Pyrroloquinoline Quinone (PQQ) Attenuates Hydrogen Peroxide-Induced Injury Through the Enhancement of Mitochondrial Function in Human Trabecular Meshwork Cells

Abstract

Mitochondrial metabolism in the trabecular meshwork (TM) plays a critical role in maintaining intraocular pressure homeostasis by supporting the energy-demanding processes involved in aqueous humour outflow. In primary open-angle glaucoma, oxidative stress impairs mitochondrial function, leading to TM dysfunction. Therefore, understanding and targeting mitochondrial health in TM cells could offer a novel therapeutic strategy. Pyrroloquinoline quinone (PQQ) is a redox cofactor with antioxidant and mitochondrial-enhancing properties. However, its effects on human TM (HTM) cells remain largely unexplored. This study examined PQQ cytoprotective effects against H₂O₂-induced oxidative stress in HTM cells. Seahorse analyses revealed that PQQ alone improves mitochondrial respiration and ATP production. Moreover, PQQ mitigates H₂O₂-induced cellular damage and preserves mitochondrial function by normalising proton leak and increasing ATP levels. Furthermore, TEM and confocal microscopy showed that PQQ can partially alleviate structural damage, restoring mitochondrial network morphology, thereby leading to reduced cell death. Although these protective effects seem not to be mediated by changes in mitochondrial content or activation of the SIRT1/PGC1-α pathway, they may involve modulation of SIRT3, a key factor of mitochondrial metabolism and homeostasis. Overall, these results suggest that PQQ may represent a promising candidate for restoring mitochondrial function and reversing oxidative damage in HTM cells.

Keywords: SIRT1/PGC1-α pathway; SIRT3 signalling; human trabecular meshwork cells; mitochondrial bioenergetic; mitochondrial network morphology; pyrroloquinoline quinone (PQQ).

Figure 1

PQQ does not alter cellular homeostasis, redox balance, and mitochondrial membrane potential (ΔΨm) in HTM cells. HTM cells were exposed or not exposed (CTR) to increasing concentrations of PQQ for 24 h. Cell growth rate (A) and viability (B) were assessed using the trypan blue exclusion assay. (C) Intracellular ROS levels (a.u., arbitrary units) were measured using the fluorescent probe DCFH2-DA. (D(i)) Representative images of ΔΨm profiles obtained through staining with the potentiometric probe JC-1 and analysed by flow cytometry. (D(ii)) The graph shows the ΔΨm, expressed as the mean of the red/green fluorescence ratio of the mean fluorescence intensity, for each different experimental group. CCCP was used as a positive control for the abolishment of ΔΨm. Data from three independent experiments are presented as the mean ± SE; one-way ANOVA.

Figure 2

PQQ enhances respiratory capacity and ATP production in HTM cells. HTM cells were exposed to increasing concentrations of PQQ (1–100 nM) for 24 h. Bioenergetic parameters were analysed using the Seahorse XF flux analyser. (A) The graph shows the time-course profiles of oxygen consumption rate (OCR) in samples treated with different concentrations of PQQ. (B) The bar graphs represent the quantification of the following key parameters: “Basal respiration”, “Proton leak”, “Spare respiratory capacity”, “Maximal respiration”, “ATP production-coupled respiration”, and “Non-mitochondrial oxygen consumption”. Data from three independent experiments are expressed as the mean ± SE. One-way ANOVA followed by the Holm–Sidak post hoc test (* p < 0.05).

Figure 3

Effects of hydrogen peroxide-induced stress on cell growth, viability, and mitochondrial function and bioenergetics in HTM cells. HTM cells were exposed to a range of H₂O₂ concentrations (100–1600 µM) for 1 h. The dose–response curve (A) shows IC20, IC50, and IC80 values evaluated 24 h after exposure. Data from three independent experiments are presented as the mean ± SE, analysed by OriginPro software version 8.5 (OriginLab Corporation, Northampton, MA, USA). (B) Graph displays ΔΨm levels, expressed as the mean of the red/green fluorescence intensity ratio in control and H₂O₂ -treated (100 µM, for 1 h) cell populations. Data are expressed as mean ± SE from three independent experiments. * p < 0.05 by unpaired t-test. ((C,C(i))) Mitochondrial bioenergetic profiling of control and H₂O₂ -treated cells. Data are shown as mean ± SE from three independent experiments and multiple paired t-tests (* p < 0.05).

Figure 4

Protective effects of PQQ against hydrogen peroxide-induced decline in mitochondrial respiratory capacity in HTM cells. Following a 23 h pretreatment with PQQ and subsequent 1 h co-exposure to H₂O₂ (100 µM), all experimental groups were subjected to analysis of mitochondrial respiratory capacity (A) using the Seahorse analyser and mitochondrial membrane potential (ΔΨm) (B) by flow cytometry. (A) Bar graphs show the mean ± SE of the measured metabolic parameters. (A(i)) Time-course profiles of OCR in the different treated samples. (B) ΔΨm is expressed as the mean of the red/green fluorescence intensity ratio of mean fluorescence intensity (MFI). One-way ANOVA followed by the Holm–Sidak post hoc test (* p < 0.05; # p < 0.05 vs. CTR).

Figure 5

PQQ pretreatment upregulates the expression of SIRT3 in HTM cells. (A) Bar graphs represent protein levels of SIRT1, PGC1-α, TOM20, SIRT3, and SOD2, after 24 h of exposure to 100 nM PQQ, analysed by Western blotting. Both PQQ alone and PQQ + H₂O₂ co-treatment induced a significant increase in SIRT3 levels, whereas PGC1-α, SIRT1, TOM20, and SOD2 levels remained mostly unchanged. H₂O₂ exposure by itself did not affect any of these protein levels. Protein levels are expressed as relative units (r.u.). (B) Representative images of Western blot membranes; β-actin was used as an internal control. Data are shown as the mean ± SE from three independent experiments. One-way ANOVA followed by the Holm–Sidak post hoc test (* p < 0.05 vs. CTR; # p < 0.05). ANOVA on ranks performed for the analyses of SOD2 protein levels.

Figure 6

PQQ alleviates H₂O₂-induced mitochondrial ultrastructural damage in HTM cells. Panel (A): representative transmission electron microscopy (TEM) images (3400×) of control and treated HTM cells. (a) Control cells; (b) cells treated with 100 nM PQQ; (c) cells treated with 100 µM H₂O₂; (d) cells co-treated with 100 µM H₂O₂ and 100 nM PQQ. Scale bar = 2 µm. Panel (B): representative TEM images (10,500×) of mitochondrial morphology in control and treated cells. (a) In control cells, the mitochondria exhibit lamellar cristae with visible cristae membranes; (b) in cells treated with PQQ, the mitochondria are comparable to those in the control group. In some zones (*), the cristae are presumably cut tangentially and therefore not visualised by electron microscopy. (c) In cells treated with H₂O₂, the mitochondria seem to be smaller. The cristae of the membrane also appear thicker. Numerous vacuoles (V) and phagolysosomes (P) are present. (d) Combined treatment with PQQ and H₂O₂ appears to counteract the mitochondrial stress induced by H₂O₂ alone, leading to restoration of normal internal architecture (*) and causing some mitochondria to divide (arrow). Scale bar = 1 µm. Panel (C): TEM images showing details at higher magnification (46,000×) of the mitochondrial ultrastructure in control and treated cells. (a) Elongated mitochondria with normal internal architecture and visible lamellar cristae in control cells; (b) mitochondria in PQQ-treated cells similar to control mitochondria; (c) altered mitochondria with swollen, electron-dense cristae (sm) associated with vacuoles (V) in H₂O₂-treated cells; (d) restored (m) and dividing mitochondria (arrows) in PQQ + H₂O₂-treated cells. Scale bar = 200 nm. Abbreviations: N, nucleus; ER, rough endoplasmic reticulum; m, mitochondria; sm, swollen and electron-dense mitochondrial cristae; V, vacuoles; P, phagolysosomes.

Figure 7

PQQ mitigates hydrogen peroxide-induced morphological alterations of the mitochondrial network in HTM cells. (A) Representative confocal microscopy images of the different experimental groups obtained by TOM20 (red) and DAPI (blue) staining. (B) Schematic representation of the computational skeletonisation workflow process performed using ImageJ (Fiji, version 1.51n, Bethesda, MD, USA). (C) Graphs show the quantification of “Branch Length Mean”, “Mitochondrial Footprint”, “Branch junctions”, and “Branch end points” parameters, analysed using MiNA and the Mitochondria Analyser plugin in ImageJ (Fiji, Bethesda, MD, USA). Data are presented as the mean of three independent experiments ± SE; statistical analysis was performed using ANOVA on ranks (Kruskal–Wallis test) followed by Dunn’s method (* p < 0.05 vs. CTR; # p < 0.05) or one-way ANOVA followed by the Holm–Sidak method (* p < 0.05 vs. CTR; # p < 0.05).

Figure 8

Protective effects of PQQ against hydrogen peroxide-induced cytotoxicity in HTM cells. (A) Representative flow cytometry images of HTM cells stained with Annexin V and Propidium Iodide, corresponding to the indicated experimental groups. Q1 = early apoptosis; Q2 + Q4 = Cell Death. (B) Bar graphs show the percentages of early apoptotic cells (Early Apoptosis) and late apoptotic/necrotic cells (Cell Death) in the different samples, assessed 24 h after H₂O₂ treatment. Data are expressed as the mean ± SE of three independent experiments. One-way ANOVA followed by the Holm–Sidak post hoc test (* p < 0.05 vs. CTR; # p < 0.05).