Protective role of pyrroloquinoline quinone against gentamicin induced cochlear hair cell ototoxicity

Gentamicin (GM) is one of the commonly used antibiotics in the aminoglycoside class but is ototoxic, which constantly impacts the quality of human life. Pyrroloquinoline quinone (PQQ) as a redox cofactor produced by bacteria was found in soil and foods that exert an antioxidant and redox modulator. It is well documented that the PQQ can alleviate inflammatory responses and cytotoxicity. However, our understanding of PQQ in ototoxicity remains unclear. We reported that PQQ could protect against GM‐induced ototoxicity in House Ear Institute‐Organ of Corti 1 (HEI‐OC1) cells in vitro. To evaluate reactive oxygen species (ROS) production and mitochondrial function, ROS and JC‐1 staining, oxygen consumption rate (OCR), and extracellular acidification rate (ECAR) measurements in living cells, mitochondrial dynamics analysis was performed. GM‐mediated damage was performed by reducing the production of ROS and inhibiting mitochondria biogenesis and dynamics. PQQ ameliorated the cellular oxidative stress and recovered mitochondrial membrane potential, facilitating the recovery of mitochondrial biogenesis and dynamics. Our in vitro findings improve our understanding of the GM‐induced ototoxicity with therapeutic implications for PQQ.

The quinoprotein cofactors include cysteine tryptophylquinone (CTQ), lysine tyrosylquinone (LTQ), 2,4,5-trihydroxyphenylalanine quinone or topaquinone (TPQ), pyrroloquinoline quinone (PQQ), and tryptophan phylloquinone (TTQ) (Anthony, 2001;Matsushita et al., 2002).Among those quinoprotein cofactors, PQQ was first reported as a cofactor for bacterial dehydrogenases in the late 1960s, and its structure and character were gradually elucidated (Anthony, 2001;Anthony & Ghosh, 1998;Hauge, 1964).PQQ is a negatively charged, water-soluble complex with redox quinones and free radical scavenging properties (Gallop et al., 1989).PQQ plays a variety of biological functions, such as promoting the growth and development of animals, cell proliferation, protecting nerve cells, and promoting the secretion of growth factors (Matsushita et al., 2002).In recent years, more researches suggest that PQQ can reduce inflammatory responses and cytotoxicity, such as ameliorating skeletal muscle atrophy via inhibition of the inflammatory, inhibiting the toxicity and inflammation of obesity in mice by early supplementation of PQQ, inhibiting LPS-induced inflammation by down-regulating inflammatory signaling pathways in microglial, inhibiting rotenoneinduced microglia inflammation by enhancing autophagy, improving the anti-cyclophosphamide induced nephrotoxicity, and restraining cytotoxicity of truncated alpha-synuclein (Kim et al., 2010;Lin et al., 2020;Lu et al., 2018;Ma et al., 2019;Yang et al., 2014).However, the role of PQQ in GM-induced capillary damage of the cochlea remains unclear.
In the present study, we employed an in vitro study to report the protective role of PQQ against GM-induced cochlea HC damage.PQQ protected the HEI-OC1 cell proliferation and viability decrease caused by GM and reduced the depolarization effect of GM.Otherwise, PQQ suppressed reactive oxygen species (ROS) production in HEI-OC1 cells.We detected the effect of PQQ on mitochondrial ATP synthesis and maximal respiration.PQQ protected against the decline of mitochondrial ATP synthesis and maximal respiration in GM-induced HEI-OC1 cell trauma.At last, confocal microscopy analysis demonstrated that PQQ can protect against GM-induced mitochondrial dynamic deteriorations in HEI-OC1 cells.

| Materials
The following reagents were used in this study: high-glucose Dulbecco's Eagle's medium was from GIBCO-BRL (Gaithersburg, MD).
Fetal bovine serum was from Invitrogen (Karlsruhe, Germany).PQQ was from Sigma-Aldrich (Merck, America), and stock solutions were prepared in water.PQQ and GM (Sigma, America) were supplemented concurrently into the medium in assay concentrations to the indicated concentrations (PQQ: 0.1, 1, 10 nM; GM: 0.1, 1, 10, 100 mg/mL).Penicillin was from Sigma-Aldrich (Merck, America).All other chemicals used were of analytical reagent grade quality or better and were obtained from the usual commercial sources.

| Cell culture
The HEI-OC1 cell line was provided by Professor F. Kalinec (UCLA, Los Angeles, CA, USA).HEI-OC1 is an auditory immortalized Corti-derived epithelial cells, which were cultured in high-glucose Dulbecco's Eagle's medium containing 10% fetal bovine serum with 100 IU/mL penicillin at 33 C, with 10% CO 2 under permissive conditions.

| Cell population doubling rate and viability assay
The HEI-OC1 auditory cells were first incubated with multiple concentrations of GM (0.1 mg/mL, 1 mg/mL, or 10 mg/mL, Shandong Lu Kang Chen Xin industrial Co.) and PQQ (0.1 nM, 1 nM or 10 nM, Sigma-Aldrich Co., Germany) separately.Then, the optimal concentration of PQQ and GM were used for further assay.Cells were divided into four groups: control group, no treatment; GM group, treated with 10 mg/mL GM for 24 h; treat I group, treated with 10 mg/mL GM 24 h followed by 0.1 nM PQQ for 24 h; and treat II group, treat with 10 mg/mL GM 24 h followed by 1.0 nM PQQ for 24 h.After being washed with Dulbecco's phosphate-buffered saline (DPBS), cells were harvested via trypsinization (0.05% trypsin, 0.53 mM EDTA for 2 min), resuspended in DPBS, and diluted in 0.4% trypan blue solution (1:1).

| Analysis of mitochondrial membrane potential (MMP) and ROS detection
We analyzed the disruption or loss of MMP by 5,5 0 ,6,6 0 -tetrachloro-1,3,3 0 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Invitrogen, California, USA), which exhibited potential-dependent accumulation in mitochondria.At a high level of MMP, JC-1 is uptaken by mitochondria and forms JC-1 dimmers aggregates with the emission of red fluorescence (590 ± 17.5 nm).On the contrary, mitochondria emit green fluorescence (530 ± 15 nm) at low MMP after forming JC-1 monomers.The HEI-OC1 cells were exposed to the conditions of the four groups and replaced with the normal medium with 200 nM JC-1 (Abcam, Cambridge, USA) for 15 min before the MMP ratio assay.A fluorescence emission ratio was analyzed with an infinite M200 PRO plate-reader (TECAN, USA), which collected green and red signals under 10% CO 2 incubation at 33 C. The JC-1 emission ratio was performed and calculated at multiple reads per well (circle, 4 Â 4).We also collected the images of JC-1 with a confocal microscope.Cell superoxide was detected using MitoSoxTM Red molecular probes (Invitrogen, California, USA).Cells were exposed to four conditions: control, GM, GM + 0.1 nM PQQ, and GM + 1.0 nM PQQ groups.All group cells were incubated in Hank's buffer with 2 μM MitoSoxTM Red for 30 min at 33 C in a 10% CO 2 atmosphere.After washing with DPBS, the cells were added with 4 μg/mL DAPI (Sigma-Aldrich, Missouri, USA), then captured with confocal microscopy.All image processing was performed using NIS-Elements (Nikon, Japan).

| Measurement of mitochondrial function
A Seahorse XF24 Analyzer (Agilent Technologies, Santa Clara, CA) was used to measure mitochondrial function in intact HEI-OC1.The cells were treated as different groups: (1) control group, no PQQ treatment; (2) GM group, treated with 10 mg/mL GM for 24 h; (3) GM + 0.1 nM PQQ group, treated with 10 mg/mL GM for 24 h followed by 0.1 nM PQQ for 1 day; and (4) GM + 0.1 nM PQQ group, treated with 10 mg/mL GM for 24 h followed by 1.0 nM PQQ for 1 day.A Seahorse XF24 Analyzer (Agilent Technologies, Santa Clara, CA) was used to measure mitochondrial function in HEI-OC1 cells.After all the groups finished, the cells were rinsed twice using an assay medium (XF assay medium, DMEM without NaHCO3, without GlutaMAX, without L-glutamine; 102,353-100, Seahorse Bioscience, Billerica, MA, USA) supplemented with 25 mM (450 mg/dL) D-glucose (Otsuka Seiyaku, Tokushima, Japan) and 1 mM sodium pyruvate (Nacalai Tesque, Kyoto, Japan) and then equilibrated for 1 h at 33 C in a non-CO 2 incubator prior to the assay.The mitochondrial respiration was detected before any pharmacological perturbation (basal respiration).
Cells were then subjected to (in sequence): (1) 2.5 μM oligomycin to inhibit ATP synthase and oxidative phosphorylation (OXPHOS); (2) 1.25 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) to induce maximal respiration; and (3) 0.625 μM rotenone to end this reaction.The XF24 analyzer determined the oxygen and proton concentration in real time, which reflects the rates of oxygen consumption and proton production speeds.Parameters are expressed as the oxygen consumption rate (OCR) in pMoles/min or the rate of extracellular acidification rate (ECAR) in mpH/min and were the key indicators of mitochondrial respiration and glycolysis.

| Measurement of mitochondrial dynamics
Cells were dyed with tetramethylrhodamine, ethyl ester (TMRE) (200 nM, Biotium, USA) mitochondrial fluorescence and captured focusing on mitochondrial-rich regions beside the cell nucleus with confocal microscopy.Images were captured at 3 s of intervals for a duration of 3 min across Z stacks of the cell.Videos provide 3D information about mitochondrial dynamics over time.All image processing and analysis were performed using NIS-Elements (Nikon, Japan), ImageJ (Rueden et al., 2017;Schneider et al., 2012) with Fiji (Schindelin et al., 2012), TrackMate (Tinevez et al., 2017), and @msdanalyzer (Tarantino et al., 2014) in MATLAB R2019b.The mean square displacement (MSD) and track displacementand mean velocity of the mitochondrial movement measurements were used for the analysis of the parameters of mitochondrial motion.

| Statistical methods
The statistical software GraphPad Prism was used in data processing and analyzed its significance.ANOVA analysis with Tukey was used to the compare groups (P < 0.05 was considered significant).

| Protective effect of PQQ against GMinduced cell proliferation and viability reduction
GM, one of the aminoglycosides, has a limited therapeutic index because of the dose-dependent ototoxic effects (Aleman et al., 2021;Jiang et al., 2017).Before analyzing any protective effect of PQQ, we should determine whether GM could lead to auditory HC death.Therefore, we evaluated the population doubling rates of HEI-OC1 cells under different concentrations of GM.The population doubling rate significantly decreased at 10 mg/mL GM after 24 h of exposure (Figure 1A).Then, we examined the appropriate concentration of PQQ to cells before toxicity and found that the cell population decreased significantly at 10 nM PQQ after 24 h of culture (Figure 1B).As a consequence, we chose 10 mg/mL GM and 0.1 nM, 1 nM PQQ for subsequent experiments.We treated GM-stimulated HEI-OC1 cells with two different concentrations of PQQ.The cell population doubling rate increased in the PQQ treatment group compared with the GM group (Figure 1C).The cell viability in the PQQ treatment group also increased compared with the GM group (Figure 1D).These results indicate that auditory HCs got ototoxicity induced by GM in a concentration-dependent manner, which could be alleviated by PQQ.

| PQQ improves mitochondrial depolarization and ROS induced by GM in HEI-OC1 cells
MMP is essential for maintenance viability, further leading to cell apoptosis.To verify PQQ protective function against MMP changes in GM-induced cell injury, we utilized JC-1 to detect mitochondrial membrane fluorescent dye in analyzing the disruption of MMP.The green emission part showed the state of the membrane with low potential whereas the red emission showed a high potential state in JC-1 MMP fluorescence.We sought to assess the change of dynamic mitochondria in different groups.We found that the control group has exhibited a deep red/green color, and the GM group exhibited mainly the green color.Whereas, in the PQQ treatment group, the fluorescence changed back to deep red and green color.The red/green ratio showed the status of the mitochondrial membrane, which decreased after GM treatment in auditory cells.However, a depolarized mitochondrion was improved after PQQ was pre-treated (Figure 2A).These results indicate that GM induces mitochondrial depolarization in HEI-OC1 cells whereas PQQ alleviated this condition.PQQ suppresses ROS production in HEI-OC1 cells (Figure 2B).
It has been recognized that mitochondrial depolarization leads to excessive production of ROS and contributes to HC apoptosis.
To confirm whether ROS mediated PQQ's protective function on GM ototoxicity, we used MitoSoxTM Red to evaluate mitochondrial ROS levels in GM-treated HEI-OC1 cells.Immunohistochemistry showed that the ROS levels were increased after GM treatment compared with the control group and is reversed after PQQ treatment (Figure 2C).This result demonstrates that PQQ can restrain GMinduced ROS production in HEI-OC1 cells (Figure 2D).

| PQQ protects against the decline of mitochondrial ATP synthesis and maximal respiration in GM-induced HEI-OC1 cell trauma
As oxidative metabolism is an important function of mitochondria, we evaluated the function of the electron transport chain (ETC) by performing oximetry on HEI-OC1 cells using a Seahorse XF24 mitochondria flux analyzer.We evaluated the OCRs (Figure 3A) and ECAR (Figure 3D), which measures the fundamental parameters of the ETC, basal OCR, ATP-linked respiration, maximal OCR, spare respiratory capacity, and proton leak.The basal ATP production speed and the maximal respiration measured following the injection of a mitochondrial uncoupling agent FCCP decreased significantly in the GM group compared with the control group whereas the basal and maximum respiration were improved significantly in the GM with 1.0 nM PQQ group compared with the GM group (Figure 3B,C).Whereas, there was no significant change in ECAR between groups (Figure 3E,F).
These results indicate that PQQ inhibits the decline of not only mitochondrial basal respiration but also mitochondrial respiratory capacity under GM exposure in HEI-OC1 cells.

| PQQ has the potential to protect against mitochondrial dynamic deteriorations in HEI-OC1 cells with exposures to GM
We evaluated the dynamic motility of mitochondria to confirm the improvements with regard to mitochondrial function after PQQ treatment.The HEI-OC1 cells were stained with TMRE fluorescence dye, which accumulates in mitochondria because of the MMP and was observed by Nikon's structured illumination microscope.The kinetics of the moving particles were evaluated quantitatively using the Track-Mate plugin in ImageJ/FIJI (Figure 4A), and the MATLAB processing platform was used to analyze the complex data derived from spatiotemporal imaging of trafficking particles (Figure 4B).

| DISCUSSION
Drugs and therapeutic agents, especially antibiotic treatment (e.g.GM), can cause inner ear structures damage, which may lead to permanent hearing, balance dysregulation, and tinnitus (Xie et al., 2011).PQQ, an important universal redox cofactor, has been proven to have radical scavenging activity and neuroprotective, cardiovascular, and anti-tumor effects as antioxidants (Cordell & Daley, 2022;Rucker et al., 2009).However, the primary cellular mechanisms of how PQQ protects GM-induced ototoxicity remain unclear.effect of GM on the respiratory activities of mitochondria and the permeability of their membranes (O'Reilly et al., 2019).PQQ attenuates clinically relevant conditions such as ischemia, inflammation, and lipotoxicity and also has nootropic properties (Jonscher et al., 2021).In this regard, genes essential for fatty acid metabolism and mitochondrial function are particularly targeted by PQQ.We selected the optimum concentration of PQQ by determining the concentration at which there were no decrease in cell numbers and MMP.We demonstrated PQQ had a mitochondrial uncoupling effect (the MMP decrease) in the high concentration condition, and it increased cell viability and worked protectively after GM-induced ototoxicity.Indeed, the decrease of MMP by PQQ has been reported in tumor cell lines at high doses of over 15 μM (Min et al., 2014).The optimum concentration analysis is crucial in evaluating the protective effect of PQQ and from the aspect of the drug screening targeting mitochondrial protection mitochondrial uncoupling effect is one of the key factors in reducing damage and protecting the mitochondria in relation to ROS.
We selected the optimum concentration of PQQ in this study by determining the concentration at which there was no decrease in cell numbers.Maintenance of membrane potential is another essential property of mitochondria for its function.Discharge of the MMP has several consequences for the cell, including apoptosis (Govindaraj et al., 2011).Decreased MMP was found in a variety of aging cell types from several mammalian species (Sugrue & Tatton, 2001).Here, we observed PQQ led to the restoration of the MMP, and ROS decreased in the GM-induced auditory cell injury.Therefore, these results suggest that PQQ can protect the mitochondria in relation to ROS and the mitochondrial uncoupling effect.
Extracellular flux analysis is a mainstream method for measuring mitochondrial function in cells and tissues, which reports the rate of ATP production, proton leak rate, coupling efficiency, maximum respiratory rate, respiratory control ratio, and spare respiratory capacity (Salabei et al., 2014;Tan et al., 2015).Oligomycin, an inhibitor of ATP synthesis, is used to prevent phosphorylating respiration.
The second injection of FCCP allows for the uninhibited movement of protons across the mitochondrial inner membrane and effectively depletes the MMP.This collapse of the MMP leads to an increase in oxygen consumption and allows a determination of the maximal oxygen consumption.Herein, we investigate mitochondrial respiratory function and indicate that GM could cause a decrease in maximal respiration, which is attenuated by PQQ.Confirming studies for GM reduction have been undertaken by others previously (O'Reilly et al., 2019).GM stimulates non-phosphorylating respiration and inhibits uncoupled respiratory rates, thereby reducing the respiratory control ratio while simultaneously causing a collapse of the MMP.
PQQ could ameliorate the collapse of the MMP via increased mitochondrial respiratory.Mitochondrial function is dependent on mitochondrial dynamics.The mitochondrial transport to discrete subcellular regions may contribute to the accumulation of ROS in the nucleus and the oxidative-based signaling function (Mattenberger et al., 2003).The anterograde mitochondrial transport delivers healthy mitochondria to peripheral sites whereas the retrograde mitochondrial transport returns damaged mitochondria to central sites, which is crucial for neural survival (Schwarz, 2013).Mitochondrial motility and dynamics both regulate and reflect mitochondrial function in auditory cells (Gao et al., 2022).The recovery of mitochondrial dynamics is an important step in the protection of mitochondrial metabolic activity in auditory cells (Zou et al., 2022).The dynamic deteriorations caused by GM exposure were alleviated by PQQ after treatment in HEI-OC1 cells, which indicates that PQQ works protectively in terms of mitochondrial function and mitochondrial condition.
With regard to the possible mechanisms by which GM cytotoxicity causes damage, GM-induced renal tubular cytotoxicity indicates that tubular cytotoxicity is the consequence of many interconnected actions, triggered by drug accumulation in epithelial tubular cells.GM accesses and accumulates in the endosomal compartment, the golgi and endoplasmic reticulum (ER), causing ER stress.GM then acts on mitochondria to unleash the intrinsic pathway of apoptosis.In addition, lysosomal cathepsins lose confinement and, depending on their new cytosolic concentration, contribute to the activation of apoptosis or produce massive proteolysis that leads to necrotic cell death (Quiros et al., 2011).Drug cytotoxicity on HEI-OC1 cells suggests that mitochondria are vulnerable to oxidative stress and damage caused by GM.GM can induce the production of ROS, triggering oxidative stress and damaging mitochondria in cochlear HCs (Kalinec et al., 2016).
Therefore, although the exact mechanisms of GM-induced ototoxicity are not fully understood, several studies suggest that mitochondrial damage plays a significant role.
In conclusion, our in vitro study demonstrated the function and protection of PQQ in GM-induced cochlear HCs ototoxicity.PQQ can improve mitochondrial depolarization and ROS, inhibit the decline of mitochondrial basal respiration and respiratory capacity and alleviate mitochondrial dynamic deteriorations.Our investigation mainly focuses on cellular study at present, whether PQQ has some protection in vivo deserves further research.Furthermore, our observation that pharmacological treatment with PQQ curtailed the toxicity of cochlear HCs suggests a new avenue for therapeutic targeting in ototoxicity.
Figure4D,E showed a significant difference in the PQQ group compared with the GM group.The AUC of the MSD plot showed significant decreases in the PQQ treated groups compared with the GM Our research revealed that PQQ has the potential to protect against GM-induced cellular oxidative stress in auditory cells by enabling the recovery of MMP, suppressing ROS production, and facilitating the recovery of mitochondrial biogenesis.Our in vitro research could improve our understanding of the possible cellular mechanisms by which PQQ treats GM-induced ototoxicity.F I G U R E 2 Membrane potential change in mitochondria with exposure to gentamicin and pre-protected with pyrroloquinoline quinone (PQQ).(A) Live cell immunofluorescent staining of the four treatment groups.Normal mitochondrial membrane potential (MMP) was shown in red with 5,5 0 ,6,6 0tetrachloro-1, 3,3 0 -tetraethyl benzimidazolyl carbocyanine iodide (JC-1) dimers and depolarized MMP shown in green in JC-1 monomers.(B) MMP determined by fluorescent plate reader.Relative fluorescence units (RFU) at 590 nm represented red color, RFU at 535 nm represented green color.(C) Four different groups of House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were labeled by Mito-SOXTM red.(D) reactive oxygen species (ROS) and 4',6-diamidino-2-phenylindole (DAPI) double staining showed the changes in four groups and its relative content analysis.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.F I G U R E 3 Protective properties of pyrroloquinoline quinone (PQQ) against gentamicin on mitochondrial respiration.(A) Seahorse XF-24 instrument analysis of oxygen consumption rate (OCR) in four different groups of a typical trial.Intact auditory cells in the presence of glucose were exposed sequentially to oligomycin (oligo), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), rotenone, and antimycin.OCR value before the time oligo added represents O 2 consumption of mitochondrial basal respiration, and the value of the time between FCCP and rotenone added represents maximum respiration.(B) OCR of basal respiration in four groups.(C) OCR of maximum respiration in four groups.(D) Extracellular acidification rate (ECAR) in four different groups of a typical trial.(E) Comparison in four groups for basal glycolysis.(F) Compensatory glycolysis comparison of four groups.All values are shown as mean ± SD, n = 5 wells per group.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.Ototoxicity usually causes functional impairment and cellular degeneration of the inner ear caused by drugs and therapeutic agents, resulting in loss of hearing and/or vestibular function (Rodriguez et al., 2007).GM has long been assumed to target mitochondria once inside the HCs of the cochlea-induced toxicity.This has largely been based upon indirect lines of evidence showing co-localization of fluorescent mitochondrial dyes with fluorescently conjugated GM, increased susceptibility to GM-induced damage due to mitochondrial DNA mutation, and inferences made based on ROS production (O'Reilly et al., 2019).Other lines of evidence have also shown a direct F I G U R E 4 Dynamic evaluation of mitochondrial motility in gentamicin (GM) ototoxicity model under quinone (PQQ) pretreatment.The changes in mitochondrial dynamics exposed to GM and treated with PQQ in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were evaluated using microscopic imaging and image processing.(A) Image of the tetramethylrhodamine, ethyl ester (TMRE) immunofluorescent staining and TrackMate plugin in ImageJ/FIJI motility analysis.(B) MATLAB processing platform motility analysis of the mitochondria.(C) the mean square displacement (MSD) of each group.(D) The track displacement analysis of the mitochondrial of each group.(E) The mean velocity analysis of each track for the mitochondrial of each group.(F) The area under the curve (AUC) of the MSD of each group.The mitochondrial motion tracking video showed in the supplement figure.The mitochondrial particle tracking lasted for 3 min in 3 s of intervals under ImageJ.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.