Main Research Findings

1) Treatment with myricetin mitigated motor disturbances and decreased neuronal ferroptosis in animal models of Parkinson’s disease.

2) Myricetin was found to regulate hypoxia-induced expression of adipokines and hypoxia-regulated pathways in adipocytes.

 

Selected Data

1) The research team of Gu et al completed a comprehensive experimental investigation into the protective effects of myricetin in a Parkinson’s disease (PD) model using both in vivo and in vitro systems. Various reagents, procedures, and assessments were used to elucidate the therapeutic potential of myricetin, with a focus on oxidative stress, ferroptosis, and neuroprotection [1].

The SH-SY5Y human neuroblastoma cell line was used for in vitro studies and cultured in standard DMEM supplemented with 10% fetal bovine serum under 5% CO₂ at 37°C. For treatment, the cells were exposed to 0.4 mM MPP⁺, a neurotoxin that mimics PD pathology, 50 μM myricetin, 1 μM erastin, as a ferroptosis inducer, or 1 μM ferrostatin-1, as a ferroptosis inhibitor, for 24 hours. The impact of these treatments was subsequently evaluated through various biochemical and molecular analyses.

A rat model of Parkinson’s disease was created using intraperitoneal injections of MPTP at a dose of 30 mg/kg/day for five days, a method known to induce dopaminergic neuron degeneration, particularly in the substantia nigra. Control rats received vehicle injections without MPTP. To test the therapeutic effect of myricetin, it was administered orally at 25 mg/kg/day from day 1 to day 14. In another experimental arm, erastin was delivered intranasally at 15 mg/kg immediately after MPTP injections. All rats were maintained under standard laboratory conditions with controlled temperature, humidity, and a 12-hour light/dark cycle. They had free access to food and water. Rats were weighed weekly from day 7, and euthanized after four weeks using an overdose of pentobarbital followed by cervical dislocation. Substantia nigra tissues were then harvested for further analysis [1].

Motor coordination and balance of the rats was assessed through behavioral tests including the rotarod, balance beam, and foot fault tests. In the rotarod test, rats were tested on their ability to stay on a rotating rod that accelerated from 4 to 40 rpm over five minutes. The time before falling was recorded across three trials. The balance beam test evaluated rats’ ability to walk across a narrow wooden beam elevated 60 cm above the ground. Rats were scored from 1 to 6 based on performance, ranging from falling immediately to walking without any issues. The foot fault test involved a mesh platform where rats’ foot slips were counted during a 1-minute ambulation period. Foot fault percentage was calculated by the ratio of missteps to total steps, multiplied by 100% [1].

Western blotting was employed to assess protein expression levels in both substantia nigra tissue and SH-SY5Y cells. Proteins were extracted using a RIPA buffer, and concentrations were measured using a Pierce BCA assay. (20 μg of proteins per sample were separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk, and incubated with specific primary antibodies overnight at 4°C. This was followed by secondary antibody incubation and signal detection using ChemiDox XRS imaging and ImageJ analysis, with β-actin and Lamin B1 serving as loading controls.
Immunohistochemical staining of substantia nigra tissues was performed to localize specific proteins. Fixed tissues were paraffin-embedded, sectioned, and processed through standard dewaxing, hydration, antigen retrieval using citrate buffer, and blocking steps. Primary antibodies were applied, followed by HRP-conjugated secondary antibodies. Visualization was achieved using an HRP DAB kit, and counterstaining was done with hematoxylin [1].

Reactive oxygen species levels were measured in both substantia nigra tissues and SH-SY5Y cells using the DCFDA fluorescent probe. Substantia nigra tissue was homogenized, incubated with DCFDA for 30 minutes, and fluorescence intensity was measured with a microplate reader. SH-SY5Y cells were similarly treated with DCFDA, fixed with paraformaldehyde, and fluorescence was visualized under a microscope and quantified using ImageJ software.

Iron content was determined using an iron assay kit. Substantia nigra tissue and SH-SY5Y cells were homogenized in an assay buffer, centrifuged, and supernatants were incubated with an iron reducer and probe, while absorbance was measured at 593 nm. This assay helps assess ferroptosis, as iron accumulation is a hallmark of this form of cell death. Lipid peroxidation, a sign of oxidative stress, was evaluated by measuring malondialdehyde levels using an MDA assay kit. Absorbance was read at 532 nm. Glutathione levels, representing antioxidant capacity, were measured using a total glutathione assay kit and read at 412 nm [1].

Statistical analyses were performed using the Student’s t-test for two-group comparisons and one-way ANOVA with Scheffé post hoc tests for multiple comparisons. All experiments were conducted in triplicate, and results were expressed as mean ± standard deviation. In summary, this study employs a set of biochemical, molecular, and behavioral assessments to evaluate the neuroprotective effects of myricetin in PD models. By using both in vitro and in vivo systems and focusing on markers of oxidative stress, ferroptosis, and neuronal integrity, it provides insights into myricetin’s therapeutic potential [1].

2) The study performed by researchers Geiger et al, aimed to investigate the effects of hypoxia and myricetin treatment on adipocyte inflammation and associated molecular pathways, using differentiated human SGBS preadipocytes as a model system. The passage provides a detailed description of the cell culture methods, treatment conditions, and the analytical techniques used to evaluate changes at the gene and protein levels in response to hypoxic stress and myricetin administration [2].

Human SGBS (Simpson-Golabi-Behmel syndrome) preadipocytes were utilized in this experiment. These cells are commonly used to model human adipocyte biology due to their high differentiation potential and relevance to metabolic research. The preadipocytes were cultured in a medium comprising a mixture of DMEM and Ham’s F12, supplemented with 10% fetal calf serum, penicillin, streptomycin, biotin, and pantothenate. Once the cells reached 80% confluence, they were differentiated into mature adipocytes using a specialized induction cocktail. This differentiation medium included transferrin, insulin, hydrocortisone, triiodothyronine, dexamethasone, IBMX, and rosiglitazone. After four days of treatment, the medium was changed to a maintenance medium lacking dexamethasone, IBMX, and rosiglitazone, and this was continued for ten more days with regular medium changes every 3–4 days [2].

To simulate the effects of hypoxia on adipocytes, fully differentiated SGBS cells were subjected to 1% oxygen concentration in a modular incubator chamber, while control cells remained in normoxic conditions with 21% oxygen. Hypoxic exposures were conducted for 4, 24, or 48 hours to assess both early and sustained responses. Myricetin was dissolved in DMSO and applied at a concentration of 25 μM and the cells were pretreated with myricetin for 2 hours before being exposed to hypoxia. Control groups received the same volume of DMSO to ensure consistency in experimental conditions.
Gene expression analysis focused on several key adipokines associated with inflammation: apelin, leptin, chemerin, asprosin, and dipeptidyl peptidase-4. RNA was extracted from the treated cells using a lipid-specific RNA isolation kit. The RNA was reverse transcribed into complementary DNA, which was then used in real-time quantitative polymerase chain reaction to determine relative gene expression. Specific primers designed to span exon-exon junctions were used to ensure accurate amplification of transcripts. The data was normalized to the housekeeping gene TBP and analyzed to calculate fold changes compared to control samples [2].

In addition to gene expression, protein levels and activation states of several signaling molecules were assessed through immunoblotting. Whole-cell and nuclear extracts were prepared using specialized reagents, with protease and phosphatase inhibitors included to preserve protein integrity. Protein concentration was measured using the Bradford assay. Proteins were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with specific primary antibodies. The panel of antibodies included markers of hypoxic response, inflammatory signaling, cell survival and transcription, metabolic signaling, and epithelial-mesenchymal transition.

After primary antibody incubation, membranes were treated with appropriate secondary antibodies conjugated to horseradish peroxidase. Detection of protein bands was achieved using various chemiluminescence substrates, and signals were visualized using an AutoChemi detection system. The intensity of the bands was quantified using ImageJ software, which allowed for precise analysis of protein expression changes under different experimental conditions [2].

To ensure the statistical reliability of the data, results were expressed as mean values with standard deviation. Differences between experimental groups were assessed using unpaired two-tailed Student’s t-tests. All statistical analyses were conducted using IBM SPSS Statistics and GraphPad Prism 8. This statistical framework provided robust validation of experimental findings, confirming the significance of observed changes due to hypoxia and/or myricetin treatment.

In summary, this study used an in vitro model of human adipocytes to explore the effects of hypoxia and myricetin on gene and protein expression related to inflammation and metabolic stress. Myricetin was administered as a potential therapeutic compound due to its known anti-inflammatory and antioxidant properties. The experiment involved inducing hypoxia in mature adipocytes, followed by rigorous assessment of both mRNA and protein responses. The key inflammatory adipokines were quantified through qPCR, while molecular signaling pathways were explored via Western blot analysis of proteins such as HIF-1α, NF-κB, CREB, Akt, and EMT markers. This comprehensive approach aimed to unravel how hypoxic stress alters adipocyte function and how myricetin might counteract these effects, potentially highlighting a new therapeutic avenue for metabolic or inflammatory conditions associated with adipose tissue dysfunction [2].

Discussion
1) This study conducted by Gu et al explores the neuroprotective effects of myricetin in PD using both in vivo and in vitro models. Specifically, the research focuses on how myricetin mitigates motor dysfunction, dopaminergic neuron loss, α-synuclein accumulation, and ferroptosis, a form of regulated cell death associated with iron overload and oxidative stress. The mechanistic pathway investigated includes the activation of the Nrf2/Gpx4 signaling axis, known for its role in counteracting ferroptosis [1].

To model PD in rats, MPTP was administered intraperitoneally to induce dopaminergic neuron degeneration. Rats were then treated orally with myricetin. MPTP treatment led to a significant reduction in body weight gain, indicating systemic and neurological stress. However, myricetin administration effectively restored normal weight gain patterns, suggesting overall health improvement. Behavioral tests including the beam balance test, foot fault test, and rotarod test were used to evaluate motor coordination and function. MPTP-treated rats exhibited impaired motor behavior across all tests, while myricetin significantly improved performance. Importantly, erastin, a specific ferroptosis inducer, partially reversed the motor improvements induced by myricetin in the foot fault test, though it had no significant effect in the beam balance and rotarod tests, indicating a partial dependence on ferroptosis regulation [1].

Figure 2: Changes in B) weight in grams, C) balance beam test score, D) percent of foot fault, and E) latency to fall assessed through the rotarod test, throughout the control group, the MPTP group, the MPTP + myricetin group, and the MPTP + myricetin + erastin group
To determine if myricetin could protect dopaminergic neurons, the study evaluated tyrosine hydroxylase, a marker of dopamine neurons, and α-synuclein, a key pathological protein in PD. Western blotting and immunohistochemistry of the substantia nigra showed that MPTP dramatically increased α-synuclein accumulation and reduced tyrosine hydroxylase expression, signifying neuronal loss. Myricetin reversed these changes, decreasing α-synuclein levels and restoring tyrosine hydroxylase protein expression. Furthermore, immunohistochemistry analysis confirmed an increase in tyrosine hydroxylase-positive cells in the substantia nigra following myricetin treatment. Again, these beneficial effects were inhibited by erastin, further indicating the involvement of ferroptosis in MPTP-induced neurotoxicity and myricetin’s protective effects.

Given that ferroptosis is characterized by iron overload and oxidative stress, the study assessed iron, reactive oxygen species, malondialdehyde, and glutathione levels in both substantia nigra tissue and serum. MPTP significantly elevated iron and reactive oxygen species levels, along with an increase in malondialdehyde, a marker of lipid peroxidation, and a decrease in GSH, an essential antioxidant. Myricetin treatment reversed all these changes, strongly indicating that it alleviates ferroptosis. When co-administered with erastin, these effects were blocked, confirming that ferroptosis inhibition is a critical mechanism of myricetin’s neuroprotection [1].

Figure 3: Changes in A) iron levels in the substantia nigra, B) iron levels in the serum, C) relative reactive oxygen species levels, D) malodialdehyde levels in the tissues, and E) GSH levels in the tissues
To understand whether motor function improvements correlated with ferroptosis inhibition, the researchers analyzed relationships between latency to fall assessed through the rotarod test, and ferroptosis biomarkers. No significant correlation was found between latency to fall and iron in the substantia nigra; however, serum iron and substantia nigra malondialdehyde levels showed a negative correlation with motor performance, while GSH levels had a positive correlation, supporting the hypothesis that oxidative stress and iron metabolism are tightly linked to motor dysfunction in PD [1].
The effects of myricetin were further validated in in vitro experiments using SH-SY5Y cells, a human neuroblastoma cell line commonly used to model PD. Exposure to MPP⁺, a toxic metabolite of MPTP, mimicked PD-like pathology by reducing cell viability and inducing ferroptosis. Treatment with Fer-1, a ferroptosis inhibitor, or myricetin restored cell viability. Myricetin also reduced iron, reactive oxygen species, and malondialdehyde levels while increasing GSH in MPP⁺-treated cells. Once again, these effects were nullified by co-treatment with erastin, reinforcing the role of ferroptosis inhibition in myricetin’s protective mechanism.

A crucial part of this study was the investigation of the Nrf2 (nuclear factor erythroid 2–related factor 2) pathway and its downstream effector Gpx4 (glutathione peroxidase 4), both of which are known to suppress ferroptosis. In MPP⁺-treated SH-SY5Y cells, total Nrf2 protein levels were not significantly altered. However, treatment with myricetin increased both total and nuclear Nrf2 levels, indicating enhanced activation and translocation of the transcription factor. Furthermore, MPP⁺ treatment caused a decrease in Gpx4 protein expression, which was reversed by myricetin, suggesting that the protective effects are mediated by Nrf2-driven upregulation of Gpx4 [1].

In conclusion, the study demonstrates that myricetin exerts strong neuroprotective effects in both in vivo and in vitro PD models. It improves motor function, protects dopaminergic neurons, and reduces α-synuclein accumulation. These effects are mechanistically linked to suppression of ferroptosis, evidenced by reduced iron and oxidative stress markers, and are mediated by activation of the Nrf2/Gpx4 pathway. The partial reversal of myricetin’s benefits by erastin underscores the critical role of ferroptosis inhibition. Taken together, these findings suggest that myricetin may be a promising candidate for therapeutic development in Parkinson’s disease and potentially other neurodegenerative conditions involving ferroptosis and oxidative stress [1].

2) This study completed by Geiger et al explores the impact of myricetin on gene expression and cellular signaling pathways in human SGBS adipocytes under hypoxic conditions. Hypoxia, or low oxygen availability, is known to trigger inflammation and alter gene expression in adipose tissue, contributing to metabolic dysfunction. The researchers aimed to assess whether myricetin could counteract these hypoxia-induced effects, particularly focusing on pro-inflammatory adipokines, transcription factors, and signaling proteins.

The researchers investigated the expression of five pro-inflammatory adipokines, apelin, leptin, chemerin, asprosin, and DPP-4, in adipocytes cultured for 48 hours under normoxic or hypoxic conditions, with or without myricetin treatment. Under normoxia, 25 μM of myricetin had little effect on most adipokines, except for a significant reduction in leptin and chemerin mRNA levels. In contrast, hypoxic conditions led to a significant upregulation of all five adipokines, with apelin showing the most dramatic increase by 25.3-fold. However, when hypoxic cells were treated with myricetin, the expression of all these adipokines was markedly suppressed. For example, leptin expression was reduced to 5.4-fold, and DPP-4 dropped to 0.5-fold. The reduction in expression was statistically significant for all adipokines except apelin, which showed a notable but non-significant decrease. These findings suggest that myricetin can mitigate hypoxia-induced inflammation by repressing adipokine expression [2].

Figure 4: Changes in hypoxia-induced gene expression of selected adipokines, including apelin, leptin, chemerlin, asprosin, and DPP4 in groups treated with DMSO or myricetin and exposed to either normoxic or hypoxic conditions
The study further examined the role of hypoxia-inducible factor 1-alpha (HIF-1α), a transcription factor central to the cellular response to low oxygen. Under hypoxic conditions, nuclear HIF-1α levels increased at both 4 and 24 hours. Myricetin treatment did not significantly affect HIF-1α levels under normoxia, except for a slight increase at 4 hours. Myricetin also strongly suppressed hypoxia-induced HIF-1α accumulation in the nucleus at 4 hours, though this inhibitory effect was not sustained at 24 hours. These results indicate that myricetin can interfere with the early hypoxic signaling cascade by blocking HIF-1α stabilization and nuclear localization [2].

Next, the study focused on the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a key regulator of inflammation. Under hypoxic conditions, total and phosphorylated NF-κB p65 subunit levels were elevated at 4 hours. Myricetin attenuated this increase, particularly reducing nuclear accumulation of the phosphorylated NF-κB p65 at 4 hours. Interestingly, no significant differences were found in total or phosphorylated NF-κB levels at 24 hours across any treatment groups, indicating the effect of myricetin may be most relevant during early hypoxic response. Furthermore, the cytoplasmic inhibitor IκB, which prevents NF-κB activation, remained unchanged in both expression and phosphorylation status under all conditions, suggesting myricetin’s effects on NF-κB occur downstream or independently of IκB modulation [2].

The researchers also analyzed cAMP response element-binding protein (CREB), a transcription factor involved in metabolic regulation and inflammation. CREB levels slightly declined under hypoxia at 4 hours and with myricetin treatment, but by 24 hours, CREB protein expression was not significantly affected by hypoxia alone. Notably, myricetin treatment increased CREB expression under both normoxic and hypoxic conditions at 24 hours. Additionally, myricetin prevented the hypoxia-induced accumulation of phosphorylated CREB in the nucleus at 24 hours. These findings suggest that myricetin may exert anti-inflammatory effects in part by modulating CREB signaling and reducing its activation under stress conditions.

Akt, a serine/threonine-specific protein kinase, is another signaling molecule examined in this study. Its phosphorylated form is associated with cell survival, metabolism, and inflammation. Under hypoxia, phosphorylation of Akt at serine 473 increased at both 4 and 24 hours. Myricetin effectively inhibited this phosphorylation at 4 hours but was ineffective at 24 hours. Importantly, the total Akt protein level remained unchanged throughout the treatments, implying that myricetin’s effect is specific to phosphorylation and activation of Akt rather than its expression [2].

Finally, the study investigated the impact of myricetin on epithelial-mesenchymal transition-related transcription factors, referred to as Snail and Slug. Under normoxia, myricetin reduced nuclear levels of these proteins at both 4 and 24 hours. This suppression was even more pronounced under hypoxia, where myricetin treatment nearly eliminated nuclear Snail and Slug signals by 24 hours. These results are significant because Snail and Slug are known to promote fibrosis, inflammation, and metabolic dysfunction, particularly in the context of hypoxic adipose tissue. By inhibiting their nuclear accumulation, myricetin may help preserve adipocyte integrity and limit hypoxia-induced pathological remodeling [2].

Overall, the findings of this study highlight the multifaceted role of myricetin in combating hypoxia-induced inflammation and metabolic disruption in human adipocytes. Myricetin significantly downregulated the expression of key pro-inflammatory adipokines, interfered with hypoxia-responsive signaling via HIF-1α, inhibited activation of NF-κB and Akt, suppressed phosphorylated CREB, and reduced nuclear accumulation of EMT-related factors Snail and Slug. These effects were most prominent during early hypoxic exposure, suggesting that myricetin may act as an early-phase modulator of hypoxia-induced cellular stress. Given its ability to target several inflammatory and metabolic pathways, myricetin emerges as a promising candidate for therapeutic intervention in hypoxia-related adipose tissue dysfunction, potentially relevant in obesity and related metabolic disorders [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Gu SC, Xie ZG, Gu MJ, et al. Myricetin mitigates motor disturbance and decreases neuronal ferroptosis in a rat model of Parkinson’s disease. Sci Rep. 2024;14(1):15107. Published 2024 Jul 2. doi:10.1038/s41598-024-62910-6

[2] Geiger K, Muendlein A, Leiherer A, et al. Myricetin attenuates hypoxia-induced inflammation in human adipocytes. Mol Biol Rep. 2023;50(12):9833-9843. doi:10.1007/s11033-023-08865-9

 

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