Tizoxanide induces autophagy by inhibiting PI3K/Akt/mTOR pathway in RAW264.7 macrophage cells.

As the main metabolite of nitazoxanide, tizoxanide (TIZ) has a broad-spectrum anti-infective effect against parasites, bacteria, and virus. In this study, we investigated the effects of TIZ on autophagy by regulating the PI3K/Akt/mTOR signaling pathway. RAW264.7 macrophage cells were treated with various TIZ concentrations. Cell viability assay, transmission electron microscope, and immunofluorescence staining were used to detect the biological function of the macrophage cells, and the expression levels of the autophagy pathway-related proteins were measured by Western blot. Results revealed that TIZ promoted the conversion of LC3-I to LC3-II, the formation of autophagy vacuoles, and the degradation of SQSTM1/p62 in a concentration- and time-dependent manner in RAW264.7 cells. Treatment with TIZ increased the Beclin-1 expression level and inhibited PI3K, Akt, mTOR, and ULK1 activation. These effects were enhanced by pretreatment with rapamycin but attenuated by pretreatment with LY294002. In addition, the conversion of LC3-I to LC3-II was observed in Vero, 293T, and HepG2 cells treated with TIZ. These data suggested that TIZ may induce autophagy by inhibiting the Akt/mTOR/ULK1 signaling pathway in macrophages and other cells.