Abstract
Puerarin (PUR), an 8-C-glucoside of daidzein extracted from Pueraria plants, is closely related to autophagy, reduced reactive oxygen species (ROS) production, and anti-inflammatory effects, but its effects on human nucleus pulposus mesenchymal stem cells (NPMSCs) have not yet been identified. In this study, NPMSCs were cultured in a compression apparatus to simulate the microenvironment of the intervertebral disc under controlled pressure (1.0 MPa), and we found that cell viability was decreased and apoptosis level was gradually increased as compression duration was prolonged. After PUR administration, apoptosis level evaluated by flow cytometry and caspase-3 activity was remitted, and protein levels of Bas as well as cleaved caspase-3 were decreased, while elevated Bcl-2 level was identified. Moreover, ATP production detection, ROS, and JC-1 fluorography as well as quantitative analysis suggested that PUR could attenuate intercellular ROS accumulation and mitochondrial dysfunction. Besides, the rat tail compression model was utilized, which indicated that PUR could restore impaired nucleus pulposus degeneration induced by compression. The PI3K/Akt pathway was identified to be deactivated after compression stimulation by western blot, and PUR could rescue the phosphorylation of Akt, thus reactivating the pathway. The effects of PUR, such as antiapoptosis, cell viability restoration, antioxidation, and mitochondrial maintenance, were all counteracted by application of the PI3K/Akt pathway inhibitor (LY294002). Summarily, PUR could alleviate compression-induced apoptosis and cell death of human NPMSCs in vitro as well as on the rat compression model and maintain intracellular homeostasis by stabilizing mitochondrial membrane potential and attenuating ROS accumulation through activating the PI3K/Akt pathway.
Highlights
Intervertebral disc degeneration (IDD) is one of the most common pathological disorders around the world, which greatly affects the life quality of patients and imposes enormous financial burden on society [1]
Cells were digested using 0.25% trypsin-0.02% ethylenediaminetetraacetic acid (EDTA, Sigma) when they reached a confluence of 80–90% and were subcultured in culture flasks at a ratio of 1 : 3. Second generation of Nucleus pulposus mesenchymal stem cells (NPMSCs) was used throughout the following experiments
We successfully isolated NPMSCs, and the cells in passage 2 were used in the present study
Summary
Intervertebral disc degeneration (IDD) is one of the most common pathological disorders around the world, which greatly affects the life quality of patients and imposes enormous financial burden on society [1]. As for its multidirection differentiation ability [6, 7] and tissue specificity, NPMSCs are potentially superior to nonintervertebral disc- (IVD-) derived MSCs for NPC-specific differentiation and might be the potential therapeutic target for IDD. PUR is greatly associated with modification of autophagy [22], decreased reactive oxygen species (ROS) production [23], and anti-inflammation effects [23]. PUR could suppress apoptosis and reduce myocardial injury induced by ischemia [24], while promoting apoptosis in cancer cells [25, 26]. The specific effects of PUR on the fate of NPMSCs in the development of IDD need to be further clarified
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