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Updated: Jun 23, 2025
Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
Tanreqing Injection Inhibits Activation of NLRP3 Inflammasome in Macrophages Infected with Influenza A Virus by
Tian-Yi Liu1, Yu Hao1, Qin Mao1
1School of Life Sciences, Beijing University of Chinese Medicine, Beijing, 102488, China.
Objective:
To investigate the inhibitory effect of Tanreqing Injection (TRQ) on the activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome in macrophages infected with influenza A virus and the underlying mechanism based on mitophagy pathway.
Methods:
The inflammatory model of murine macrophage J774A.1 induced by influenza A virus [strain A/Puerto Rico/8/1934 (H1N1), PR8] was constructed and treated by TRQ, while the mitochondria-targeted antioxidant Mito-TEMPO and autophagy specific inhibitor 3-methyladenine (3-MA) were used as controls to intensively study the anti-inflammatory mechanism of TRQ based on mitophagy-mitochondrial reactive oxygen species (mtROS)-NLRP3 inflammasome pathway. The levels of NLRP3, Caspase-1 p20, microtubule-associated protein 1 light chain 3 II (LC3II) and P62 proteins were measured by Western blot. The release of interleukin-1β (IL-1β) was tested by enzyme linked immunosorbent assay, the mtROS level was detected by flow cytometry, and the immunofluorescence and co-localization of LC3 and mitochondria were observed under confocal laser scanning microscopy.
Results:
Similar to the effect of Mito-TEMPO and contrary to the results of 3-MA treatment, TRQ could significantly reduce the expressions of NLRP3, Caspase-1 p20, and autophagy adaptor P62, promote the expression of autophagy marker LC3II, enhance the mitochondrial fluorescence intensity, and inhibit the release of mtROS and IL-1β (all P<0.01). Moreover, LC3 was co-localized with mitochondria, confirming the type of mitophagy.
Conclusion:
TRQ could reduce the level of mtROS by promoting mitophagy in macrophages infected with influenza A virus, thus inhibiting the activation of NLRP3 inflammasome and the release of IL-1β, and attenuating the inflammatory response.
Insights
Tanreqing Injection (TRQ) reduces inflammation in influenza A virus-infected macrophages by promoting mitophagy. This process decreases mitochondrial reactive oxygen species (mtROS), inhibiting NLRP3 inflammasome activation and IL-1β release.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- Influenza A virus infection triggers inflammatory responses in macrophages.
- Nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation is a key driver of this inflammation.
- Mitophagy, the selective degradation of damaged mitochondria, plays a role in regulating inflammasome activation.
Purpose of the Study:
- To investigate the inhibitory effect of Tanreqing Injection (TRQ) on NLRP3 inflammasome activation in influenza A virus-infected macrophages.
- To elucidate the underlying mechanism involving the mitophagy pathway.
Main Methods:
- Established an influenza A virus-infected murine macrophage model (J774A.1).
- Treated cells with TRQ, Mito-TEMPO (antioxidant control), and 3-methyladenine (3-MA, autophagy inhibitor).
- Assessed NLRP3, Caspase-1, LC3II, and P62 protein levels via Western blot; IL-1β release via ELISA; mtROS via flow cytometry; and mitophagy via confocal microscopy.
Main Results:
- TRQ treatment significantly reduced NLRP3, Caspase-1 p20, and P62 expression, while increasing LC3II expression, similar to Mito-TEMPO.
- TRQ inhibited mtROS and IL-1β release, consistent with mitophagy promotion.
- Confirmed mitophagy through co-localization of LC3 and mitochondria.
Conclusions:
- TRQ promotes mitophagy in influenza A virus-infected macrophages.
- This mitophagy induction reduces mtROS levels.
- Consequently, TRQ inhibits NLRP3 inflammasome activation and IL-1β release, attenuating the inflammatory response.
