Benzo[a]pyrene Induces Cell Death in MLO-Y4 Osteocytes via a ROS-Dependent Mitophagy Signaling Pathway
JIN Yuxin1#, ZHANG Tao2#, ZHENG Yuxi1, HONG Shuyu1, LIU Jiacheng1, XU Ningsheng1, NI Jiaxuan1, ZHANG Yun1*
This study aimed to investigate the effect of BaP (benzo[a]pyrene) on mitophagy in MLO-Y4 osteocytes and to explore its underlying molecular mechanism. MTT assay and colorimetric analysis were performed to evaluate the effects of different concentrations of BaP on cell viability and LDH (lactate dehydrogenase) release. Hoechst 33342/PI double staining was used to observe cell death. JC-1 staining was applied to detect mitochondrial membrane potential, and Mito-tracker staining was performed to observe mitochondrial morphological changes. The probes of DCFH-DA and MitoSOX were used to determine the levels of ROS (reactive oxygen species) and mtROS (mitochondrial ROS), respectively. Intracellular MDA (malondialdehyde) and ATP contents were quantified using a colorimetric assay and luciferase luminescence assay, respectively. Western blot was used to detect the protein expressions of Drp-1 (dynamin-related protein 1), TOM20 (translocase of outer mitochondrial membrane 20), MFN1 (mitofusin 1), LC3 (microtubule-associated protein 1 light chain 3), p62, PINK1 (PTEN induced putative kinase 1), E3 ubiquitin-protein ligase Parkin, Nrf2 (nuclear factor erythroid 2-related factor 2), HO-1 (heme oxygenase-1) and NQO1 [NAD(P)H quinone dehydrogenase 1). Immunofluorescence staining was conducted to visualize the colocalization and aggregation of LC3B with mitochondria. Results showed that compared with control group, cell viability, mitochondrial membrane potential, ATP content, and protein levels of TOM20, MFN1, p62, Nrf2, and HO-1 were significantly decreased in the BaP-treated group; whereas, LDH release, intracellular levels of ROS, mtROS and MDA, protein levels of Drp-1, PINK1, Parkin, and NQO1, as well as the LC3-II/LC3-I ratio were markedly increased; meanwhile, the colocalization and aggregation of LC3B with mitochondria were obviously enhanced. Compared with BaP (100 μmol/L) group, Baf A1 treatment remarkably promoted the accumulation of LC3-II and p62; 3-MA effectively restored cell viability and reduced cell death. In addition, Mdivi-1 (mitochondrial division inhibitor 1) and NAC (N-acetylcysteine) significantly downregulated the expression of PINK1 and Parkin, decreased the LC3-II/LC3-I ratio and upregulated p62 expression, as well as improving cell viability and suppressed cell death. Collectively, BaP induces cell death in MLO-Y4 cells via a ROS dependent mitophagy signaling pathway.
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