Mechanisms of in Vitro Chondrogenic Differentiation of Human BMSCs Induced by Piperlongumine

Abstract

Background: Piperlongumine (PL) regulates the production of cellular reactive oxygen species (ROS), and ROS can affect the differentiation of mesenchymal stem cells. In order to add to the medicinal value of PL, this study aimed to explore whether and how PL induces chondrogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs). Methods: To determine the PL concentrations for drug intervention, BMSCs were exposed to varying concentrations of PL, the effects on cell viability were measured by Cell Counting Kit (CCK)-8. After the IC₅₀ (intervention concentration) of PL was determined, the BSMCs were analyzed for changes in morphology (microscopy), degree of chondrogenic differentiation (Alcian blue staining), oxidative stress levels (reactive oxygen species (ROS) assay) and mRNA and protein expressions (quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot) at varying times of treatment. To prove the reliability of the results, the effects of PD98059, an extracellular-signal-regulated kinase (ERK)1/2 inhibitor, and N-acetylcysteine (NAC), an inhibitor of ROS production were also studied. Results: The IC₅₀ of PL was <50%, when PL concentration was greater than 5 μmol/L (p < 0.001) and 5 μmol/L PL used in subsequent experiments. PL promoted the chondrogenic differentiation of BMSCs and increased glycosaminoglycan deposition and ROS production (p < 0.001). At the molecular level, PL increased the expressions of chondrogenic differentiation-related genes and activated the phosphorylation of ERK1/2 (p < 0.001). These effects of PL were partially prevented by NAC (p < 0.01). PD98059 exerted similar effects as NAC (p < 0.001) but did not affect ROS production. Conclusions: ROS and ERK1/2 pathway may be part of the mechanism by which PL enhances chondrogenic differentiation of BMSCs.