Pyrroloquinoline quinone (PQQ) protects mitochondrial function of HEI-OC1 cells under premature senescence

Abstract

The aim of this study was to investigate the effects of pyrroloquinoline quinone (PQQ), an oxidoreductase cofactor, on the H₂O₂-induced premature senescence model in HEI-OC1 auditory cells and to elucidate its mechanism of action in vitro. Cells were treated with PQQ for 1 day before H₂O₂ (100 μM) exposure. Mitochondrial respiratory capacity was damaged in this premature senescence model but was restored in cells pretreated with PQQ (0.1 nM or 1.0 nM). A decrease in mitochondrial potential, the promotion of mitochondrial fusion and the accelerated movement of mitochondria were all observed in PQQ-pretreated cells. The protein expression of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) were significantly decreased under H₂O₂ exposure while they were increased with PQQ pretreatment, and PGC-1α acetylation was significantly decreased. In conclusion, PQQ has a protective effect on the premature senescence model of HEI-OC1 auditory cells and is associated with the SIRT1/PGC-1α signaling pathway, mitochondrial structure, and mitochondrial respiratory capacity.

Fig. 1. Effects of PQQ on cell proliferation, cell metabolic activity, and the mitochondrial membrane potential in HEI-OC1 cells evaluated using multiple assays.

A Cell proliferation rate analyzed by cell counting after PQQ pretreatment for 1 day. B Cell metabolic activity measured with the WST-8 formazan assay after PQQ pretreatment for 1 day, shown in optical density (O.D.). The higher value indicates higher metabolic activity. C Mitochondrial membrane potential (MMP) measured with JC-1 fluorescence staining after PQQ pretreatment for 1 day. The ratio of red signal (relative fluorescence units (RFU) at 590 nm; mitochondrial polarized cells) to green signal (RFU at 535 nm; mitochondrial depolarized cells) is shown (n = 5 per group). Box plot shows statistical parameters as follows; central line: median; box limits: first and third quartile; whiskers: minimum and maximum. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 2. Cell proliferation, cell metabolic activity, senescence markers, and mitochondrial membrane potential (MMP) in HEI-OC1 cells with premature cellular senescence induced by H₂O₂ exposure with and without PQQ pretreatment.

The effects of PQQ under H₂O₂ exposure are evaluated using multiple assays in the same manner as shown in Fig. 1. A Cell count 1 day after H₂O₂ exposure. B Cell viability 1 day after H₂O₂ exposure. C Cell proliferation rate using total cell count for 3 days after H₂O₂ exposed. D Cell proliferation rate using live-cell count for 3 days after H₂O₂ exposure. E Cell metabolic activity measured with WST-8 formazan assay. F Relative mRNA expressions of Cdkn1a (p21), Cdkn2a (p16), and Trp53 (p53) using β-actin as an internal control. RQ relative quantification. G The changes of senescence-associated β-galactosidase (SA-β-Gal) using SPiDER-βGal staining in flow cytometry analysis. H The changes of dipeptidyl peptidase DPP4 (CD26) cell surface protein marker in flow cytometry analysis. I The increased ratio in positivity rate over control of SPiDER-βGal positive cells. J The increased ratio in positivity rate over control of CD26 positive cells. K Imaging of mitochondria stained with JC-1. Green signal indicates low MMP and red signal indicates high MMP. Scale bar, 10 µm. L MMP measured with JC-1. (A–D, L: n = 6 per group, E, I, J: n = 5 per group, F: n = 3 per group). Box plot shows statistical parameters as follows; central line: median; box limits: first and third quartile; whiskers: minimum and maximum. RQ data are shown as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3. Mitochondrial ultrastructure in HEI-OC1 cells exposed to H₂O₂ (100 µM) with and without PQQ pretreatments (0.1 nM and 1.0 nM), and control cells.

A Short exposure to H₂O₂ induces fine structural damage to mitochondria, which is alleviated with PQQ pretreatment. From left to right: control cells, cells exposed to H₂O₂, cells exposed to H₂O₂ after 0.1 nM PQQ pretreatment, and cells exposed to H₂O₂ after 1.0 nM PQQ pretreatment. AP autophagosome, DM damaged mitochondria, E endosome, ER endoplasmic reticulum, GL Golgi body, L lysosome, M mitochondria, N nucleus. Scale bar, 500 nm in top row, 200 nm bottom row. B The numerical analysis of TEM images. The endosome ratio, damaged mitochondria number, autophagosome number, and lysosome number were calculated by visual inspection (n = 5 per group). Box plot shows statistical parameters as follows; central line: median; box limits: first and third quartile; whiskers: minimum and maximum. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4. Mitochondrial biogenesis in the premature cellular senescence model with and without PQQ treatment.

The effects of PQQ pretreatment in HEI-OC1 cells exposed to H₂O₂ were evaluated using a XF24 flux analyzer. A Oxygen consumption rate (OCR) diagram of the XF assay, normalized by the number of cells. B Extracellular acidification rate (ECAR) diagram of the XF assay. C ATP production rate quantified with OCR measured following the injection of oligomycin, maximal respiration rates measured following injection of FCCP, and cell energy phenotype profile of the XF assay. D Basal ATP production rate and ATP rate index of the XF assay (n = 5 per group). Data are shown as mean ± standard deviation. *P < 0.05.

Fig. 5. Substrate-specific mitochondrial biogenesis in the premature cellular senescence model with and without PQQ treatment.

The substrate-specific effects of PQQ pretreatment in HEI-OC1 cells exposed to H₂O₂ were evaluated using a XF24 flux analyzer; A Oxygen consumption rate (OCR) under palmitic acid supplementation. B OCR under Cpt (Carnitine palmitoyltransferase) inhibition by Etomoxir. C OCR under glutamine supplementation. D OCR under Gls (Glutaminase) inhibition by BPTES ((bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide). E Relative mRNA expressions of Cpt1a, Cpt1c, Cpt2, Gls1, and Gls2 using β-actin as an internal control. F OCR under glutamine supplementation in HEI-OC1 cells in the control group or H₂O₂-exposed group (n = 5 per group). Data are shown as mean ± standard deviation. *P < 0.05.

Fig. 6. The morphological evaluation of mitochondria in the premature cellular senescence model under PQQ pretreatment.

The morphological changes with PQQ pretreatment in HEI-OC1 cells exposed to H₂O₂ to induce premature cellular senescence were evaluated using microscopic imaging. A Mitochondrial images stained with MitoTracker Orange CMTMRos. Scale bar, 10 µm. B The image processing flow of the skeletonized image analysis. C The different types of dynamic mitochondrial regulations including fusion and fission mechanisms involving network nodes (tip-to-tip, tip-to-side, or side-to-side). Graph representation of the mitochondrial reticulum using four node types: 1 (magenta), 2 (green), 3 (dark blue), and 4 (blue). D The descriptions of parameters in the mitochondrial network morphology. E The number of branches, junctions, and the average length of branches in each image (E: n = 5 per group; repeated experiments). Box plot shows statistical parameters as follows; central line: median; box limits: first and third quartile; whiskers: minimum and maximum. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7. The dynamic evaluation of mitochondrial motility in the premature cellular senescence model under PQQ pretreatment.

The changes in mitochondrial dynamics with PQQ pretreatment in HEI-OC1 cells exposed to H₂O₂ to induce premature cellular senescence were evaluated using microscopic imaging and image processing. A Image preprocessing flow of the motility analysis. B Motility analysis of the mitochondria. C The average of the mean square displacement (MSD) of each group. D The area under curve (AUC) of the MSD of each group. E Mitochondrial motion tracking video. The mitochondrial particle tracking lasted for 3 min in 3 s of intervals under ImageJ (D: n = 5 per group; repeated experiments). Box plot shows statistical parameters as follows; central line: median; box limits: first and third quartile; whiskers: minimum and maximum. *P < 0.05.

Fig. 8. Analysis of protein expression and protein acetylation in the premature cellular senescence model under PQQ pretreatment.

A SIRT1 and PGC-1α protein expression was analyzed by Western blotting (upper, middle) using β-actin as a loading control (lower). B Relative protein expression level of SIRT1 normalized by β-actin. n = 10 per group. C Relative protein expression level of PGC-1α normalized by β-actin. D Acetylation of PGC-1α analyzed by immunoprecipitation. E Relative acetylation level of PGC-1α normalized by PGC-1α. F Relative mRNA expressions of GAPDH, SIRT1, and PGC-1α using β-actin as an internal control. G The changes of SIRT1 expression in flow cytometry analysis. H The rate of SIRT1 positive cells (B, C, E, H: n = 5 per group, F: n = 3 per group; repeated experiments). Box plot shows statistical parameters as follows; central line: median; box limits: first and third quartile; whiskers: minimum and maximum. RQ data are shown as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, *****P < 0.00001.

Fig. 9. Schematic diagram of the mechanism of HEI-OC1 mitochondrial biogenesis modulation under H₂O₂ exposure and PQQ protection.

A Sirtuin 1 (SIRT1) expression and deacetylation activity are decreased in HEI-OC1 cells by H₂O₂ exposure. PGC-1α mediates the protective effect of SIRT1 expression and mitochondrial biogenesis and function. PQQ improves mitochondrial biogenesis and function by restoring SIRT1 expression and deacetylation activity under H₂O₂ exposure in HEI-OC1 cells. B Schematic diagram of mitochondrial morphological changes.