Minocycline attenuates colistin-induced neurotoxicity via suppression of apoptosis, mitochondrial dysfunction and oxidative stress

Abstract

Background: Neurotoxicity is an adverse effect patients experience during colistin therapy. The development of effective neuroprotective agents that can be co-administered during polymyxin therapy remains a priority area in antimicrobial chemotherapy. The present study investigates the neuroprotective effect of the synergistic tetracycline antibiotic minocycline against colistin-induced neurotoxicity.

Methods: The impact of minocycline pretreatment on colistin-induced apoptosis, caspase activation, oxidative stress and mitochondrial dysfunction were investigated using cultured mouse neuroblastoma-2a (N2a) and primary cortical neuronal cells.

Results: Colistin-induced neurotoxicity in mouse N2a and primary cortical cells gives rise to the generation of reactive oxygen species (ROS) and subsequent cell death via apoptosis. Pretreatment of the neuronal cells with minocycline at 5, 10 and 20 μM for 2 h prior to colistin (200 μM) exposure (24 h), had an neuroprotective effect by significantly decreasing intracellular ROS production and by upregulating the activities of the anti-ROS enzymes superoxide dismutase and catalase. Minocycline pretreatment also protected the cells from colistin-induced mitochondrial dysfunction, caspase activation and subsequent apoptosis. Immunohistochemical imaging studies revealed colistin accumulates within the dendrite projections and cell body of primary cortical neuronal cells.

Conclusions: To our knowledge, this is first study demonstrating the protective effect of minocycline on colistin-induced neurotoxicity by scavenging of ROS and suppression of apoptosis. Our study highlights that co-administration of minocycline kills two birds with one stone: in addition to its synergistic antimicrobial activity, minocycline could potentially ameliorate unwanted neurotoxicity in patients undergoing polymyxin therapy.

© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Figures

Figure 1

Protective effect of minocycline against colistin-induced neurotoxicity in mouse neuronal N2a and primary cortical cells. (a) Impact of minocycline (Mino) pretreatment (5, 10 and 20 μM for 2 h) on colistin (200 μM)-induced cytotoxicity in N2a cells (24 h incubation). (b) The neuroprotective effect of minocycline pretreatment (5, 10 and 20 μM for 2 h) in mouse primary cortical neurons against colistin (200 μM)-induced cell death. The cell viability data were normalized and calculated as a percentage of untreated vehicle control values. All cell viability data shown represent the mean ± SD from five independent experiments. *P < 0.05, **P < 0.01, compared with the untreated control; #P < 0.05, ##P < 0.01, compared with colistin treatment. (c) The chemical structures of the clinically used polymyxins and minocycline. Leu, leucine; Phe, phenylalanine; Dab, α,γ-diaminobutyric acid.

Figure 2

Confocal fluorescence microscopy images of colistin (400 μM)-treated mouse primary cortical neurons stained with anti-polymyxin monoclonal antibody (green channel), phalloidin (red channel) and DAPI nuclear stain (blue channel). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 3

Protective effect of minocycline against colistin-induced apoptosis in N2a cells. (a) Apoptosis of Na2 cells was analysed by flow cytometry following annexin V-FITV/PI staining. Q1, necrosis cells; Q2, later apoptotic cells; Q3, live cells; Q4, early apoptotic cells. Cells were pretreated with minocycline for 2 h at 37 °C. After removing the medium containing minocycline, the cells were then incubated in media containing colistin for 24 h at 37 °C. (b) Apoptotic rate in N2a cells in response to colistin and minocycline pretreatment. (c, d) Caspase-9 and caspase-3 activities in N2a cells in response to colistin and minocycline pretreatment were examined using ELISA. All the data shown represent the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, compared with the untreated control; #P < 0.05, ##P < 0.01, compared with colistin treatment.

Figure 4

Minocycline attenuates colistin-induced mitochondrial dysfunction. (a) N2a cells were plated onto 12-well plates at a density of 2×105 cells/well and pretreated with minocycline at a final concentration of 20 μM at 37 °C for 2 h (n = 3). The cells were treated with colistin (200 μM) for an additional 24 h. The change in mitochondrial membrane potential (MTP) was evaluated using the cationic fluorescent indicator JC-1. The JC-1 aggregate form, indicating normal MTP function, appears red. The JC-1 monomeric form, indicating disrupted MTP, appears green; Magnification ×40. (b) Quantitative analysis of the confocal data presented as the ratio between fluorescence intensity in the green (low membrane potential) and red (high membrane potential) channels. An increase in the ratio was interpreted as the loss of ψm. Values are presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, compared with the untreated control; #P < 0.05, ##P < 0.01, compared with colistin treatment. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 5

Minocycline protects N2a cells against colistin-induced oxidative stress. (a) Cellular ROS levels were detected using confocal microscopy imaging following staining of N2a cells with the ROS-sensitive dye 2,7-dichlorofluorescein diacetate; Magnification ×40. (b) ROS generated relative to control were quantified. (c, d) The impact of minocycline pretreatment (5, 10 and 20 μM for 2 h) on cellular SOD and CAT activities in N2a cells treated with colistin (200 μM for 24 h). The data shown represent the mean ± SD from three independent experiments. **P < 0.01, compared with the untreated control; #P < 0.05 and ##P < 0.01, compared with colistin treatment. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.