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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
The ROS/TXNIP/NLRP3 pathway mediates LPS-induced microglial inflammatory response
Qianlei Zhao1, Guanhao Liu2, Qiang Ding2
1Department of Pediatric Neurology, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, PR China.
Background:
Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction activated by microglia. The potential pathological changes of SAE are complex, and the cellular pathophysiological characteristics remains unclear. This study aims to explore the ROS/TXNIP/NLRP3 pathway mediated lipopolysaccharide (LPS)-induced inflammatory response in microglia.
Methods:
BV-2 cells were pre-incubated with 10 μM N-acetyl-L-cysteine (NAC) for 2 h, which were then reacted with 1 μg/mL LPS for 24 h. Western blot assay examined the protein levels of IBA1, CD68, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. The contents of inflammatory factor were detected by ELISA assay. The co-immunoprecipitation assay examined the interaction between TXNIP and NLRP3.
Results:
LPS was confirmed to promote the positive expressions of IBA1 and CD68 in BV-2 cells. The further experiments indicated that LPS enhanced ROS production and NLRP3 inflammasome activation in BV-2 cells. Moreover, we also found that NAC partially reversed the facilitation of LPS on the levels of ROS, IL-1β, IL-18, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. NAC treatment also notably alleviated the interaction between TXNIP and NLRP3 in BV-2 cells.
Conclusion:
ROS inhibition mediated NLRP3 signaling inactivation by decreasing TXNIP expression.
Insights
Sepsis-associated encephalopathy involves microglia activation. This study shows N-acetyl-L-cysteine (NAC) inhibits the ROS/TXNIP/NLRP3 pathway, reducing inflammation in microglia during sepsis.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Sepsis-associated encephalopathy (SAE) is a complex brain dysfunction initiated by microglia.
- The precise cellular mechanisms underlying SAE pathogenesis remain incompletely understood.
- This research investigates the role of the ROS/TXNIP/NLRP3 pathway in lipopolysaccharide (LPS)-induced microglial activation.
Purpose of the Study:
- To elucidate the involvement of the reactive oxygen species (ROS)/thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome pathway in LPS-induced microglial inflammatory responses.
- To evaluate the therapeutic potential of N-acetyl-L-cysteine (NAC) in modulating this pathway.
Main Methods:
- BV-2 microglial cells were treated with LPS and NAC.
- Western blot analysis was used to assess protein expression levels of key inflammatory markers and pathway components (IBA1, CD68, TXNIP, NLRP3, ASC, Cleaved Caspase-1).
- Enzyme-linked immunosorbent assay (ELISA) quantified inflammatory cytokine levels, and co-immunoprecipitation examined TXNIP-NLRP3 interactions.
Main Results:
- LPS treatment increased microglial activation markers (IBA1, CD68), ROS production, and NLRP3 inflammasome activation.
- NAC treatment partially reversed LPS-induced increases in ROS, inflammatory cytokines (IL-1β, IL-18), TXNIP, NLRP3, ASC, and Cleaved Caspase-1.
- NAC also attenuated the interaction between TXNIP and NLRP3.
Conclusions:
- Reactive oxygen species (ROS) inhibition is a key mechanism by which NAC inactivates the NLRP3 inflammasome signaling pathway.
- Decreased TXNIP expression is central to ROS inhibition-mediated NLRP3 inflammasome inactivation.
- Targeting the ROS/TXNIP/NLRP3 pathway presents a potential therapeutic strategy for SAE.
