Effect of pyrroloquinoline quinone on lipopolysaccharide-induced autophagy in HAPI microglia cells

Pyrroloquinoline quinone (PQQ) is involved in various physiological and biochemical processes, including antioxidant, cell proliferation, and mitochondrial formation. It plays a vital role in protecting neurons. However, the effect of PQQ on microglia, an inflammatory cell of the central nervous system (CNS), is still unclear. This study aimed to investigate the biological role and neuroprotective mechanism of PQQ in HAPI microglial cells exposed to lipopolysaccharide (LPS).

Western blot (WB) was used to detect apoptosis and autophagy-related molecules Bax, Bcl2, active-caspase-3, caspase-3, LC3, lysosomal associated membrane protein 2 (LAMP2), AKT, tumor necrosis factor receptor (TNFR) 1, and TNFR2 expression. The phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor LY294002 was used to block the Akt pathway. WB detected the effects of PI3K on autophagy and TNFR1 and TNFR2 expression. The localization of active-caspase-3, caspase-3, LC3, LAMP2, TNFR1, and TNFR2 in cells was observed by immunofluorescence staining. The effect of PQQ on the cell cycle was examined by flow cytometry. We used 5-Ethynyl-2'-deoxyuridine (EdU) assay to detect cell proliferation. The migration ability of cells under different conditions was detected by scratch test and Transwell assay.

Our results showed that there were different effects on the apoptosis-related molecules Bcl2/Bax and active-caspase-3/caspase in HAPI microglial cells treated with PQQ at different times. PQQ had no significant effect on the LC3b/a ratio in the early stage, which was upregulated in the later stage. The expression of LAMP2 was significantly increased in both early and late stages after PQQ treatment. At the same time, we found that PQQ can reverse the translocation of LAMP2 from the cytoplasm to the nucleus in LPS-induced HAPI microglia. After PQQ treatment, TNFR1 was significantly decreased, but TNFR2 increased in LPS-induced HAPI microglia. It may be that PQQ works through the PI3K/Akt signaling pathway to up-regulate LC3, LAMP2, and TNFR1 and down-regulate TNFR2 in LPS-induced HAPI microglia. However, PQQ has little effect on LPS-induced proliferation, cell cycle, and migration of HAPI microglia.

In LPS-induced HAPI microglia, PQQ reduces the apoptosis level and increases that of autophagy. In addition, PQQ changes the distribution of LAMP2 in the cytoplasm and nucleus, which is regulated through the PI3K/Akt signaling pathway.