Diclofenac impairs autophagic flux via oxidative stress and lysosomal dysfunction: Implications for hepatotoxicity

Abstract

Treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with various side effects, including cardiovascular and hepatic disorders. Studies suggest that mitochondrial damage and oxidative stress are important mediators of toxicity, yet the underlying mechanisms are poorly understood. In this study, we identified that some NSAIDs, including diclofenac, inhibit autophagic flux in hepatocytes. Further detailed studies demonstrated that diclofenac induced a reactive oxygen species (ROS)-dependent increase in lysosomal pH, attenuated cathepsin activity and blocked autophagosome-lysosome fusion. The reactivation of lysosomal function by treatment with clioquinol or transfection with the transcription factor EB restored lysosomal pH and thus autophagic flux. The production of mitochondrial ROS is critical for this process since scavenging ROS reversed lysosomal dysfunction and activated autophagic flux. The compromised lysosomal activity induced by diclofenac also inhibited the fusion with and degradation of mitochondria by mitophagy. Diclofenac-induced cell death and hepatotoxicity were effectively protected by rapamycin. Thus, we demonstrated that diclofenac induces the intracellular ROS production and lysosomal dysfunction that lead to the suppression of autophagy. Impaired autophagy fails to maintain mitochondrial integrity and aggravates the cellular ROS burden, which leads to diclofenac-induced hepatotoxicity.

Keywords: Diclofenac; Hepatotoxicity; Lysosomal dysfunction; Mitophagy; Nonsteroidal anti-inflammatory drugs; Reactive oxygen species.

Graphical abstract

Fig. 1

Diclofenac induces autophagosome accumulation in hepatoma cells and mouse primary hepatocytes (MPH). HepG2 cells (A, B) or MPH (C) were treated with diclofenac as indicated and subjected to Western blot analysis with antibodies against LC3-II, SQSTM1 and NBR1 (A, C). Total RNA isolated from the cells treated with diclofenac (500 μM; 24 h) was subjected to qRT-PCR analysis to quantify the relative expression of mapllc3b, sqstm1, and nbr1 (B). (D) HepG2 cells transfected with GFP-LC3 were treated with diclofenac (500 μM; 24 h) or chloroquine (CQ, 50 μM; 4 h). Representative images are shown (scale bar: 4 μm). GFP-LC3 spots were calculated using Image J software. (E) Representative transmission electron microscopy images of the cells treated with diclofenac (300 or 500 μM; 24 h) or CQ (50 μM; 4 h). The enlarged images are magnified from the boxed areas of the upper images. All data are the mean ± SD of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2

Diclofenac suppresses autophagic flux in human hepatoma cells. (A) HepG2 cells were treated with diclofenac (500 μM; 24 h) in the presence or absence of chloroquine (CQ, 50 μM; 4 h) and subjected to Western blot analysis with antibodies against LC3-II and NBR1. Representative Western blot images and the relative quantification of the protein are shown. (B) HepG2 cells transfected with GFP-mCherry-LC3 were incubated under starved conditions and treated with diclofenac (500 μM; 24 h) or BafA1 (100 nM; 8 h). Representative fluorescent images of the cells are shown. The zoomed images in the upper right or left corners were magnified from the small boxed areas in each image (scale bars: 20 μm; magnification, 2 μm). (C) Quantification of autophagosomes (yellow dots) and autolysosomes (red dots) in cells transfected with GFP-mCherry-LC3 and (D) the percentage of mCherry puncta exhibiting autolysosomes per cell were analyzed. All data are the mean ± SD of at least 3 independent experiments. ***p < 0.001 and ns, not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Autophagy impairment by NSAIDs is independent of COX inhibition. (A) Chemical structures of (S)- and (R)-ibuprofen. HepG2 cells were incubated with (S)-ibuprofen (1, 2, 4 mM) or (R)-ibuprofen (0.8, 1.6, 3.2 mM) for 24 h. The protein levels of LC3-II, NBR1 and SQSTM1 were measured by Western blot analysis. Representative Western blot images and the relative quantification of proteins are shown. (B) HepG2 cells expressing GFP-mCherry-LC3 were incubated for 24 h with (S)- ibuprofen (4 mM) or (R)-ibuprofen (3.2 mM). Representative fluorescent images of the cells are shown. The zoom images were magnified from the boxed areas in each image (scale bars: 20 μm; magnification, 4 μm). Quantitative data represent the number of autophagosomes (yellow dots) and autolysosomes (red dots) in cells transfected with GFP-mCherry-LC3 (n > 20 cells per experiment). All data are the mean ± SD of at least 3 independent experiments. **p < 0.01, ***p < 0.001 and ns, not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Diclofenac inhibits lysosomal activity and fusion with autophagosomes. (A) HepG2 cells were treated with diclofenac or Cathepsin B inhibitor (CTBi) for 24 h. Cathepsin B activity was measured using a cathepsin B activity fluorometric kit. (B, C) The cells were treated with diclofenac (500 μM; 24 h), and the lysosomal activity and acidity were analyzed by Magic Red (B), DQ-BSA Red and LysoSensor Green DND-189 (scale bars: 20 μm) (C), respectively. Nuclei were stained with DAPI (blue). The fluorescence intensity is calculated using Image J software. (D) HepG2 cells expressing GFP-LC3 (green) were incubated with diclofenac (500 μM; 24 h) or bafilomycin A1 (BafA1, 100 nM; 8 h). The fixed cells were subjected to immunofluorescence analysis with antibodies against LAMP1 (red). Nuclei were stained with DAPI (blue). Representative confocal microscopy images are shown. The zoomed images in the upper right corners were magnified from the small boxed areas in each image (scale bars: 20 μm; magnification, 2 μm). The relative number of GFP-LC3 spots in lysosomes versus that of total GFP-LC3 spots was plotted. All data are the mean ± SD of at least 3 independent experiments. **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Lysosomal reactivation restores diclofenac-induced autophagy impairment. (A) HepG2 cells pretreated with clioquinol (ClioQ, 10 μM) for 1 h or transfected with pENTR-CMV-TFEB or empty control vector were exposed to diclofenac (500 μM; 24 h) and subjected to Western blot analysis with antibodies against LC3-II and NBR1. Representative Western blot images and the relative quantification of protein expression are shown. (B) The cells transfected with GFP-mCherry-LC3 were treated with diclofenac for 24 h in the absence or presence of ClioQ (10 μM). The zoomed images are magnified from the boxed areas in the overlay images (scale bars: 20 μm; magnification, 2 μm). Quantification of autophagosomes (yellow dots) and autolysosomes (red dots) in cells transfected with GFP-mCherry-LC3 was analyzed. (C) Lysosomal acidity was analyzed by LysoSensor Green DND-189 (scale bar: 10 μm). Representative fluorescent images of the cells are shown. The fluorescence intensity is calculated using Image J software. All data are the mean ± SD of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Diclofenac-induced ROS are responsible for the impairment of lysosomal activity and autophagy. (A) HepG2 cells were treated with diclofenac for 16 h at the indicated concentrations, and the cellular and mitochondrial ROS levels were measured by quantifying the fluorescence of CM-H₂DCFDA, dihydroethidium, MitoSOX and Mito-PY1 using flow cytometry. (B–D) The cells were pretreated with N-acetylcysteine (NAC; 2 mM) or Mito-Tempo (20 μM) for 1 h, and further incubated with diclofenac (500 μM; 24 h). The cells were stained with LysoSensor Green DND-189 (scale bar: 20 μm) (B) or subjected to Western blot analysis with antibodies against LC3-II and NBR1. Representative Western blot images and the relative quantification of proteins are shown (C). HepG2 cells expressing GFP-mCherry-LC3 were incubated as described in (B) and were analyzed by confocal microscopy. The zoomed images are magnified from the boxed areas in the overlay images (scale bars: 20 μm; magnification, 2 μm). Representative images are shown and quantitative data represent the number of autophagosomes (yellow dots) and autolysosomes (red dots) in cells transfected with GFP-mCherry-LC3 (n > 20 cells per experiment) (D). All data are the mean ± SD of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Diclofenac induces oxidative mitochondrial damage and mitochondrial dysfunction in human hepatoma cells. (A, B) HepG2 cells were cultured with diclofenac for 16 h. (A) Total RNA was isolated and subjected to qRT-PCR analysis to measure cytochrome c oxidase mRNA expression. (B) The oxidation levels of ROS-sensitive mitochondrial proteins were measured by blot analysis using HRP-conjugated streptavidin. Oxidation of mitochondrial lipid cardiolipin and mitochondrial membrane potential were analyzed by flow cytometry following staining with nonyl acridine orange and tetramethylrhodamine ethyl ester (TMRE), respectively. (C) The cells were incubated with diclofenac (500 μM) or rotenone (ROT, 100 μM) for 8 h, and the mitochondria were stained with MitoTracker Red. Images were obtained by confocal microscopy and analyzed with ImageJ with the Mitochondrial Network Analysis (MiNA) program. Red lines indicate mitochondrial networks. Mitochondrial networks were analyzed using the mean branch length and median branch length supplied by the MiNA program. (D) The level of fission protein DRP1 was measured by Western blot analysis in the cells treated with diclofenac. (E) Mitochondrial function was analyzed by measuring OCR using a Seahorse XF Cell Mito Stress Test Kit. Each indicator was determined by the Seahorse XF Cell Mito Stress Test Report Generator. (F) ATP levels were measured using the CellTiter-Glo assay. All data are the mean ± SD of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Diclofenac inhibits lysosomal colocalization with mitochondria and mitophagy in hepatocytes. (A) Representative images of mitochondria in HepG2 cells treated with diclofenac were analyzed by transmission electron microscopy. The zoomed images are magnified from the boxed areas of the images. (B) HepG2 cells expressing dsRed-mito (red, mitochondria) were incubated with diclofenac (500 μM) or BafA1 (100 nM) for 8 h under starved conditions. The fixed cells were subjected to immunofluorescence analysis with antibodies against LAMP1 (green). Nuclei were stained with DAPI (blue). Representative confocal microscopy images are shown, with the zoomed images magnified from the boxed areas in the overlay images. White arrows indicate mitochondria in lysosomes (scale bars: 10 μm; magnification, 2 μm). (C) Hep3B cells stably expressing mt-Keima were pretreated with N-acetylcysteine (NAC, 2 mM) or clioquinol (ClioQ, 10 μM) for 1 h and then exposed to diclofenac for 8 h. Confocal microscopy was analyzed to detect the mt-Keima located in mitochondria (mitochondria at neutral pH, green) and the mt-Keima puncta delivered to lysosomes (mitochondria at acidic pH, red). The zoom images were magnified from boxed areas in overlay images (scale bars: 20 μm; magnification, 2 μm). Mitophagy index was evaluated by analyzing the ratio of 561/458 nm. (D, E) HepG2 cells pretreated with ClioQ (10 μM) for 1 h were incubated with diclofenac (500 μM) for 24 h. Mitochondrial function was analyzed by measuring oxygen consumption rate (OCR) using the Seahorse XF Cell Mito Stress Test Kit (D). Each indicator was determined by the Seahorse XF Cell Mito Stress Test Report Generator (E). All data are the mean ± SD of at least 3 independent experiments. *p < 0.05, **p < 0.01. ***p < 0.001 and ns, not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

Rapamycin inhibits diclofenac-induced cell death and liver injury in vitro and in vivo. (A) HepG2 cells were pretreated with or without rapamycin (500 nM) for 24 h, and then incubated with diclofenac (500 μM) for 48 h. Cell confluency was determined using a Cytation 3 cell imaging microplate reader. (B) After culture in the absence or presence of rapamycin (500 nM) for 48 h, the cells were further incubated with diclofenac (500 μM) for 16 h and subjected to FACS analysis (left panel). The percentage of cell death was plotted as the sum of the percentage of Annexin-positive, PI-positive and Annexin/PI-positive cells (right panel). (C) HepG2 cells were pretreated with or without rapamycin (500 nM) for 24 h and further incubated with diclofenac (500 μM) for 24 h. Expression of the proteins was analyzed by Western blot analysis using antibodies against LC3-II, NBR1, and SQSTM1 in HepG2 cells incubated as indicated. (D–F) Male C57BL/6 mice were injected intraperitoneally with rapamycin 0.5 h before diclofenac administration and sacrificed after 6 h. (D) Serum ALT, AST and LDH measurements were performed using an automated Chemistry Analyzer (Tokyo Boeki Medical system, Prestige 24I). (E) Liver histology was analyzed using H&E staining. Original magnification, 10X. Arrows indicate the region of sinusoidal telangiectasia (scale bar: 100 μm). (F) Protein extracts were prepared from the liver homogenates and subjected to Western blot analysis with antibodies against LC3-II and SQSTM1. Representative Western blot images and the relative quantification of proteins are shown. All data are mean ± SD of at least 3 in vitro and 4–5 in vivo experiments. *p < 0.05, **p < 0.01. ***p < 0.001.

Fig. 10

The underlying mechanism of NSAID-induced liver injury. Mitochondrial damage and ROS production induce lysosomal dysfunction and autophagy flux impairment. The failure to efficiently degrade damaged mitochondria by mitophagy induces additional ROS production, leading to hepatotoxicity. See the text for details.