

RESEARCH ARTICLE Year : 2018 | Volume : 13 | Issue : 3 | Page : 449-455

Quinacrine pretreatment reduces microwave-induced neuronal damage by stabilizing the cell membrane



Xue-feng Ding1, Yan Wu1, Wen-rui Qu2, Ming Fan Ph.D. 1 , Yong-qi Zhao Ph.D. 1

1 Department of Cognitive Sciences, Beijing Institute of Basic Medical Sciences, Beijing, China

2 Hand & Foot Surgery and Reparative & Reconstructive Surgery Center, Orthopedic Hospital of the Second Hospital of Jilin University, Changchun, Jilin Province, China



Date of Acceptance 15-Feb-2017 Date of Web Publication 4-Apr-2018

Correspondence Address:

Ming Fan

Department of Cognitive Sciences, Beijing Institute of Basic Medical Sciences, Beijing

China

Yong-qi Zhao

Department of Cognitive Sciences, Beijing Institute of Basic Medical Sciences, Beijing

China

Source of Support: This work was supported by the Integrated Drug Discovery Technology Platform of National Science and Technology Major Projects for “Major New Drugs Innovation and Development”, No. 2012ZX09J12201-005; the National Natural Science Foundation of China, No. 31071042, 31200822; and a grant of Beijing Natural Science Foundation, No. 5122033, Conflict of Interest: The authors declare that the study was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest Check

DOI: 10.4103/1673-5374.228727



Abstract



Quinacrine, widely used to treat parasitic diseases, binds to cell membranes. We previously found that quinacrine pretreatment reduced microwave radiation damage in rat hippocampal neurons, but the molecular mechanism remains poorly understood. Considering the thermal effects of microwave radiation and the protective effects of quinacrine on heat damage in cells, we hypothesized that quinacrine would prevent microwave radiation damage to cells in a mechanism associated with cell membrane stability. To test this, we used retinoic acid to induce PC12 cells to differentiate into neuron-like cells. We then pretreated the neurons with quinacrine (20 and 40 mM) and irradiated them with 50 mW/cm2 microwaves for 3 or 6 hours. Flow cytometry, atomic force microscopy and western blot assays revealed that irradiated cells pretreated with quinacrine showed markedly less apoptosis, necrosis, and membrane damage, and greater expression of heat shock protein 70, than cells exposed to microwave irradiation alone. These results suggest that quinacrine stabilizes the neuronal membrane structure by upregulating the expression of heat shock protein 70, thus reducing neuronal injury caused by microwave radiation.





Keywords: nerve regeneration; quinacrine; microwave; irradiation; heat shock; cell apoptosis; cell necrosis; thermal effect; cell membrane; heat shock protein 70; nerve cells; neural regeneration

How to cite this article:

Ding Xf, Wu Y, Qu Wr, Fan M, Zhao Yq. Quinacrine pretreatment reduces microwave-induced neuronal damage by stabilizing the cell membrane. Neural Regen Res 2018;13:449-55

How to cite this URL:

Ding Xf, Wu Y, Qu Wr, Fan M, Zhao Yq. Quinacrine pretreatment reduces microwave-induced neuronal damage by stabilizing the cell membrane. Neural Regen Res [serial online] 2018 [cited 2020 Sep 23];13:449-55. Available from: http://www.nrronline.org/text.asp?2018/13/3/449/228727

Xue-feng Ding, Yan Wu. These authors contributed equally to this paper.

Introduction

Materials and Methods

2

Results

Figure 1: PC12 cells were successfully induced into neuronal cells.

Before induction with retinoic acid, PC12 cells were cultured in Dulbecco’s modified Eagle’s medium containing 1% fetal bovine serum and 1% horse serum. After induction, the differentiated cells were characterized using immunohistochemistry with NSE (neuron) and NF (neuron) polyclonal antibody. (A) Morphology of PC12 cells before and after induction. Neurites (arrow) in the induced PC12 cells are observed. (B) Compared with the control, large numbers of neurites (arrows) were observed in NSE- and NF-immunoreactive cells. Scale bars: 50 μm. NSE: Neuron-specific enolase; NF: neurofilament.



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Figure 2: Quinacrine reduced microwave-induced neuronal cell apoptosis and necrosis.

The induced PC12 cells were exposed to microwaves (MW) with or without quinacrine pretreatment at low (20 mM; QA-L) or high (40 mM; QA-H) concentration for 3 and 6 hours separately. The treated cells were examined using flow cytometry. (A) Apoptosis and necrosis rates of control (3 h), MW group (3 h), MW + QA-L (3 h), and MW + QA-H (3 h). (B, C) Statistical analysis of apoptosis (B) and necrosis (C) in the three groups (3 h). (D) Apoptosis and necrosis rates of control (6 h), MW (6 h), MW + QA-L (6 h), and MW + QA-H (6 h) groups. (E, F) Statistical analysis of apoptosis and necrosis in the three groups (6 h). **P < 0.01 (mean ± SEM, n = 3, one-way analysis of variance analysis followed by Tukey’s post-hoc tests). Experiments were performed in triplicate. Con, Normal control; MW (3, 6 h), MW exposure for 3 and 6 h, respectively; MW + QA-H (3, 6 h): 40 mM quinacrine pretreatment followed by MW exposure for 3 or 6 h, respectively; MW + QA-L (3, 6 h), 20 mM quinacrine pretreatment followed by MW exposure for 3 or 6 h, respectively. MW: microwave; QA: quinacrine; QA-L: low-dose quinacrine; QA-H: high-dose quinacrine; h: hours.



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Figure 3: Quinacrine pretreatment reduced membrane injury caused by microwave exposure.

Atomic force microscopy images showing membrane surface ultrastructure of microwave-exposed PC12 cells. Darker areas represent more severe membrane damage. At 3 and 6 hours after microwave exposure, cells pretreated with high-concentration quinacrine showed markedly less membrane injury than non-pretreated cells. Relative injury intensity was analyzed using Image-Pro Plus software. The scale represents the height of the outermost surface: the lighter the color, the greater the height of the surface. MW (3, 6 h): microwave exposure for 3 or 6 hours, respectively; QA-H: 40 mM quinacrine pretreatment; QA-L: 20 mM quinacrine pretreatment.



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Figure 4: Quinacrine pretreatment reduced microwave-induced neuronal membrane injury by increasing HSP70 expression.

(A) Western blot assay of HSP70 expression level among different groups. (B) Statistical analysis of HSP70 expression level using Quantity One software. Cells pretreated with 40 mM quinacrine before 3-hour microwave exposure (QA-H+MW (3 h)) showed greater HSP70 expression than those not pretreated (MW (3 h)) or pretreated with 20 mM quinacrine (QA-L+MW (3 h)). In cells exposed to 6 hours of microwave irradiation, both 20 and 40 mM quinacrine pretreatment groups (QA-L+MW (6 h) and QA-H+MW (6 h)) showed greater HSP70 expression than those not pretreated (MW (6 h)). *P < 0.05, **P < 0.01 (mean ± SEM, n = 3, one-way analysis of variance analysis followed by Tukey’s post-hoc tests). Experiments were performed in triplicate. HSP70: Heat shock protein 70; h: hours.



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Discussion

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