Epigallocatechin-3-Gallate (EGCG) Inhibits Neuroinflammation in BV2 Cells by Regulating the ROS/TXNIP/NLRP3 Pathway

Research Background

Alzheimer’s Disease (AD) is a degenerative brain disorder primarily affecting the elderly, characterized by persistent cognitive dysfunction and behavioral impairment. The neuropathological changes in AD include β-amyloid (Aβ) deposition, abnormal neurofibrillary tangles (NFTs), and neuronal loss in the brain. Studies have shown that chronic Aβ deposition activates microglia, leading to chronic inflammation and ultimately resulting in neuronal death and cognitive dysfunction. Furthermore, the activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is closely associated with microglial inflammation and AD. Therefore, preventing inflammasome activation may be a potential intervention for treating AD.

Research Motivation

Current research indicates that many NLRP3 inflammasome stimulants can induce the production of reactive oxygen species (ROS), and the increase in ROS is crucial for inflammasome activation (Dominic et al. 2022). ROS is considered a key factor in triggering the formation and activation of NLRP3 inflammasomes (Billingham et al. 2022), with mitochondrial dysfunction being the main cause of elevated ROS levels (Angelova and Abramov 2018). TXNIP (thioredoxin-interacting protein) is an endogenous inhibitor of the ROS scavenging protein thioredoxin (Trx) and serves as a bridge between oxidative stress and NLRP3 inflammasome activation. In this context, this study focuses on the anti-inflammatory mechanism of epigallocatechin-3-gallate (EGCG), a polyphenol compound found in green tea.

Research Source

This study was conducted by the following authors: Yanyan Xiao, Chenglin Yang, Nana Si, Tao Chu, Jiahui Yu, Xintong Yuan, and Xiang-Tao Chen, all from the School of Pharmacy, Anhui Medical University. The paper was published in the Journal of Neuroimmune Pharmacology, with the official acceptance date of June 8, 2024, and can be found at https://doi.org/10.1007/s11481-024-10131-z.

Research Process

Scientific Experiment Section

To verify the inhibitory effect of EGCG on inflammation in BV2 cells, the experiment adopted the following steps:

1. Cell Culture and Treatment: BV2 cells (Beijing, China) were cultured in incomplete high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, incubated at 37°C with 5% CO2.

2. Inflammation Induction and Drug Treatment: The experimental procedure included:

   • Exposing BV2 cells to EGCG solutions (5 µM, 10 µM, and 20 µM) or MCC950 (10 µM), NAC (20 mM), or Mitoquinone (MitoQ, 0.2 µM) for 1 hour.
   • Subsequently treating with LPS (1 µg/ml) for 1 hour, followed by Aβ1-42 oligomers (Aβo) (10 µg/ml) for 6 hours to induce inflammatory responses (Zhong et al. 2019).

3. Cell Viability Detection: The survival rate of BV2 cells was determined using the CCK-8 kit, and the absorbance was measured at 450nm wavelength using a microscope.

4. Western Blot Analysis and RT-PCR: Analyzed protein levels in BV2 cells (such as IL-1β, TNF-α, IBA-1, NLRP3, and Caspase-1), and determined changes in inflammatory indicators between cells through experiments.

5. Immunofluorescence Detection and Intracellular ROS Detection: ROS production in BV2 cells was observed through fluorescence microscopy, and intracellular ROS levels were detected using the DCFH-DA staining method.

6. Mitochondrial Membrane Potential Detection: Changes in mitochondrial membrane potential of BV2 cells were detected using the JC-1 fluorescent probe, analyzing ROS production under mitochondrial damage.

Research Results

Inhibitory Effect of EGCG on Inflammatory Responses in LPS/Aβo-induced BV2 Cells

After EGCG treatment, the mRNA expression levels of related inflammatory factors such as IL-1β, IL-6, and TNF-α in BV2 cells were significantly reduced (Fig. 1a-c), and the corresponding protein expression levels were also significantly decreased (Fig. 1d-i). Immunofluorescence staining further confirmed that EGCG also reduced the initiation of inflammatory responses and Aβ deposition (Fig. 1j).

EGCG Inhibits LPS/Aβo-induced Inflammatory Responses by Suppressing NLRP3 Inflammasome Activation

When exploring the mechanism of EGCG’s inhibition of inflammation in BV2 cells, it was found that EGCG reduced the expression levels of inflammasome-related molecules such as NLRP3, ASC, and Caspase-1 (Fig. 2a-e). In particular, the expression of IL-1β was significantly reduced after EGCG treatment, confirming its inhibitory effect on inflammasome activation. Additionally, the inhibitory mechanism of EGCG on NLRP3 inflammasome was verified using the inflammasome inhibitor MCC950 and activator ATP (Fig. 2f-h).

EGCG Inhibits LPS/Aβo-induced NLRP3 Inflammasome Activation in BV2 Cells by Reducing Oxidative Stress

The increase in endogenous ROS production is an important factor in NLRP3 inflammasome activation. This study found through fluorescent probe staining that EGCG significantly reduced LPS/Aβo-induced intracellular ROS levels, and verified the effect through ROS inhibitor NAC and ROS activator tBHP (Fig. 3a-b).

EGCG Inhibits ROS Due to Mitochondrial Dysfunction

The experiment further explored whether EGCG inhibits ROS generation through mitochondrial dysfunction. Results showed that EGCG and the mitochondria-specific ROS inhibitor MitoQ exhibited similar effects (Fig. 4a). Using the JC-1 fluorescent probe, it was found that both EGCG and MitoQ could reduce the proportion of green fluorescence induced by mitochondrial damage (Fig. 4b-f).

The Role of TXNIP in EGCG Regulating NLRP3 Inflammasome Activation

As a key signaling molecule, TXNIP plays a crucial role in oxidative stress and NLRP3 inflammasome activation. This study found that EGCG could inhibit the expression of TXNIP in LPS/Aβo-induced BV2 cells, and further confirmed the inhibitory effect of EGCG through siRNA knockdown and overexpression experiments (Fig. 5a-i).

Conclusion and Significance

This study demonstrates the potential mechanism of EGCG in inhibiting neuroinflammation through the ROS/TXNIP/NLRP3 pathway, providing new insights for AD treatment. Although the pharmacological mechanism was verified in an in vitro model, its pharmacokinetics and interactions with other molecules in in vivo models still require further research. By emphasizing the new mechanism of EGCG in inhibiting inflammasome activation, this study provides new potential targets for AD prevention and treatment and reveals new pathways for anti-inflammatory therapy.

Research Highlights

• First revelation of the mechanism by which EGCG inhibits neuroinflammation through the mitochondrial ROS/TXNIP/NLRP3 pathway.
• EGCG shows significant anti-inflammatory and neuroprotective effects, offering a potential new approach for AD treatment.
• Provides a possible targeted therapeutic approach with important guiding significance for subsequent research.

Limitations and Future Directions

Although EGCG has shown good anti-inflammatory effects in in vitro studies, its efficacy in in vivo models still needs further verification, especially its bioavailability in the brain and specific signaling mechanisms. Additionally, the potential for combining EGCG with other anti-inflammatory or neuroprotective drugs is worth exploring.