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The protective effect of nitronyl nitroxide radical on peroxidation of A549 cell damaged by iron overload
Journal Pre-proof The protective effect of nitronyl nitroxide radical on peroxidation of A549 cell damaged by iron overload
Shao-Min He, Yu-Hua Lei, Jian-Min Wang, Li-Na Geng, ShuPing Wang, Juan Zhao, Yi-Fang Hou PII:
S0928-4931(18)32146-5
DOI:
https://doi.org/10.1016/j.msec.2019.110189
Reference:
MSC 110189
To appear in:
Materials Science & Engineering C
Received date:
22 July 2018
Revised date:
9 September 2019
Accepted date:
10 September 2019
Please cite this article as: S.-M. He, Y.-H. Lei, J.-M. Wang, et al., The protective effect of nitronyl nitroxide radical on peroxidation of A549 cell damaged by iron overload, Materials Science & Engineering C (2019), https://doi.org/10.1016/j.msec.2019.110189
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof The Protective Effect of Nitronyl Nitroxide Radical on Peroxidation of A549 Cell Damaged by Iron Overload Shao-Min He1#, Yu-Hua Lei2#, Jian-Min Wang1, Li-Na Geng1*, Shu-Ping Wang1*, Juan Zhao2, Yi-Fang Hou1 1
College of Chemistry and Material Science, Hebei Normal University, Shijiazhuang 050024, China 2 College of Basic Medicine, Hebei Medical University, Shijiazhuang 050017, China # These authors contributed equally to this study. *Corresponding authors. Email: [email protected]; [email protected]
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Abstract: Particulate pollution in the air has strong links with increased morbidity of cardiopulmonary diseases. Iron is one of the major carcinogens in air pollution and can produce hydroxyl radical which induce oxidative stress, lead to cell damage and even to cancer. In this work, a novel nitronyl nitroxide radical NITPh(OMe)2 (2-(2,4-dimethoxyphenyl) -4,4,5,5tetramethylimidazoline- 1- oxyl-3- oxide) was prepared and characterized by electron spin-resonance spectroscopy (ESR), X-ray crystal diffraction, Fourier transform infrared (IR), X-ray powder diffraction (XRD), elemental analysis, ultraviolet and visible spectra (UV-Vis), and the electronic transition processes was also calculated by time-dependent density functional theory (TDDFT) to analysis UV-Vis spectrum. In vitro cell model of oxidative damage was established by ferric ammonium citrate (FAC) overload, and NITPh(OMe)2 was studied as a free radical scavenger to protect peroxidation of A549 cells. Results showed that NITPh(OMe)2 could significantly alleviate the damage of A549 cells by iron overload in cell morphology, cell viability, cell proliferation and cell apoptosis. The apoptotic signaling pathway of A549 cells induced by FAC and the protection mechanism of NITPh(OMe)2 were all discussed through the expression of three relating proteins, Bcl-2, Bax and DDIT3. This work confirms that nitroxide radicals are effective antioxidants, and have potential application in clinical practice as therapeutic agents.
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To further investigate the correlation of the UV-Vis spectrum and electronic transition processes theoretically, the time-dependent density functional theory (TDDFT) calculation was performed with Gaussian 09 program. The calculation was carried out based on the X-ray crystal structure of NITPh(OMe)2, and the excited states was calculated at the B3LYP/6-31G* level. Solvent effect (dichloromethane) was considered in TDDFT calculation, where the PCM model was implemented using Gaussian 09.
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Figure 2 Cell viability and morphology treated with NITPh(OMe)2 or/and FAC for 24 h. MTT assay of A549 cells treated with (A) NITPh(OMe)2; (B) FAC and (C) NITPh(OMe)2 + FAC for 24 h. Data = mean ± SD, n=8. *P<0.05 and **P<0.01 versus control, #P<0.05 and ##P<0.01 versus FAC. (D) Photographs of bright field (200×). Scale bar = 50 μm. (E) Photographs of fluorescence field (200×). Scale bar = 50 μm.
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Scheme 3 The single electron spin density distribution (A) transformation of the REDOX form (B) of NITPh(OMe)2
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Figure 3 The effects of NITPh(OMe)2 on the cell cycle distribution. (A) Cells were incubated with NITPh(OMe)2 or/and FAC for 24 h, then determined the distribution of cells by FCM. (B) Percentage of cells at different stage. Data = mean ± SD, n = 3.
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Figure 4 The detection of A549 cells’ apoptosis rate by FCM. (A) A549 cells were incubated with NITPh-2, 4-(OMe)2 or/and FAC for 24 h, stained, and analyzed by FCM. (B) The rate of different
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phase of apoptosis in A549 cells, induced by NITPh-2,4- (OMe)2 or/and FAC. Data = mean ± SD, n = 3. *P<0.05 versus control, #P<0.05 versus FAC.
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analyzed MMP by FCM ,
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Figure 6 Proteins expression in A549 cells. (A, C) The protein expression tested by Western blot, A549 cells incubated with NITPh(OMe)2 or/and FAC for 24 h. (B, D) Columns show the data compared with equal loading control. Data = mean ± SD, n=3. *P<0.05 versus control, #P<0.05 versus FAC.
Reference
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Graphical Abstract
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a novel nitronyl nitroxide radical NITPh(OMe)2 (2-(2,4-dimethoxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3oxide) was prepared and characterized by electron spin-resonance spectroscopy (ESR), X-ray crystal diffraction, Fourier transform infrared (IR), X-ray powder diffraction (XRD), elemental analysis, ultraviolet and visible spectra (UV-Vis). Its protective effect on peroxidation of A549 cell damaged by iron overload was also studied. Results showed that NITPh(OMe)2 could significantly alleviate the damage of A549 cells in cell morphology, cell viability, cell proliferation and cell apoptosis. The apoptotic signaling pathway of A549 cells and the protection mechanism of NITPh(OMe)2 were all related with the three proteins, Bcl-2, Bax and DDIT3.
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Highlights
A novel nitronyl nitroxide radical NITPh(OMe)2 was prepared and characterized.
NITPh(OMe)2 could significantly alleviate the damage of A549 cells by iron overload.
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The apoptotic signaling pathway and the protection mechanism of were studied.
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Shao-Min He, Yu-Hua Lei, Jian-Min Wang, Li-Na Geng, ShuPing Wang, Juan Zhao, Yi-Fang Hou PII:
S0928-4931(18)32146-5
DOI:
https://doi.org/10.1016/j.msec.2019.110189
Reference:
MSC 110189
To appear in:
Materials Science & Engineering C
Received date:
22 July 2018
Revised date:
9 September 2019
Accepted date:
10 September 2019
Please cite this article as: S.-M. He, Y.-H. Lei, J.-M. Wang, et al., The protective effect of nitronyl nitroxide radical on peroxidation of A549 cell damaged by iron overload, Materials Science & Engineering C (2019), https://doi.org/10.1016/j.msec.2019.110189
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof The Protective Effect of Nitronyl Nitroxide Radical on Peroxidation of A549 Cell Damaged by Iron Overload Shao-Min He1#, Yu-Hua Lei2#, Jian-Min Wang1, Li-Na Geng1*, Shu-Ping Wang1*, Juan Zhao2, Yi-Fang Hou1 1
College of Chemistry and Material Science, Hebei Normal University, Shijiazhuang 050024, China 2 College of Basic Medicine, Hebei Medical University, Shijiazhuang 050017, China # These authors contributed equally to this study. *Corresponding authors. Email: [email protected]; [email protected]
Jo ur
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lP
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-p
ro
of
Abstract: Particulate pollution in the air has strong links with increased morbidity of cardiopulmonary diseases. Iron is one of the major carcinogens in air pollution and can produce hydroxyl radical which induce oxidative stress, lead to cell damage and even to cancer. In this work, a novel nitronyl nitroxide radical NITPh(OMe)2 (2-(2,4-dimethoxyphenyl) -4,4,5,5tetramethylimidazoline- 1- oxyl-3- oxide) was prepared and characterized by electron spin-resonance spectroscopy (ESR), X-ray crystal diffraction, Fourier transform infrared (IR), X-ray powder diffraction (XRD), elemental analysis, ultraviolet and visible spectra (UV-Vis), and the electronic transition processes was also calculated by time-dependent density functional theory (TDDFT) to analysis UV-Vis spectrum. In vitro cell model of oxidative damage was established by ferric ammonium citrate (FAC) overload, and NITPh(OMe)2 was studied as a free radical scavenger to protect peroxidation of A549 cells. Results showed that NITPh(OMe)2 could significantly alleviate the damage of A549 cells by iron overload in cell morphology, cell viability, cell proliferation and cell apoptosis. The apoptotic signaling pathway of A549 cells induced by FAC and the protection mechanism of NITPh(OMe)2 were all discussed through the expression of three relating proteins, Bcl-2, Bax and DDIT3. This work confirms that nitroxide radicals are effective antioxidants, and have potential application in clinical practice as therapeutic agents.
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，
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biological activities, including antinociceptive effect, antitumor activity,
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anti-inflammatory,
ON+ N
O
.
R N O
TEMTO
.
NITR
neuroprotective
effect,
Journal Pre-proof CH3 O-
O
N+
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O
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To further investigate the correlation of the UV-Vis spectrum and electronic transition processes theoretically, the time-dependent density functional theory (TDDFT) calculation was performed with Gaussian 09 program. The calculation was carried out based on the X-ray crystal structure of NITPh(OMe)2, and the excited states was calculated at the B3LYP/6-31G* level. Solvent effect (dichloromethane) was considered in TDDFT calculation, where the PCM model was implemented using Gaussian 09.
λ
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Figure 2 Cell viability and morphology treated with NITPh(OMe)2 or/and FAC for 24 h. MTT assay of A549 cells treated with (A) NITPh(OMe)2; (B) FAC and (C) NITPh(OMe)2 + FAC for 24 h. Data = mean ± SD, n=8. *P<0.05 and **P<0.01 versus control, #P<0.05 and ##P<0.01 versus FAC. (D) Photographs of bright field (200×). Scale bar = 50 μm. (E) Photographs of fluorescence field (200×). Scale bar = 50 μm.
、 SOD (U/mg·pro)
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Scheme 3 The single electron spin density distribution (A) transformation of the REDOX form (B) of NITPh(OMe)2
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Figure 3 The effects of NITPh(OMe)2 on the cell cycle distribution. (A) Cells were incubated with NITPh(OMe)2 or/and FAC for 24 h, then determined the distribution of cells by FCM. (B) Percentage of cells at different stage. Data = mean ± SD, n = 3.
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Figure 4 The detection of A549 cells’ apoptosis rate by FCM. (A) A549 cells were incubated with NITPh-2, 4-(OMe)2 or/and FAC for 24 h, stained, and analyzed by FCM. (B) The rate of different
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phase of apoptosis in A549 cells, induced by NITPh-2,4- (OMe)2 or/and FAC. Data = mean ± SD, n = 3. *P<0.05 versus control, #P<0.05 versus FAC.
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analyzed MMP by FCM ,
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Figure 6 Proteins expression in A549 cells. (A, C) The protein expression tested by Western blot, A549 cells incubated with NITPh(OMe)2 or/and FAC for 24 h. (B, D) Columns show the data compared with equal loading control. Data = mean ± SD, n=3. *P<0.05 versus control, #P<0.05 versus FAC.
Reference
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Jo ur
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Graphical Abstract
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a novel nitronyl nitroxide radical NITPh(OMe)2 (2-(2,4-dimethoxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3oxide) was prepared and characterized by electron spin-resonance spectroscopy (ESR), X-ray crystal diffraction, Fourier transform infrared (IR), X-ray powder diffraction (XRD), elemental analysis, ultraviolet and visible spectra (UV-Vis). Its protective effect on peroxidation of A549 cell damaged by iron overload was also studied. Results showed that NITPh(OMe)2 could significantly alleviate the damage of A549 cells in cell morphology, cell viability, cell proliferation and cell apoptosis. The apoptotic signaling pathway of A549 cells and the protection mechanism of NITPh(OMe)2 were all related with the three proteins, Bcl-2, Bax and DDIT3.
Journal Pre-proof
Highlights
A novel nitronyl nitroxide radical NITPh(OMe)2 was prepared and characterized.
NITPh(OMe)2 could significantly alleviate the damage of A549 cells by iron overload.
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-p
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The apoptotic signaling pathway and the protection mechanism of were studied.
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