Research on the Effects of Amorfrutin B on Human Microglia under Hypoxia/Ischemia Conditions: Anti-inflammatory Action, Proliferation Potential, and Mitochondrial State Based on the PPARγ Pathway

Research Background

Hypoxia/ischemia is a major cause of brain injury in both neonatal and adult populations. Perinatal asphyxia and ischemic stroke are leading causes of high mortality in newborns and adults, respectively, and these diseases remain significant challenges in modern medicine. The primary treatment strategy for perinatal asphyxia is hypothermia therapy; however, about 40% of newborns receiving this treatment experience adverse reactions such as hypotension and cardiopulmonary hemodynamic instability. The standard treatment for ischemic stroke is recombinant tissue plasminogen activator (rtPA), but due to its narrow treatment window, only 1-5% of patients can receive treatment within 4.5 hours of symptom onset. Inflammatory processes play an important role in the pathogenesis of perinatal asphyxia and stroke, and regulating the function of microglia in the brain can inhibit neuroinflammation, thereby promoting recovery. Therefore, research on therapeutic approaches that can effectively regulate microglial function is currently a hot topic.

Research Source

This study was conducted by scientists including Karolina Przepiórska-Drónska, Agnieszka Wnuk, Bernadeta Angelika Pietrzak-Wawrzyńska, Andrzej Łach, Weronika Biernat, Anna Katarzyna Wójtowicz, and Małgorzata Kajta, from institutions including the Institute of Pharmacology, Polish Academy of Sciences, and the University of Agriculture in Krakow. The paper was published in the Journal of Neuroimmune Pharmacology in 2024.

Research Objectives and Significance

The main objective of the study was to investigate the effects of Amorfrutin B, a plant-derived selective PPARγ modulator, on human microglia under hypoxia/ischemia conditions, with a particular focus on whether it exerts anti-inflammatory effects through the PPARγ pathway and influences mitochondrial state and proliferation potential. Previous research has mainly focused on the neuroprotective potential of Amorfrutin B in animal models, but its effects on microglia have not been clearly defined. Given the importance of microglia in brain injury response, exploring the effects of Amorfrutin B on microglia has both theoretical and practical significance.

Research Methods

Experimental Model Construction

To simulate hypoxia/ischemia conditions, the research team used the human microglial cell line HMC3 and constructed hypoxia and ischemia models. Specific experimental steps included:

1. Hypoxia Model: HMC3 cells were exposed to an environment of 95% nitrogen and 5% carbon dioxide for 6 hours, then returned to normal oxygen concentration for 18 hours of reoxygenation.
2. Ischemia Model: HMC3 cells were exposed to glucose-free medium and incubated in an environment of 95% nitrogen and 5% carbon dioxide for 6 hours, then returned to normal oxygen and glucose concentrations for 18 hours of reoxygenation.

The research team also used methylene blue as a positive control to analyze the anti-inflammatory and neuroprotective effects of Amorfrutin B.

Main Experimental Methods

1. Cell Viability Assay: Using AlamarBlue™ reagent to assess cell survival rates after hypoxia/ischemia and Amorfrutin B treatment.
2. Neuronal Degeneration Assay: Using Fluoro-Jade C staining to assess the degree of neuronal degeneration.
3. Immunofluorescence Staining: Using Iba1 antibody to detect microglial activation state.
4. Mitochondrial Membrane Potential Detection: Using JC-1 dye to assess mitochondrial membrane potential.
5. Inflammatory Marker Detection: Using real-time quantitative PCR and ELISA to detect mRNA and protein levels of IL-1β, IL-10, and TNF-α.
6. Cell Proliferation Assay: Using BrdU incorporation to assess microglial proliferation potential.
7. Mitochondrial Function Assay: Using MTT assay to evaluate mitochondrial enzyme activity and cellular metabolic activity.

Data Analysis Methods

Data were statistically analyzed using analysis of variance (ANOVA) with Newman-Keuls test for multiple comparisons. A P-value less than 0.05 was considered statistically significant.

Research Results

Anti-inflammatory Effects

Amorfrutin B significantly reduced Iba1 fluorescence intensity in microglia under hypoxia/ischemia conditions, decreased caspase-1 activity, and regulated the expression of inflammatory factors. Specifically: - Decreased IL-1β and TNF-α expression: Under hypoxic conditions, IL-1β mRNA expression was reduced to 0.77-fold (1 µm Amorfrutin B) and 0.50-fold (5 µm Amorfrutin B). TNF-α expression decreased to 4.90-fold under hypoxic conditions with 5 µm Amorfrutin B. - Increased IL-10 expression: IL-10 significantly increased under both hypoxic and ischemic conditions, indicating the anti-inflammatory effect of Amorfrutin B.

Mitochondrial Function and Proliferation Potential

Amorfrutin B could reverse hypoxia/ischemia-induced changes in mitochondria-related parameters and reduce microglial proliferation potential. Specifically: - Mitochondrial Membrane Potential: Under hypoxic conditions, mitochondrial membrane potential increased to 175% and decreased to 138% with 5 µm Amorfrutin B. Under ischemic conditions, it increased to 184% and decreased to 162% with 5 µm Amorfrutin B. - Bcl-2 Expression: Under hypoxic conditions, Bcl-2 expression significantly increased to 8.88-fold and decreased to 2.18-fold and 1.06-fold with 1 µm and 5 µm Amorfrutin B, respectively. Under ischemic conditions, it increased to 9.88-fold and decreased to 5.51-fold and 2.28-fold with 1 µm and 5 µm Amorfrutin B, respectively. - Cell Proliferation Potential: Under hypoxic conditions, proliferation potential increased to 127% and decreased to 110% and 98% with 1 µm and 5 µm Amorfrutin B, respectively. Under ischemic conditions, it increased to 163% and decreased to 135% with 5 µm Amorfrutin B.

Neuroprotective Effects

Amorfrutin B significantly improved neuronal viability under hypoxia/ischemia conditions and reduced neuronal degeneration. Specifically: - Cell Viability: Under hypoxic conditions, cell viability recovered to 86% and 88% with 1 µm and 5 µm Amorfrutin B, respectively. Under ischemic conditions, it recovered to 55% and 53% with 1 µm and 5 µm Amorfrutin B, respectively. - Neuronal Degeneration: Under hypoxic conditions, neuronal degeneration increased to 124% and decreased to 93% and 94% with 1 µm and 5 µm Amorfrutin B, respectively. Under ischemic conditions, it increased to 120% and decreased to 85% and 82% with 1 µm and 5 µm Amorfrutin B, respectively.

Research Conclusions and Significance

This study demonstrates for the first time that Amorfrutin B regulates the activation state of human microglia under hypoxia/ischemia conditions through the PPARγ pathway, significantly affecting inflammation, mitochondrial function, and proliferation potential. These data expand the protective potential of Amorfrutin B in drug therapy for hypoxic/ischemic brain injury, not only targeting neurons but also activated microglia. The results suggest that Amorfrutin B has important scientific value and application prospects, especially in the treatment of hypoxic/ischemic brain injury.

Highlights

Amorfrutin B not only regulates the inflammatory response of microglia under hypoxia/ischemia conditions but also improves mitochondrial function and reduces cell proliferation potential. Additionally, Amorfrutin B has significant neuroprotective effects, markedly improving cell viability and reducing neuronal degeneration.

Other Valuable Information

Before clinical application, further research is needed on the effects of Amorfrutin B in other cell types and its potential side effects. Additionally, animal experiments and clinical trials are necessary to confirm its safety and efficacy in clinical applications.

This study provides new ideas and possibilities for future brain injury treatment. By regulating microglial function, Amorfrutin B has the potential to become an effective neuroprotective agent for treating various brain injuries.