Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Dec 26, 2020; 12(12): 1603-1622
Published online Dec 26, 2020. doi: 10.4252/wjsc.v12.i12.1603
6-gingerol protects nucleus pulposus-derived mesenchymal stem cells from oxidative injury by activating autophagy
Li-Ping Nan, Feng Wang, Yang Liu, Zhong Wu, Xin-Min Feng, Jun-Jian Liu, Liang Zhang
Li-Ping Nan, Department of Orthopedic, Tongji University School of Medicine, Shanghai Tenth People’s Hospital, Tenth People’s Hospital of Tongji University, Shanghai 200072, China
Feng Wang, Department of Spine Surgery, Tongji University School of Medicine, Shanghai East Hospital, Shanghai 200120, China
Yang Liu, Department of Orthopedic, West China Hospital of Sichuan University, Chengdu 610000, Sichuan Province, China
Zhong Wu, Jun-Jian Liu, Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China
Xin-Min Feng, Liang Zhang, Department of Orthopedics, Clinical Medical College of Yangzhou University, Yangzhou 225000, Jiangsu Province, China
Author contributions: All authors helped to perform the research; Nan LP and Wang F wrote the paper and contributed equally to this work; Liu Y, Wu Z, and Feng XM performed the procedures and data analysis and interpretation; Zhang L and Liu JJ are co-corresponding authors.
Supported by National Natural Science Foundation of China, No. 81972136; National Natural Science Foundation for Young Scholars of China, No. 81401830; Guangxi Natural Science Foundation General Project, No. 2018JJA14775; Young Medical Scholars Major Program of Jiangsu Province, No. QNRC2016342; Innovation Team Project of Jiangsu Province, No. CXTDB2017004; and Key Funding Project of Maternal and Child Health Research of Jiangsu Province, No. F201801.
Institutional review board statement: The study was reviewed and approved by the Research Ethics Committee of Clinical Medical College of Yangzhou University Institutional Review Board (No. SBYY2019-023).
Conflict-of-interest statement: The authors declare no conflicts of interest in regard to the present manuscript. The authors alone are responsible for the content and writing of the article.
Data sharing statement: The data used to support the findings of this study are available from the corresponding authors upon request.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Liang Zhang, MD, PhD, Academic Research, Assistant Professor, Associate Chief Physician, Chief Doctor, Research Scientist, Surgeon, Department of Orthopedics, Clinical Medical College of Yangzhou University, No. 98 Nantong West Road, Yangzhou 225001, Jiangsu Province, China. zhangliang6320@sina.com
Received: April 3, 2020
Peer-review started: April 3, 2020
First decision: August 9, 2020
Revised: August 30, 2020
Accepted: September 22, 2020
Article in press: September 22, 2020
Published online: December 26, 2020
Processing time: 267 Days and 17.1 Hours
Abstract
BACKGROUND

To date, there has been no effective treatment for intervertebral disc degeneration (IDD). Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) showed encouraging results in IDD treatment, but the overexpression of reactive oxygen species (ROS) impaired the endogenous repair abilities of NPMSCs. 6-gingerol (6-GIN) is an antioxidant and anti-inflammatory reagent that might protect NPMSCs from injury.

AIM

To investigate the effect of 6-GIN on NPMSCs under oxidative conditions and the potential mechanism.

METHODS

The cholecystokinin-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of 6-GIN. ROS levels were measured by 2´7´-dichlorofluorescin diacetate analysis. Matrix metalloproteinase (MMP) was detected by the tetraethylbenzimidazolylcarbocyanine iodide assay. TUNEL assay and Annexin V/PI double-staining were used to determine the apoptosis rate. Additionally, autophagy-related proteins (Beclin-1, LC-3, and p62), apoptosis-associated proteins (Bcl-2, Bax, and caspase-3), and PI3K/Akt signaling pathway-related proteins (PI3K and Akt) were evaluated by Western blot analysis. Autophagosomes were detected by transmission electron microscopy in NPMSCs. LC-3 was also detected by immunofluorescence. The mRNA expression of collagen II and aggrecan was evaluated by real-time polymerase chain reaction (RT-PCR), and the changes in collagen II and MMP-13 expression were verified through an immunofluorescence assay.

RESULTS

6-GIN exhibited protective effects against hydrogen peroxide-induced injury in NPMSCs, decreased hydrogen peroxide-induced intracellular ROS levels, and inhibited cell apoptosis. 6-GIN could increase Bcl-2 expression and decrease Bax and caspase-3 expression. The MMP, Annexin V-FITC/PI flow cytometry and TUNEL assay results further confirmed that 6-GIN treatment significantly inhibited NPMSC apoptosis induced by hydrogen peroxide. 6-GIN treatment promoted extracellular matrix (ECM) expression by reducing the oxidative stress injury-induced increase in MMP-13 expression. 6-GIN activated autophagy by increasing the expression of autophagy-related markers (Beclin-1 and LC-3) and decreasing the expression of p62. Autophagosomes were visualized by transmission electron microscopy. Pretreatment with 3-MA and BAF further confirmed that 6-GIN-mediated stimulation of autophagy did not reduce autophagosome turnover but increased autophagic flux. The PI3K/Akt pathway was also found to be activated by 6-GIN. 6-GIN inhibited NPMSC apoptosis and ECM degeneration, in which autophagy and the PI3K/Akt pathway were involved.

CONCLUSION

6-GIN efficiently decreases ROS levels, attenuates hydrogen peroxide-induced NPMSCs apoptosis, and protects the ECM from degeneration. 6-GIN is a promising candidate for treating IDD.

Keywords: Nucleus pulposus-derived mesenchymal stem cells; 6-Gingerol; Intervertebral disc degeneration; Oxidative stress; Autophagy; Apoptosis

Core Tip: To date, there has been no effective treatment for intervertebral disc degeneration (IDD). Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) showed encouraging results in IDD treatment, but the overexpression of reactive oxygen species (ROS) impaired the endogenous repair abilities of NPMSCs. 6-gingerol efficiently decreased ROS levels, attenuated hydrogen peroxide-induced NPMSC apoptosis, and protected the ECM from degeneration, representing a promising candidate for treating IDD.