Silver Nanoparticle-Induced Apoptosis in ARPE-19 Cells Is Inhibited by Toxoplasma gondii Pre-Infection Through Suppression of NOX4-Dependent ROS Generation

Int J Nanomedicine. 2020 May 26:15:3695-3716. doi: 10.2147/IJN.S244785. eCollection 2020.

Abstract

Purpose: External and internal stimuli easily affect the retina. Studies have shown that cells infected with Toxoplasma gondii are resistant to multiple inducers of apoptosis. Nanoparticles (NPs) have been widely used in biomedical fields; however, little is known about cytotoxicity caused by NPs in the retina and the modulators that inhibit nanotoxicity.

Materials and methods: ARPE-19 cells from human retinal pigment epithelium were treated with silver nanoparticles (AgNPs) alone or in combination with T. gondii. Then, the cellular toxicity, apoptosis, cell cycle analysis, autophagy, ROS generation, NOX4 expression, and MAPK/mTOR signaling pathways were investigated. To confirm the AgNP-induced cytotoxicity in ARPE-19 cells and its modulatory effects caused by T. gondii infection, the major experiments carried out in ARPE-19 cells were performed again using human foreskin fibroblast (HFF) cells and bone marrow-derived macrophages (BMDMs) from NOX4-/ - mice.

Results: AgNPs dose-dependently induced cytotoxicity and cell death in ARPE-19 cells. Apoptosis, sub-G1 phase cell accumulation, autophagy, JNK phosphorylation, and mitochondrial apoptotic features, such as caspase-3 and PARP cleavages, mitochondrial membrane potential depolarization, and cytochrome c release into the cytosol were observed in AgNP-treated cells. AgNP treatment also increased the Bax, Bik, and Bim protein levels as well as NOX4-dependent ROS generation. However, T. gondii-infected ARPE-19 cells inhibited AgNP-induced apoptosis, JNK phosphorylation, sub-G1 phase cell accumulation, autophagy, NOX4-mediated ROS production, and mitochondrial apoptosis. Furthermore, mitochondrial apoptosis was found in AgNP-treated HFF cells and BMDMs, and AgNP-induced mitochondrial apoptosis inhibition via NOX4-dependent ROS suppression in T. gondii pre-infected HFF cells and BMDMs was also confirmed.

Conclusion: AgNPs induced mitochondrial apoptosis in human RPE cells combined with cell cycle dysregulation and autophagy; however, these effects were significantly inhibited by T. gondii pre-infection by suppression of NOX4-mediated ROS production, suggesting that T. gondii is a strong inhibitory modulator of nanotoxicity in in vitro models.

Keywords: NADPH oxidase 4; Toxoplasma gondii; human retinal pigment epithelium; mitochondrial apoptosis; reactive oxygen species; silver nanoparticles.

MeSH terms

  • Animals
  • Apoptosis / drug effects*
  • Autophagy / drug effects
  • Cell Line
  • Cell Shape / drug effects
  • Disease Models, Animal
  • Fibroblasts / drug effects
  • Fibroblasts / parasitology
  • G1 Phase / drug effects
  • Humans
  • MAP Kinase Signaling System / drug effects
  • Male
  • Metal Nanoparticles / chemistry*
  • Mice
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Models, Biological
  • NADPH Oxidase 4 / metabolism*
  • Phosphorylation / drug effects
  • Reactive Oxygen Species / metabolism*
  • Retinal Pigment Epithelium / metabolism*
  • Retinal Pigment Epithelium / parasitology*
  • Silver / pharmacology*
  • Toxoplasmosis / pathology*

Substances

  • Reactive Oxygen Species
  • Silver
  • NADPH Oxidase 4
  • NOX4 protein, human

Grants and funding

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2017R1A2B4012822 and 2019R1A2C1088346) at Chungnam National University, and the National Natural Science Foundation of China (81771612 and 81971389), the Science Foundation of Guangdong Medical University (GDMUZ201801), the Natural Science Foundation of Guangdong Province (2019A1515011888 and 2019A1515011715). This work was also supported by research fund of Chungnam National University.