[Legionella pneumophila activates Nrf2-Keap1 pathway to inhibit autophagy of mouse RAW264.7 macrophages].

Objective To investigate the effect of Legionella pneumophila (LP) on the autophagy flux of RAW264.7 macrophages and explore the molecular mechanism of the expression changes of autophagy-related factors. Methods Live LP and inactivated LP (MOI=10, 50, 100) were separately used to affect RAW264.7 for 1, 2 and 3 hours so as to screen the optimum condition of LP infection. The optimal condition for LP infection was MOI=50 and the infection time was 2 hours. After affected by rapamycin (RAPA) for 12 hours, RAW264.7 cells were then treated by live and inactivated LP for another 2 hours. Normal control group, RAPA group, live LP group, inactivated LP group, RAPA-treated live LP group, RAPA-treated inactivated LP group were designed. The pmCherry-C1-EGFP-LC3B double fluorescent labeling protein method was used to monitor the changes of autophagy flux. The relevant factor CLN3, histone deacetylase 6 (HDAC6), regulator of G protein signaling 19 (RGS19), tumor necrosis factor (TNF), cathepsin B (CTSB), GABA type A receptor associated protein like 2 (GABARAPL2), P62, microtubule-related protein 1 light chain 3 (LC3) were screened by gene array analysis. In order to validate the results of gene array, real-time quantitative PCR (RT-qPCR) was used to detect the mRNA levels of nuclear factor erythroid derived 2 like 2 (Nrf2), beclin1 and kelch like ECH associated protein 1 (Keap1); Western blot analysis was performed to measure the protein levels of Nrf2, beclin1 and Keap1. Results Both the live LP group and the inactivated LP group inhibited the autophagy flux compared with the normal control group and the RAPA group. Gene array analysis showed that in the live LP and inactivated LP groups, LC3 expression was down-regulated and P62 expression was up-regulated. The results of RT-qPCR and Western blot analysis were consistent with the gene array. The mRNA and protein levels of Keap1, beclin1, and Nrf2 significantly decreased, while the mRNA and protein levels of Nrf2 significantly increased. Conclusion LP can inhibit the autophagy of macrophage via activating Nrf2-Keap1 signaling pathway.