4-hydroxynonenal regulates mitochondrial function in human small airway epithelial cells

Abstract

Prolonged exposure to oxidative stress causes Acute Lung Injury (ALI) and significantly impairs pulmonary function. Previously we have demonstrated that mitochondrial dysfunction is a key pathological factor in hyperoxic ALI. While it is known that hyperoxia induces the production of stable, but toxic 4-hydroxynonenal (4-HNE) molecule, it is unknown how the reactive aldehyde disrupts mitochondrial function. Our previous in vivo study indicated that exposure to hyperoxia significantly increases 4-HNE-Protein adducts, as well as levels of MDA in total lung homogenates. Based on the in vivo studies, we explored the effects of 4-HNE in human small airway epithelial cells (SAECs). Human SAECs treated with 25 μM of 4-HNE showed a significant decrease in cellular viability and increased caspase-3 activity. Moreover, 4-HNE treated SAECs showed impaired mitochondrial function and energy production indicated by reduced ATP levels, mitochondrial membrane potential, and aconitase activity. This was followed by a significant decrease in mitochondrial oxygen consumption and depletion of the reserve capacity. The direct effect of 4-HNE on the mitochondrial respiratory chain was confirmed using Rotenone. Furthermore, SAECs treated with 25 μM 4-HNE showed a time-dependent depletion of total Thioredoxin (Trx) proteins and Trx activity. Taken together, our results indicate that 4-HNE induces cellular and mitochondrial dysfunction in human SAECs, leading to an impaired endogenous antioxidant response.

Keywords: 4-HNE; Immune response; Immunity; Immunology and Microbiology Section; ROS; acute lung injury; hyperoxia; mitochondrial dysfunction.

Figure 1. Formation of 4-HNE-Protein Adducts and MDA levels in mice

Male and female C57BL/6J mice (N = 6) were exposed to normoxia and hyperoxia for 24, 48, and 72 hours. A. Mice lung homogenates were isolated and 4-HNE-Protein adduct levels were measured by ELISA with primary anti-HNE His antibody and secondary horseradish peroxidase antibody. Values are expressed as percent of control. B. Total lung MDA levels (nmol/g protein) were determined by a MDA Lipid Peroxidation and quantified by reading optical density at 532 nm. Data are shown as means ± SEM. **p-value < 0.01 vs. control, ***p-value < 0.001 vs. control.

Figure 2. Effects of 4-HNE on SAEC viability and cleaved caspase-3

Cell viability was assessed by A. MTT Cell Proliferation and B. Trypan Blue Exclusion Assay. SAECs were treated with 5, 10, or 25 μM 4-HNE and incubated for 1 hour, then compared to vehicle controls. For MTT Assay, SAECs were then treated with yellow MTT (0.5 mg/ml) to measure absorbance of purple formazan products at 570 nm. For Trypan Blue Assay, SAECs were then treated with Trypan blue dye (0.04% in PBS), and viable and dead (blue) cells were counted, using a total of 200 cells for standardization. Values are presented as percentage of cell viability. C. Caspase-3 activity (Unit/mg protein) was determined by a colorimetric assay in SEACs treated with 5-25 μM 4-HNE, with vehicle cells for comparison. Data is reported as means ± SEM. *p-value < 0.05 vs. control, **p-value < 0.01 vs. control.

Figure 3. 4-HNE reduces SAEC ATP% levels and aconitase activity

Cultured SAECs were treated with 5, 10, and 25 μM of 4-HNE and compared to vehicle controls. A. Percent ATP levels were determined by lucerifase assay and detection of light production by a luminometer. B. Mitochondrial dysfunction was assessed by Aconitase assay. Aconitase activity was determined by measuring NADPH production from conversion of μU of citrate to isocitrate per minute per mg of mitochondrial protein, with absorbance measured at 340 nm. Values are expressed as percent of control. Data is reported as means ± SEM. *p-value < 0.05 vs. control, **p-value < 0.01 vs. control, ***p-value < 0.0001 vs. control.

Figure 4. Bioenergetic and oxygen consumption evaluation of SAEC mitochondria

SAECs were treated with 25 μM 4-HNE and compared to vehicle controls. A. Basal, B. non-ATP linked, C. reserve capacity, and D. non-mitochondrial OCR (pmol/O₂/min) were measured in SAECs by XF analysis. After basal OCR was measured, oligomycin (1μg/ml), FCCP (1μM), and antimycin A (10 μM) were injected consecutively through Seahorse Flux Pak cartridges, with resulting OCR measured after each injection. Data is reported as means ± SEM. **p-value < 0.01 vs. control, ***p-value < 0.001 vs. control.

Figure 5. Assessment of mitochondrial dysfunction by JC-1 staining of 4-HNE treated SAECs

SAECs were plated at a density of 2 × 10⁵ cells/ml in a 12 mm Nunc glass bottom dish and treated with various concentration of 4-HNE (10-100 μM) or vehicle at 37°C for 15 min. The medium was replaced and cells were incubated with JC-1 probe (10 μg/ml) at 37°C for 15 min, washed and live imaging was performed using confocal microscope. A. = Vehicle (0.1% ethanol). B.-D. = 10, 30 and 100 μM 4-HNE. In vehicle and 10 μM 4-HNE treated sample, predominantly orange-red fluorescence is seen in the mitochondria (arrows in A. and B.), whereas with 30 and 100 μM 4-HNE treatment, mitochondria showed higher green fluorescent signal relative to vehicle or 10 μM 4-HNE (arrows in C. and D.). E. The JC-1 aggregates were quantified using Image J software and the mean intensity is expressed as Arbitrary Units. ***p < 0.001 Vehicle (0.1% Ethanol) vs. 4-HNE (30 and 100 μM), ***p < 0.001 10 μM 4-HNE and 30 and 100 μM 4-HNE, respectively. Scale bar = 10 μm, Magnification = 60X.

Figure 6. 4-HNE induces generation of ROS within SAECs and is specific to the SAEC mitochondrial respiratory chain

A. SAECs were treated with 5, 10, and 25 μM 4-HNE and compared to vehicle controls. Cells were cultured and treated with 10 μM DCFH-DA for 30 min. The oxidation of DCFH-DA to DCF by ROS was determined by measuring fluorescence intensity at 480 nm (excitation) and 530 nm (emission). B. SAECs were then directly treated with 25 μM 4-HNE and compared to vehicle controls. Mitochondrial production of ROS was measured by addition of the inhibitors allopurinol (100μM), rotenone (1 μM), and apocynin (1 mM). Site of ROS production (xanthine oxidase, mitochondrial chain complex I, and NADPH-oxidase) was evaluated. Values are expressed as percent control. Data is reported as means ± SEM. **p-value < 0.01 vs. control, ***p-value < 0.001 vs. control, † p-value < 0.01, ††p-value < 0.001.

Figure 7. 4-HNE affects the function of Trx within SAEC mitochondria

A. Total Trx depletion and B. activity suppression were measured within SAECs. Cells were treated with 25 μM 4-HNE and effects were measured at 10, 20, 30, and 60 min time intervals, and compared to vehicle controls. Cells were lysed with 10.1% Triton X-100/PBS (100 μl). Total Trx activity was then determined by insulin-reducing assay and NADPH oxidation was measured by spectrophotometric analysis at 340 nm over 5 minutes. Values are presented as percentage of controls. Data are expressed as means ± SEM. *p-value < 0.05 vs. control, **p-value < 0.01 vs. control.