Objective:

2

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:

2

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:

2

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:

2

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.