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Apelin-13 Protects Neurons by Attenuating Early-Stage Postspinal Cord Injury Apoptosis In Vitro

Apelin is a 77-amino-acid peptide that is an endogenous ligand for the G protein-coupled receptor APJ (Apelin receptor, APJ). Apelin-13, as the most bioactive affinity fragment of apelin, plays a role in energy metabolism, myocardial ischemia-reperfusion injury, and the regulation of the inflammator...

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Autores principales: Lin, Taotao, Zhao, Yujie, Guo, Shengyu, Wu, Zhengru, Li, Wenwen, Wu, Rongcan, Wang, Zhenyu, Liu, Wenge
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9688050/
https://www.ncbi.nlm.nih.gov/pubmed/36358441
http://dx.doi.org/10.3390/brainsci12111515
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author Lin, Taotao
Zhao, Yujie
Guo, Shengyu
Wu, Zhengru
Li, Wenwen
Wu, Rongcan
Wang, Zhenyu
Liu, Wenge
author_facet Lin, Taotao
Zhao, Yujie
Guo, Shengyu
Wu, Zhengru
Li, Wenwen
Wu, Rongcan
Wang, Zhenyu
Liu, Wenge
author_sort Lin, Taotao
collection PubMed
description Apelin is a 77-amino-acid peptide that is an endogenous ligand for the G protein-coupled receptor APJ (Apelin receptor, APJ). Apelin-13, as the most bioactive affinity fragment of apelin, plays a role in energy metabolism, myocardial ischemia-reperfusion injury, and the regulation of the inflammatory response during oxidative stress, but its role in spinal cord injury is still unclear. This research identified and verified the differential expression of apelin in rat spinal cord injured tissues and normal spinal cord tissues by transcriptome sequencing in vivo and proved that apelin-13 protects neurons by strengthening autophagy and attenuating early-stage postspinal cord injury apoptosis in vitro. After constructing the model concerning a rat spinal cord hemisection damage, transcriptome sequencing was performed on the injured and normal spinal cord tissues of rats, which identified the differentially expressed gene apelin, with qRT-PCR detecting the representative level of apelin. The oxygen-glucose deprivation (OGD) model of PC12 cells was constructed in vitro to simulate spinal cord injury. The OGD injury times were 2 h, 4 h, 6 h, 8 h, and 12 h, and the non-OGD injury group was used as the control. The expression of apelin at each time point was observed by Western blotting. The expression of apelin was the lowest in the 6 h OGD injury group (p < 0.05). Therefore, the OGD injury time of 6 h was used in subsequent experiments. The noncytotoxic drug concentration of apelin-13 was determined with a Cell Counting Kit-8 (CCK-8) assay. An appropriate dose of apelin-13 (1 μM) significantly improved cell survival (p < 0.05). Thus, subsequent experiments selected a concentration of 1 μM apelin-13 as it significantly increased cell viability. Finally, we divided the experimental groups into four groups according to whether they received drugs (1 μM apelin-13, 24 h) or OGD (6 h): (1) control group: without apelin-13 or OGD injury; (2) apelin-13 group: with apelin-13 but no OGD injury; (3) OGD group: with OGD injury but without apelin-13; and (4) OGD + apelin-13 group: with apelin-13 and OGD injury. The TUNEL assay and flow cytometry results showed that compared with the OGD group, apoptosis in the OGD+Apelin-13 group was significantly reduced (p < 0.001). Determination of cell viability under different conditions by CCK-8 assay results displays that Apelin-13 can significantly improve the cell viability percentage under OGD conditions (p < 0.001). Western blotting results showed that apelin-13 decreased the expression ratios of apoptosis-related proteins Bax/Bcl-2 and cleaved-caspase3/caspase3 (p < 0.05), increasing the key to Beclin1-dependent autophagy pathway expression of the protein Beclin1. This finding indicates that apelin-13 protects neurons by strengthening autophagy and attenuating early-stage postspinal cord injury apoptosis in vitro.
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spelling pubmed-96880502022-11-25 Apelin-13 Protects Neurons by Attenuating Early-Stage Postspinal Cord Injury Apoptosis In Vitro Lin, Taotao Zhao, Yujie Guo, Shengyu Wu, Zhengru Li, Wenwen Wu, Rongcan Wang, Zhenyu Liu, Wenge Brain Sci Article Apelin is a 77-amino-acid peptide that is an endogenous ligand for the G protein-coupled receptor APJ (Apelin receptor, APJ). Apelin-13, as the most bioactive affinity fragment of apelin, plays a role in energy metabolism, myocardial ischemia-reperfusion injury, and the regulation of the inflammatory response during oxidative stress, but its role in spinal cord injury is still unclear. This research identified and verified the differential expression of apelin in rat spinal cord injured tissues and normal spinal cord tissues by transcriptome sequencing in vivo and proved that apelin-13 protects neurons by strengthening autophagy and attenuating early-stage postspinal cord injury apoptosis in vitro. After constructing the model concerning a rat spinal cord hemisection damage, transcriptome sequencing was performed on the injured and normal spinal cord tissues of rats, which identified the differentially expressed gene apelin, with qRT-PCR detecting the representative level of apelin. The oxygen-glucose deprivation (OGD) model of PC12 cells was constructed in vitro to simulate spinal cord injury. The OGD injury times were 2 h, 4 h, 6 h, 8 h, and 12 h, and the non-OGD injury group was used as the control. The expression of apelin at each time point was observed by Western blotting. The expression of apelin was the lowest in the 6 h OGD injury group (p < 0.05). Therefore, the OGD injury time of 6 h was used in subsequent experiments. The noncytotoxic drug concentration of apelin-13 was determined with a Cell Counting Kit-8 (CCK-8) assay. An appropriate dose of apelin-13 (1 μM) significantly improved cell survival (p < 0.05). Thus, subsequent experiments selected a concentration of 1 μM apelin-13 as it significantly increased cell viability. Finally, we divided the experimental groups into four groups according to whether they received drugs (1 μM apelin-13, 24 h) or OGD (6 h): (1) control group: without apelin-13 or OGD injury; (2) apelin-13 group: with apelin-13 but no OGD injury; (3) OGD group: with OGD injury but without apelin-13; and (4) OGD + apelin-13 group: with apelin-13 and OGD injury. The TUNEL assay and flow cytometry results showed that compared with the OGD group, apoptosis in the OGD+Apelin-13 group was significantly reduced (p < 0.001). Determination of cell viability under different conditions by CCK-8 assay results displays that Apelin-13 can significantly improve the cell viability percentage under OGD conditions (p < 0.001). Western blotting results showed that apelin-13 decreased the expression ratios of apoptosis-related proteins Bax/Bcl-2 and cleaved-caspase3/caspase3 (p < 0.05), increasing the key to Beclin1-dependent autophagy pathway expression of the protein Beclin1. This finding indicates that apelin-13 protects neurons by strengthening autophagy and attenuating early-stage postspinal cord injury apoptosis in vitro. MDPI 2022-11-08 /pmc/articles/PMC9688050/ /pubmed/36358441 http://dx.doi.org/10.3390/brainsci12111515 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lin, Taotao
Zhao, Yujie
Guo, Shengyu
Wu, Zhengru
Li, Wenwen
Wu, Rongcan
Wang, Zhenyu
Liu, Wenge
Apelin-13 Protects Neurons by Attenuating Early-Stage Postspinal Cord Injury Apoptosis In Vitro
title Apelin-13 Protects Neurons by Attenuating Early-Stage Postspinal Cord Injury Apoptosis In Vitro
title_full Apelin-13 Protects Neurons by Attenuating Early-Stage Postspinal Cord Injury Apoptosis In Vitro
title_fullStr Apelin-13 Protects Neurons by Attenuating Early-Stage Postspinal Cord Injury Apoptosis In Vitro
title_full_unstemmed Apelin-13 Protects Neurons by Attenuating Early-Stage Postspinal Cord Injury Apoptosis In Vitro
title_short Apelin-13 Protects Neurons by Attenuating Early-Stage Postspinal Cord Injury Apoptosis In Vitro
title_sort apelin-13 protects neurons by attenuating early-stage postspinal cord injury apoptosis in vitro
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9688050/
https://www.ncbi.nlm.nih.gov/pubmed/36358441
http://dx.doi.org/10.3390/brainsci12111515
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