Melatonin alleviates oxidative stress-induced injury to nucleus pulposus-derived mesenchymal stem cells through activating PI3K/Akt pathway

Ze-Nan Huang; Ze-Yu Wang; Xiao-Fei Cheng; Zhao-Zhang Huang; Yan-Ling Han; Ya-Zhou Cui; Bo Liu; Wei Tian

doi:10.1016/j.jot.2023.10.002

Melatonin alleviates oxidative stress-induced injury to nucleus pulposus-derived mesenchymal stem cells through activating PI3K/Akt pathway

J Orthop Translat. 2023 Dec 2:43:66-84. doi: 10.1016/j.jot.2023.10.002. eCollection 2023 Nov.

Authors

Ze-Nan Huang^{1

2}, Ze-Yu Wang³, Xiao-Fei Cheng⁴, Zhao-Zhang Huang⁵, Yan-Ling Han⁶, Ya-Zhou Cui¹, Bo Liu², Wei Tian²

Affiliations

¹ Department of Orthopedics, Shandong First Medical University & Shandong Academy of Medical Science, Shandong, 200072, China.
² Department of Spine Surgery, Beijing Jishuitan Hospital, The Fourth Clinical Hospital Affiliated to Peking University, No. 37 Xinjiekou East, Road, Beijing, 100035, China.
³ Department of Orthopedics, The Affiliated BenQ Hospital of Nanjing Medical University, 210019, Nanjing, Jiangsu Province, China.
⁴ Department of Orthopedic Surgery, Shanghai Key Laboratory of Orthopedics Implants, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200011, China.
⁵ Taixing Medical Center, Taixing People's Hospital, Taixing, 225400, Jiangsu Province, China.
⁶ Medical Experimental Research Center, Yangzhou University, Yangzhou, 225001, China.

Abstract

Background: The changes in the microenvironment of degenerative intervertebral discs cause oxidative stress injury and excessive apoptosis of intervertebral disc endogenous stem cells. The purpose of this study was to explore the possible mechanism of the protective effect of melatonin on oxidative stress injury in NPMSCs induced by H₂O₂.

Methods: The Cell Counting Kit-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of melatonin. ROS content was detected by 2'7'-dichlorofluorescin diacetate (DCFH-DA). Mitochondrial membrane potential (MMP) was detected by the JC-1assay. Transferase mediated d-UTP Nick end labeling (TUNEL) and Annexin V/PI double staining were used to determine the apoptosis rate. Additionally, apoptosis-associated proteins and PI3K/Akt signaling pathway-related proteins were evaluated by immunofluorescence, immunoblotting and PCR. ECMs were evaluated by RT‒PCR and immunofluorescence. In vivo, X-ray, Magnetic resonance imaging (MRI) and Histological analyses were used to evaluate the protective effect of melatonin.

Results: Melatonin had an obvious protective effect on NPMSCs treated with 0-10 μM melatonin for 24 h. In addition, melatonin also had obvious protective effects on mitochondrial dysfunction, decreased membrane potential and cell senescence induced by H₂O₂. More importantly, melatonin could significantly reduce the apoptosis of nucleus pulposus mesenchymal stem cells induced by H₂O₂ by regulating the expression of apoptosis-related proteins and decreasing the rate of apoptosis. After treatment with melatonin, the PI3K/Akt pathway was significantly activated in nucleus pulposus mesenchymal stem cells, while the protective effect was significantly weakened after PI3K-IN-1 treatment. In vivo, the results of X-ray, MRI and histological analyses showed that therapy with melatonin could partially reduce the degree of intervertebral disc degeneration.

Conclusion: Our research demonstrated that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of nucleus pulposus mesenchymal stem cells induced by oxidative stress via the PI3K/Akt pathway, which provides a novel idea for the therapy of intervertebral disc degeneration.

The translational potential of this article: This study indicates that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of NPMSCs through activating the PI3K/Akt pathway. Melatonin might serve as a promising candidate for the prevention and treatment of Intervertebral disc degeneration disease (IVDD) in the future.

Keywords: Intervertebral disc degeneration; Melatonin; Nucleus pulposus-derived mesenchymal stem cells; Oxidative stress; PI3K/Akt pathway.