NOVOS Core Reduces Oxytosis/Ferroptosis and Inflammation, Processes Associated with Aging

The study, conducted at the Salk Institute for Biological Studies, examined how a NOVOS formulation interacts with in vitro cellular models of oxytosis/ferroptosis and inflammatory signaling—biological processes commonly studied in aging-related research. Understanding the study is easier with a few foundational concepts.

Important note on interpretation: All NOVOS-related findings described in this article were generated in vitro using cellular models. These results are exploratory, do not establish effects in humans, and do not demonstrate relevance to health or disease outcomes. This article is provided for scientific and educational purposes only.

Foundational Concepts

Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation. It is distinct from apoptosis, necrosis, and other cell death programs, and can involve processes such as glutathione depletion, reactive oxygen species (ROS) accumulation, and lipid peroxidation. Ferroptosis has been studied across a range of experimental systems, including cancer biology, neurodegeneration-related models, and ischemia-reperfusion research, making it an active area of investigation in basic and translational science (Li et al., 2020; Xie et al., 2016; Stockwell et al., 2017).

Oxytosis is a regulated cell death pathway first described over 30 years ago, primarily in neuronal contexts, as a pathway for glutamate-associated oxidative cell death in certain experimental systems. Key steps can include glutathione depletion, lipoxygenase activation, ROS accumulation, and calcium influx. Oxytosis and ferroptosis share several overlapping cellular features, including oxidative stress and lipid peroxidation-related processes, and are often discussed together in the context of neuronal cell stress and survival (Maher et al., 2020).

Relationship Between Oxytosis and Ferroptosis

Oxytosis and ferroptosis are distinct but related pathways that can converge on overlapping cellular mechanisms (e.g., glutathione depletion and oxidative damage). Differences between them often include the specific triggers used in experiments and the dependency of ferroptosis on iron-driven lipid peroxidation. The literature continues to explore where these pathways overlap and where they diverge, particularly in neuronal cell models (Maher et al., 2020; Dixon et al., 2012).

Required to Understand Table 1 (Why Glutamate, Erastin, and RSL3 Are Used)

In vitro assays that use glutamate, erastin, and RSL3 are widely used to probe ferroptosis/oxytosis-related mechanisms and to test how experimental compounds influence these pathways under controlled laboratory conditions.

Glutamate (oxytosis-like stress in certain neuronal models): In some neuronal cell models, glutamate can deplete intracellular glutathione via effects on cystine availability, leading to oxidative stress and downstream lipid peroxidation. Studying this response helps researchers understand redox vulnerability in neuronal cells and evaluate how formulations influence cell survival under oxidative stress conditions (Maher et al., 2020).

Erastin (ferroptosis-related trigger): Erastin inhibits the cystine/glutamate antiporter (system Xc−), reducing cystine import, depleting glutathione, and promoting lipid peroxidation-associated ferroptosis. It is commonly used to induce ferroptosis-like stress in experimental systems and to examine how compounds modulate this pathway (Dixon et al., 2012).

RSL3 (ferroptosis-related trigger): RSL3 inhibits GPX4 (glutathione peroxidase 4), a key enzyme that limits lipid peroxidation. GPX4 inhibition can drive rapid lipid peroxidation and ferroptosis-like cell death. Screening compounds against RSL3 challenges helps identify mechanisms that influence lipid peroxidation defenses (Yang et al., 2016).

Required to Understand Table 2 (Why HT22 and BV2 Cells Are Used)

HT22 mouse hippocampal neuronal cells

HT22 cells are widely used in experimental work on oxidative stress and cell death pathways because they are sensitive to redox disruption and glutathione depletion under certain conditions.

• Neuronal origin: HT22 cells are derived from mouse hippocampal neuronal tissue, a region relevant to learning and memory research (Liu et al., 2009).
• Sensitivity to oxidative stress: In this model system, glutamate exposure can reduce glutathione and increase oxidative stress, creating a controlled environment for studying oxytosis/ferroptosis-related mechanisms (Henke et al., 2013).
• Utility as a laboratory model: These properties make HT22 cells useful for mechanistic studies of cellular stress responses and survival pathways.

BV2 microglial cells + LPS + nitric oxide readout

BV2 cells are an immortalized mouse microglial cell line frequently used to study inflammatory signaling in vitro. LPS (lipopolysaccharide) is commonly used to activate inflammatory pathways in microglial models, and nitric oxide (NO) can be used as one readout of inflammatory activation in these systems.

• Microglial activation model: LPS stimulation activates inflammatory signaling pathways in BV2 cells, which is useful for studying microglia-like immune responses in the CNS.
• NO as an inflammatory readout: Excess NO production (often via iNOS induction) is commonly measured as part of the inflammatory response in these in vitro models.
• Connection to oxidative stress research: Inflammation-linked mediators can interact with oxidative stress pathways, so researchers sometimes examine inflammatory readouts alongside ferroptosis/oxytosis-related stressors in mechanistic work.

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 separately and in combination in assays to observe their effects on cellular responses to experimentally induced oxytosis/ferroptosis-like stress in HT22 mouse hippocampal neuronal cells, and on inflammatory signaling in LPS-stimulated BV2 microglial cells.

Table 1 reports how the mixtures influenced HT22 cell survival following exposure to glutamate, erastin, and RSL3—commonly used triggers of oxidative stress and ferroptosis-related pathways in laboratory settings.

The study quantified effects using EC50 values (the concentration/amount required to achieve 50% of the measured effect in that assay). Lower EC50 values indicate stronger effects under the specific experimental conditions.

• Water (H₂O) soluble mix (1x): Showed measurable effects across the tested stressors at the reported volumes.
• DMSO soluble mix (1x): Showed stronger effects than the water-soluble mix in some conditions, consistent with the inclusion of DMSO-soluble components (e.g., fisetin and pterostilbene).
• Total mix (1x): Combining both mixtures produced the strongest effects in the assay, reflected by substantially lower EC50 values across the tested stressors. This pattern is consistent with synergy or additivity among components under these experimental conditions.

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). Table 2 reports EC50 values for effects on nitric oxide (NO) production in LPS-stimulated BV2 cells—an in vitro model commonly used to study inflammatory signaling.

Total mix (1x) and DMSO mix (1x): Both showed measurable effects on NO production at the reported EC50 values (3.2 μl per 10^5 cells and 2.4 μl per 10^5 cells, respectively).

Water-soluble mix: Showed no measurable effect up to the highest concentration tested (>1 μl per 10^4 cells), suggesting the DMSO-soluble components played a larger role in the observed NO-related effects under these conditions.

Discussion

Synergistic Reduction in Oxytosis/Ferroptosis

Across both experimental systems, the combined “total mix” produced stronger effects than either mixture alone. Under these in vitro conditions, that pattern is consistent with synergistic or additive interactions among formulation components, especially given that water-soluble and lipid-soluble compounds can influence different parts of cellular stress and signaling pathways.

This framing aligns with broader scientific discussions about multi-target approaches when studying complex pathways such as ferroptosis, which can involve iron handling, antioxidant defenses, and lipid peroxidation dynamics (Li et al., 2020; Xie et al., 2016).

Inflammatory signaling in BV2 cells

In the BV2 model, the formulation reduced NO production in LPS-stimulated cells under the tested conditions. Because NO is one commonly used readout of inflammatory activation in this model, these results add mechanistic context to how the formulation may influence inflammatory signaling pathways in vitro.

Interpreting EC50 values

The EC50 comparisons suggest the formulation produced measurable effects at relatively low volumes within these assay systems. Importantly, EC50 values in cell culture are assay-specific and are best interpreted as describing relative potency within the experimental model, not as a proxy for dosing or effects in humans.

Contextualizing the Findings in Aging and Aging Research

Aging biology research often focuses on cellular stress responses, oxidative damage, and inflammation-related signaling because these processes can influence how cells maintain function under challenge. This study adds mechanistic data—generated in cellular models—showing how a multi-ingredient formulation interacts with experimental systems used to probe oxytosis/ferroptosis-related stress and inflammatory signaling.

In particular, the stronger effects observed for the combined formulation suggest that multi-component approaches may influence multiple nodes in cellular stress pathways, which is a common rationale in aging-related mechanistic research (Franceschi and Campisi, 2014; Kennedy et al., 2014).

This study conducted on NOVOS Core by the Salk Institute’s neuroscientist Dr. Pamela Maher provides compelling evidence for its potential.

Conclusion

This in vitro study provides mechanistic evidence that the combined NOVOS Core formulation influenced (1) cellular responses to experimentally induced oxytosis/ferroptosis-like stress in HT22 neuronal cells and (2) nitric oxide–associated inflammatory signaling in LPS-stimulated BV2 microglial cells. The results also suggest the combined mixture produced stronger effects than component mixtures alone under the tested conditions, consistent with additive or synergistic interactions within these assay systems.

As with all cellular studies, these findings are exploratory and serve as a basis for further research. They do not establish effects in humans and should not be interpreted as evidence of disease-related outcomes.