Cytotoxicity and Autophagy Induced by Ivermectin via AMPK/mTOR Signaling Pathway in RAW264.7 Cells

Abstract

The widespread and excessive use of ivermectin (IVM) will not only cause serious environmental pollution, but will also affect metabolism of humans and other mammals that are exposed. IVM has the characteristics of being widely distributed and slowly metabolized, which will cause potential toxicity to the body. We focused on the metabolic pathway and mechanism of toxicity of IVM on RAW264.7 cells. Colony formation and LDH detection assay showed that IVM significantly inhibited the proliferation of and induced cytotoxicity in RAW264.7 cells. Intracellular biochemical analysis using Western blotting assay showed that LC3-B and Beclin-1 were upregulated and p62 was down-regulated. The combination of confocal fluorescence, calcein-AM/CoCl₂, and fluorescence probe results showed that IVM could induce the opening of the mitochondrial membrane permeability transition pore, reduce mitochondrial content, and increase lysosome content. In addition, we focused on induction of IVM in the autophagy signal pathway. The Western blotting results showed that IVM increased expression of p-AMPK and decreased p-mTOR and p-S6K expression in protein levels, indicating that IVM activated the AMPK/mTOR signaling pathway. Therefore, IVM may inhibit cell proliferation by inducing cell cycle arrest and autophagy.

Keywords: AMPK/mTOR pathway; autophagy; cell cycle arrest; cytotoxicity; ivermectin.

Figure 1

IVM induces cytotoxicity in RAW264.7 cells. (A) Colony formation of cells after IVM treatment of cells for 6 h. (B) The number of colonies was significantly decreased in cells treated with IVM for 6 h. (C) Release rate of LDH in the treatment of various concentrations of IVM for 6 h. Data were expressed as means ± SD (standard deviation) of three separate sets of independent experiments. * p < 0.05, ** p < 0.01 and indicate the significant differences from the control.

Figure 2

IVM induces cell cycle arrest in RAW264.7 cells. (A) Percentage of cells in G1-, G2-, and S-phases after IVM treatment of RAW264.7 cells for 24 h. (B) Cyclin D1 and CDK 4 expression in RAW264.7 cells treated with IVM for 6 h. (C) Densitometric analysis of the expression levels of cyclin D1 and CDK 4. Data were expressed as means ± SD (standard deviation) of three separate sets of independent experiments. * p < 0.05, ** p < 0.01, and indicate the significant differences from the control.

Figure 3

IVM induces autophagy in RAW264.7 cells. (A) Representative fluorescence photomicrograph (200×) of RAW264.7 cells stained with MDC after treatment with different concentrations for 6 h. (B) LC3-B, Beclin-1, and p62 expression in RAW264.7 cells treated with IVM for 6 h. (C) Densitometric analysis of the expression levels of LC3-B, Beclin-1, and p62. Data were expressed as means ± SD (standard deviation) of three separate sets of independent experiments. ** p < 0.01, and indicate the significant differences from the control.

Figure 4

The effect of IVM on ATP content in RAW264.7 cells. (A) After IVM treatment of cells for 6 h, the mPTP opening was detected by co-loading with calcein-AM and CoCl₂ (200×). (B) ATP content was measured after IVM treatment of cells for 6 h. Data were expressed as means ± SD (standard deviation) of three separate sets of independent experiments. * p < 0.05, ** p < 0.01, and indicate significant differences from the control.

Figure 5

IVM induces mitophagy in RAW264.7 cells. After being treated with IVM for 6 h, cells were stained with MitoTracker Green and LysoTracker Red and observed with a fluorescence microscope (200×).

Figure 6

The effect of IVM on AMPK/mTOR signaling pathways in RAW264.7 cells. (A) Phosphorylated mTOR, AMPK, and p70s6k expression in RAW264.7 cells treated with IVM for 6 h. (B) Densitometric analysis of the expression levels of phosphorylated mTOR, AMPK, and p70s6k. Data were expressed as means ± SD (standard deviation) of three separate sets of independent experiments. ** p < 0.01 and indicate significant differences from the control.