Objectives Inflammatory cells exhibit elevated level of protoporphyrin IX (PpIX) after the administration of 5-aminolevulinic acid (ALA). This study aims to investigate the sonodynamic effects of ALA derived PpIX (ALA-PpIX) on macrophages, which are the pivotal inflammatory cells in atherosclerosis.
Methods THP-1 derived macrophages were incubated with diffferent concentrations of ALA (0-10.0 mM), and then exposed to the ultrasound for 0-15 minutes. Fluorescence microscope and fluorescence spectrometer were performed to detect intracellular PpIX concentration. MTT assay was used for cell viability measurment. Hoechst 33342 and propidium iodide assay and flow cytometry analysis were performed to measure cell apoptosis and necrosis. Intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) loss were assessed using fluorescence prober DCFH-DA and jc-1, respectively.
Results Intracellular PpIX increased with the concentration of ALA in the incubation solution in a time dependent manner, and the most intracellular PpIX was observed at 3 hours incubation. Lower concentrations (less than 2 mM) of ALA has no influence on cell viability (more than 90% of cells survived), but sonodynamic therapy (SDT) with low concentration ALA significantly decreased the survival rate of cells, and the effect increased with both ALA concentration and ultrasound exposure time. ALA-SDT induced both cell apoptosis and necrosis, and the maximum apoptosis/necrosis ratio was observed at 6 hours after SDT with 1 mM of ALA and 5 minutes of ultrasound exposure. Flow cytometry analysis showed that ALA-SDT significantly increased late stage apoptotic cells (about 10 folds of control). Furthermore, ALA-SDT induced reactive oxygen species (ROS) generation in THP-1 macrophages immediately after the treatment and a conspicuous loss of mitochondrial membrane potential (MMP) at 6 hours, compared with that of control, ALA alone and ultrasound alone groups.
Conclusions ALA-SDT exhibited synergistic apoptotic effects on THP-1 macrophages, involving excessive intracellular ROS generation and MMP loss. Therefore, ALA-SDT can be a potential treatment for atherosclerosis.