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Apoptotic effects of norfloxacin on corneal endothelial cells

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Abstract

Norfloxacin, a frequently used ocular antibiotic, might have cytotoxic effect on human corneal endothelial cells (HCECs), subsequently damage the cornea and finally impair human vision. However, the possible mechanisms of cytotoxicity of norfloxacin to HCEC line are unclear. Herein, we investigated the cytotoxicity of norfloxacin and its underlying cellular and molecular mechanisms using in vitro cultured non-transfected HCECs and verified the cytotoxicity with cat corneal endothelium in vivo. In the present study, the cytotoxicity of norfloxacin in the in vitro cultured HCECs was recognized by causing abnormal morphology such as cell shrinkage and detachment from plate bottom, and decline of viability of in vitro cultured HCECs. Then, its cytotoxicity was verified by inducing reduction of cell density and morphological abnormality of in vivo cat corneal endothelial cells. Furthermore, the cytotoxicity of norfloxacin in HCECs was corroborated as apoptosis by elevation of plasma membrane permeability, S phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation in in vitro cultured HCECs and apoptosis-like swollen cells in the in vivo model. Moreover, norfloxacin induced extrinsic death receptor-mediated apoptosis pathway by activating caspase-2/-8/-3 and intrinsic mitochondrion-dependent apoptosis pathway by downregulating anti-apoptotic Bcl-2 and upregulating of pro-apoptotic Bad, which disrupted mitochondrial transmembrane potential, subsequently upregulated cytoplasmic cytochrome c and apoptosis-inducing factor and finally activated caspase-9/-3. Generally, norfloxacin induces HCE cell apoptosis via a death receptor-mediated and mitochondrion-dependent signaling pathway.

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Abbreviations

HCE:

Human corneal endothelium

HCEC:

Human corneal endothelial cell

CCEC:

Cat corneal endothelial cell

DMEM/F12:

Dulbecco’s modified Eagle medium/Ham’s nutrient mixture F-12 (1:1) medium

FBS:

Fetal bovine serum

MTT:

Methyl thiazolyl tetrazolium

FCM:

Flow cytometry

PI:

Propidium iodide

ECD:

Corneal endothelial cell density

CCT:

Central corneal thickness

PM:

Plasma membrane

PS:

Phosphatidylserine

AO/EB:

Acridine orange/ethidium bromide

PBS:

Phosphate-buffered saline

TEM:

Transmission electron microscope

ΔΨm:

Mitochondrial transmembrane potential

PMSF:

Phenylmethylsulfonyl fluoride

PBST:

PBS containing 0.05% Tween-20

HRP:

Horseradish peroxidase

RIPA:

Radio immunoprecipitation assay

ELISA:

Enzyme-linked immunosorbent assay

Cyt. c:

Cytochrome c

AIF:

Apoptosis-inducing factor

JC-1:

5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide

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Funding

This study was supported by the National Key Research and Development Program of China (2018YFC1106000/2018YFC1106001) from the Ministry of Science and Technology of the People’s Republic of China.

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Authors and Affiliations

  1. Key Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Yushan Road No. 5, Qingdao, 266003, China

    Ting-Jun Fan, Shu-Xian Wu & Guo-Jian Jiang

Authors
  1. Ting-Jun Fan
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  2. Shu-Xian Wu
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  3. Guo-Jian Jiang
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Contributions

TF planned the experiment, provided financial support, and performed data analysis. SW executed the experiment and performed data analysis. GJ planned the experiment, performed data analysis, and wrote the manuscript.

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Correspondence to Guo-Jian Jiang.

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Fan, TJ., Wu, SX. & Jiang, GJ. Apoptotic effects of norfloxacin on corneal endothelial cells. Naunyn-Schmiedeberg's Arch Pharmacol 393, 77–88 (2020). https://doi.org/10.1007/s00210-019-01711-5

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  • DOI: https://doi.org/10.1007/s00210-019-01711-5

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