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

Background: Intervertebral disc degenerative disease(IVDD)is one of the main causes of low back pain, causing serious economic burden to the society. The changes of microenvironment in the degenerative intervertebral disc cause oxidative stress injury and excessive apoptosis of intervertebral disc endogenous stem cells, which weakens their ability to repair intervertebral disc. 0.01-1µM melatonin has been shown to reduce excessive apoptosis of nucleus pulposus mesenchymal stem cells (NPMSCs) caused by oxidative stress injury. The purpose of this study is to explore the possible mechanism of the protective effect of melatonin on oxidative stress injury of NPMSC induced by hydrogen peroxide(H 2 O 2 ). Methods The cell counting kit-8 (CCK-8) assay was used to evaluate the cytotoxicity of hydrogen peroxide and protective effects of melatonin. ROS content was detected by 2´7´-dichlorofluorescin diacetate (DCFH-DA). Mitochondrial membrane potential (MMP) was detected by the Tetraethylbenzimidazolylcarbocyanine iodide (JC-1) assay. A TUNEL cell apoptosis detection kit and Annexin V/PI double staining was conducted to determine apoptosis rate. Additionally, apoptosis-associated proteins and PI3K/Akt signaling pathway related proteins were evaluated by immunofluorescence, immunoblotting and PCR. (ECM) were evaluated by RT-PCR and immunofluorescence. In vivo, X-ray, MRI and histological analysis were used to evaluate the protective effect of melatonin in vivo. Results melatonin had obvious protective effect on NPMSCs treated with 0–10 µM for 24 hours. In addition, melatonin also had obvious protective effects on mitochondrial dysfunction, decrease of membrane potential and cell senescence induced by hydrogen peroxide. More importantly, melatonin could significantly reduce the apoptosis of nucleus pulposus mesenchymal stem cells induced by hydrogen peroxide, such as 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 analysis 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 PI3K/Akt pathway, which provides a novel idea for the therapy of intervertebral disc degeneration.