Tanreqing Injection Inhibits Activation of NLRP3 Inflammasome in Macrophages Infected with Influenza A Virus by Promoting Mitophagy

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(mt ROS)-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 mt ROS 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 mt ROS 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 mt ROS 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.