptosis-inducing treatments or protect healthy cells from unwanted ferroptotic death (Dixon et al., 2012).

RSL3-induced Ferroptosis: RSL3 directly inhibits glutathione peroxidase 4 (GPX4), an enzyme essential for limiting lipid peroxidation within cells. By inhibiting GPX4, RSL3 triggers ferroptosis characterized by overwhelming lipid peroxidation. Screening for agents that confer protection against RSL3-induced ferroptosis can help identify molecules that either boost the antioxidant defenses of cells or provide insights into alternative pathways that can be modulated to prevent or induce ferroptosis, potentially offering new angles for cancer therapy (Yang et al., 2016).

Required to Understand Table 2

HT22 mouse hippocampal neuronal cells are widely utilized in assays studying ferroptosis and oxytosis due to several key characteristics that make them a valuable model for neuroscientific research.

Neuronal Origin and Characteristics: HT22 cells are derived from mouse hippocampal neurons, an area of the brain critically involved in memory and learning. These cells retain many neuronal properties, making them an excellent model to study neuronal behavior, including cell death mechanisms specific to neurons (Liu et al., 2009).

Sensitivity to Oxidative Stress: HT22 cells are particularly sensitive to oxidative stress induced by glutamate, which depletes intracellular glutathione by blocking the cystine/glutamate antiporter. This depletion of GSH, a critical antioxidant, leads to increased reactive oxygen species (ROS) and lipid peroxidation, hallmarks of oxytosis and ferroptosis. This model allows for the study of oxidative stress mechanisms and the testing of neuroprotective compounds against such cell death pathways (Henke et al., 2013).

Model for Neurodegenerative Disease Research: Given their origin and sensitivity to oxidative stress, HT22 cells are used to model aspects of neurodegenerative diseases where oxidative stress and cell death pathways like ferroptosis and oxytosis are implicated.

The rationale for assaying compounds for anti-inflammatory activity using mouse BV2 cells treated with bacterial lipopolysaccharide (LPS), with the read-out for inflammatory activity being the production of nitric oxide (NO), is multifaceted and rooted in the understanding of neuroinflammatory processes, particularly in the context of ferroptosis/oxytosis. Here’s a detailed explanation based on the findings from various studies:

Role of BV2 Microglial Cells

Microglial Activation: BV2 cells are an immortalized mouse microglial cell line that retains many characteristics of primary microglia, including responsiveness to LPS, a component of the outer membrane of Gram-negative bacteria. LPS is a well-known inducer of inflammation, capable of activating microglia, the resident immune cells of the central nervous system (CNS)​​.

Modeling Neuroinflammation: The activation of BV2 cells by LPS serves as a model to study neuroinflammation, which is implicated in the progression of neurodegenerative diseases and is characterized by the overproduction of pro-inflammatory cytokines and mediators like NO​​.

Production of Nitric Oxide

Indicator of Inflammation: In the CNS, excessive production of NO by activated microglia is a hallmark of inflammation. NO is produced by inducible nitric oxide synthase (iNOS) and plays a dual role as both a neuroprotective and a neurotoxic molecule. Its overproduction contributes to oxidative stress and cell death, including mechanisms relevant to ferroptosis and oxytosis​​.

Assessing Anti-inflammatory Activity: Measuring NO production in LPS-stimulated BV2 cells allows for the evaluation of the anti-inflammatory potential of compounds. A decrease in NO production in the presence of test compounds indicates their potential to mitigate neuroinflammatory responses​​.

Connection to Ferroptosis/Oxytosis

Oxidative Stress and Cell Death: Both ferroptosis and oxytosis are forms of regulated cell death associated with oxidative stress. Neuroinflammation, characterized by the overproduction of NO and other reactive oxygen species (ROS), can exacerbate these processes. Therefore, compounds that show anti-inflammatory activity by reducing NO production may also confer protection against ferroptosis/oxytosis-induced cell death​​.

Therapeutic Potential: By identifying compounds that can modulate the inflammatory response in microglial cells, researchers can explore potential therapeutic strategies for diseases where ferroptosis and oxytosis play crucial roles, such as neurodegenerative disorders. The use of BV2 cells treated with LPS to assay for NO production serves as a preliminary screening tool to identify compounds with neuroprotective and anti-inflammatory properties​​.

In summary, the use of BV2 cells treated with LPS to assay compounds for their ability to inhibit NO production is a strategic approach to identifying potential neuroprotective agents that can ameliorate neuroinflammatory conditions and possibly reduce the susceptibility to cell death pathways like ferroptosis and oxytosis. This methodology leverages the central role of microglia in CNS inflammation and the critical involvement of NO in neuroinflammatory processes.

Oxytosis/Ferroptosis Study Methodology and Results

The scientists utilized two mixtures based on the NOVOS Core formulation: a water-soluble mix and a non-water soluble mix prepared in dimethyl sulfoxide (DMSO), containing fisetin and pterostilbene. These mixtures were tested both separately and combined in assays to assess their protective effects against induced oxytosis/ferroptosis in HT22 mouse hippocampal neuronal cells and their anti-inflammatory activity in LPS-activated BV2 microglial cells. This dual approach aims to evaluate the formulation’s neuroprotective and anti-inflammatory potentials, key areas of interest in aging and neurodegenerative disease research.

The results from Table 1 demonstrate the efficacy of different mixtures of the NOVOS Core formulation in protecting HT22 mouse hippocampal neuronal cells against cell death induced by glutamate, erastin, and RSL3, which are agents known to initiate the oxytosis/ferroptosis pathway. This pathway is relevant to the study of aging and age-related neurodegenerative diseases. The protection afforded by the mixtures is quantified as the effective concentration (EC50) required to protect 50% of the cells.

Water (H2O) Soluble Mix 1x: This mixture provided moderate protection against the cell death agents, requiring 20 μl per 10^4 cells to protect against glutamate and erastin-induced cell death, and 25 μl per 10^4 cells against RSL3-induced cell death. This suggests that the water-soluble components have some protective effect against oxytosis/ferroptosis.

DMSO Soluble Mix 1x: This mixture, containing fisetin and pterostilbene, showed improved protection compared to the water-soluble mix, especially against glutamate-induced cell death, requiring only 7 μl per 10^5 cells. This indicates a stronger protective effect of the non-water soluble components, possibly due to their antioxidative properties, which are critical in combating oxidative stress linked with ferroptosis/oxytosis.

Total Mix 1x: Combining both the water-soluble and DMSO-soluble components resulted in significantly enhanced protection across all tests, with the required volume dropping to as low as 1 μl per 10^5 cells for glutamate-induced cell death.

Anti-Inflammation Study Methodology and Results

The results from Table 2 of the study reveal insightful findings regarding the anti-inflammatory activity of the NOVOS Core formulation in BV2 microglial cells treated with lipopolysaccharide (LPS). These outcomes are pivotal for understanding the formulation’s potential in modulating neuroinflammation, a key factor in the pathogenesis of various neurodegenerative diseases and a contributor to aging-related cognitive decline.

Differential Anti-inflammatory Efficacy: The total 1x mix and the 1x DMSO mix exhibited significant anti-inflammatory activity, as evidenced by their effective concentration (EC50) values of 3.2 μl per 10^5 and 2.4 μl per 10^5 cells, respectively. This suggests that the components soluble in DMSO, for example fisetin and pterostilbene, play a critical role in mitigating the inflammatory response induced by LPS. These compounds are known for their anti-inflammatory and antioxidative properties, which likely contribute to their effectiveness in this context (Tsai et al., 2011; Pan et al., 2018).

Lack of Activity in the Water-Soluble Mix: The water-soluble mix showed no anti-inflammatory activity up to the highest concentration tested (>1 μl per 10^4 cells), indicating that the water-soluble components of the NOVOS Core formulation may not directly influence the pathways involved in LPS-induced NO production in BV2 cells. This underscores the importance of the DMSO-soluble components in exerting the formulation’s anti-inflammatory effects and suggests that not all components contribute equally to mitigating inflammation.

Discussion

Synergistic Reduction in Oxytosis/Ferroptosis

The study’s findings present intriguing insights into the protective effects of the NOVOS Core formulation against cell death induced by glutamate, erastin, and RSL3 in HT22 cells, alongside its anti-inflammatory potential in LPS-stimulated BV2 microglial cells.

Enhanced Protection Against Oxytosis/Ferroptosis: The complete NOVOS Core formulation (total mix), demonstrated remarkable protective effects against the induction of oxytosis/ferroptosis by glutamate, erastin, and RSL3. The total mix’s enhanced efficacy suggests a synergistic interaction between its components, surpassing the protective capabilities of the separate water-soluble and DMSO-soluble mixes. This synergy indicates that the formulation’s components work together more effectively than individually, a critical insight for developing neuroprotective treatments. 

The finding aligns with previous research emphasizing the importance of multifactorial approaches in combating complex pathways like ferroptosis, where cellular iron homeostasis and lipid peroxidation play significant roles (Li et al., 2020; Xie et al., 2016). 

Furthermore, the differences in EC50 values across the different cell death inducers (glutamate, erastin, RSL3) also suggest variability in how these components interact with the specific pathways activated by each inducer, reflecting the complexity of the oxytosis/ferroptosis pathway and the multifaceted protective mechanism of the NOVOS Core formulation.

Anti-inflammatory Activity in BV2 Microglial Cells

The formulation’s anti-inflammatory potential was highlighted by its ability to attenuate nitric oxide (NO) production in LPS-stimulated BV2 cells. The modulation of neuroinflammation by the NOVOS Core formulation, particularly through the suppression of NO, a key proinflammatory mediator, underscores the importance of targeting microglial activation in neurodegenerative disease contexts. This finding is consistent with the growing body of literature that identifies neuroinflammation as a critical target in the prevention and treatment of diseases like Alzheimer’s and Parkinson’s (Mirzoeva et al., 1999; Ngo et al., 2012).

Dose-Dependent Efficacy

The study meticulously quantifies the effective concentrations (EC50) for protection against cell death inducers, offering precise insights into the formulation’s potency. The substantial reduction in EC50 values with the total mix across all inducers—glutamate, erastin, and RSL3—highlights the formulation’s robust neuroprotective capacity at potentially low doses, making it an attractive candidate due to the remote likelihood of side effects.

Chronic inflammation is a hallmark of aging [link] and is implicated in the progression of neurodegenerative diseases. The ability of the NOVOS Core formulation, particularly its DMSO-soluble components, to reduce LPS-induced NO production in microglial cells highlights its potential as a therapeutic agent in aging research and neuroprotection. Targeting microglial activation and the subsequent inflammatory cascade can be crucial in developing strategies to mitigate the impact of aging on the brain and prevent or slow the progression of neurodegenerative diseases (Heneka et al., 2015). The results from the anti-inflammatory activity assay provide valuable insights into the components of the NOVOS Core formulation that are most effective in reducing neuroinflammation. This has significant implications for aging and neurodegenerative disease research, emphasizing the need for targeted interventions that can modulate the inflammatory response in the CNS.

In summary, the results of this study not only affirm the NOVOS Core formulation’s neuroprotective and anti-inflammatory capabilities but also emphasize the synergistic potential of combining water-soluble and lipid-soluble components. This multifaceted approach to neuroprotection and neuroinflammation mitigation could pave the way for novel interventions in aging and age-related neurodegenerative diseases.

Contextualizing the Findings in Aging and Aging Research

This study conducted on NOVOS Core by the Salk Institute’s neuroscientist Dr. Pamela Maher provides compelling evidence for its potential as a therapeutic intervention against mechanisms implicated in aging and age-related neurodegenerative diseases. The research’s implications extend far beyond the laboratory, offering hopeful prospects for aging populations and individuals at risk of neurodegenerative conditions.

Addressing the Complexity of Aging

Aging is a multifactorial process characterized by the gradual decline in physiological functions, including the brain’s ability to maintain homeostasis and respond to stress. The NOVOS Core formulation’s ability to protect against oxytosis/ferroptosis and reduce neuroinflammation touches on two pivotal aspects of aging at the cellular level: the accumulation of damage due to oxidative stress and the chronic inflammation often referred to as “inflammaging” (Franceschi and Campisi, 2014). By targeting these pathways, the formulation could contribute to preserving neuronal integrity and function, potentially delaying the onset or progression of age-related cognitive decline.

Synergistic Approach to Neuroprotection

The study highlights the importance of a synergistic approach to neuroprotection, resonating with the complexity of aging-related cellular damage pathways. The enhanced efficacy of the complete NOVOS Core formulation suggests that a multi-targeted strategy may be more effective in countering the broad range of challenges faced by aging cells. This aligns with current trends in aging research, which advocate for interventions that can simultaneously address multiple aging hallmarks (Kennedy et al., 2014).

Implications for Neurodegenerative Disease Prevention and Therapy

Implications for Neurodegenerative Disease Prevention and Therapy
Neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, are among the most daunting challenges of aging, significantly affecting quality of life. The protective effects of the NOVOS Core formulation against cell death and inflammation provide a foundation for exploring its use as a preventative or therapeutic agent. By potentially mitigating the cellular dysfunctions that precede clinical symptoms, the formulation could offer a novel strategy for disease modification or risk reduction (Cummings et al., 2016).

Conclusion

This NOVOS Core study represents a step forward in the quest to understand and intervene in the aging process and its associated neurodegenerative conditions. By providing evidence of protection against oxidative stress-induced cell death and inflammation, this research contributes to a growing body of evidence supporting the unique longevity enhancing capabilities and synergistic nature of the NOVOS Core formulation. As aging research continues to evolve, studies such as this underscore the potential for innovative, synergistic formulations to address the complex challenges of aging, offering hope for healthier, more resilient aging populations.