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. 2022 Jan 7:2022:4266214.
doi: 10.1155/2022/4266214. eCollection 2022.

Coenzyme Q0 Inhibits NLRP3 Inflammasome Activation through Mitophagy Induction in LPS/ATP-Stimulated Macrophages

You-Cheng Hseu  1   2   3   4 , Yu-Fang Tseng  5 , Sudhir Pandey  1 , Sirjana Shrestha  1 , Kai-Yuan Lin  6   7 , Cheng-Wen Lin  8 , Chuan-Chen Lee  2 , Sheng-Teng Huang  3   4   9 , Hsin-Ling Yang  5
Affiliations

Coenzyme Q0 Inhibits NLRP3 Inflammasome Activation through Mitophagy Induction in LPS/ATP-Stimulated Macrophages

You-Cheng Hseu et al. Oxid Med Cell Longev. .

Abstract

Coenzyme Q (CoQ) analogs with a variable number of isoprenoid units have exhibited as anti-inflammatory as well as antioxidant molecules. Using novel quinone derivative CoQ0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone, zero side chain isoprenoid), we studied its molecular activities against LPS/ATP-induced inflammation and redox imbalance in murine RAW264.7 macrophages. CoQ0's non- or subcytotoxic concentration suppressed the NLRP3 inflammasome and procaspase-1 activation, followed by downregulation of IL1β expression in LPS/ATP-stimulated RAW264.7 macrophages. Similarly, treatment of CoQ0 led to LC3-I/II accumulation and p62/SQSTM1 activation. An increase in the Beclin-1/Bcl-2 ratio and a decrease in the expression of phosphorylated PI3K/AKT, p70 S6 kinase, and mTOR showed that autophagy was activated. Besides, CoQ0 increased Parkin protein to recruit damaged mitochondria and induced mitophagy in LPS/ATP-stimulated RAW264.7 macrophages. CoQ0 inhibited LPS/ATP-stimulated ROS generation in RAW264.7 macrophages. Notably, when LPS/ATP-stimulated RAW264.7 macrophages were treated with CoQ0, Mito-TEMPO (a mitochondrial ROS inhibitor), or N-acetylcysteine (NAC, a ROS inhibitor), there was a significant reduction of LPS/ATP-stimulated NLRP3 inflammasome activation and IL1β expression. Interestingly, treatment with CoQ0 or Mito-TEMPO, but not NAC, significantly increased LPS/ATP-induced LC3-II accumulation indicating that mitophagy plays a key role in the regulation of CoQ0-inhibited NLRP3 inflammasome activation. Nrf2 knockdown significantly decreased IL1β expression in LPS/ATP-stimulated RAW264.7 macrophages suggesting that CoQ0 inhibited ROS-mediated NLRP3 inflammasome activation and IL1β expression was suppressed due to the Nrf2 activation. Hence, this study showed that CoQ0 might be a promising candidate for the therapeutics of inflammatory disorders due to its effective anti-inflammatory as well as antioxidant properties.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Coenzyme Q0 (CoQ0) suppresses NO production in LPS- or LPS/ATP-stimulated RAW264.7 macrophages. (a) Structure of Coenzyme Q0 (CoQ0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone). (b) MTT assay carried out by treating RAW264.7 cells with CoQ0 (2.5-20 μM) for 24 h. (c, d) Production of NO was estimated by measuring the nitrite formation and stable end-metabolic of NO in the culture medium. Prior to NO estimation, cells were treated with different doses of CoQ0 ranging from 2.5 to 12.5 μM for 1 h, and then, LPS (1 μg/mL) was stimulated for 18 h or LPS (1 μg/mL) for 17 h followed by 5 mM ATP treatment for 1 h. The results were calculated as the mean ± SD of three independent experiments. ∗∗p < 0.01;  ∗∗∗p < 0.001, compared with untreated control cells, and ##p < 0.01; ###p < 0.001 compared with LPS or LPS/ATP-stimulated cells assigned as statistically significant.
Figure 2
Figure 2
CoQ0 inhibits NLRP3 inflammasome activation in LPS/ATP-stimulated RAW264.7 macrophages. Cells were first treated with CoQ0 (2.5-10 μM) and later stimulated with LPS (1 μg/mL) for 5 h followed by ATP (5 mM) treatment for 1 h. (a) The expression of NLRP3 and procaspase-1 protein was determined by Western blotting. (b) Caspase-1 activity was measured using a caspase-1 activity assay kit. (c) Pro-IL1β expressions in both lysis and supernatant and mature-IL1β in the supernatant were determined by Western blot using β-actin as internal control as well as band intensities were calculated by AlphaEaseFCTM (Genetic Technologies, Inc., Florida, USA) software. The data were calculated from the mean ± standard deviation (SD) of three independent experiments and ∗∗∗p < 0.001, compared with untreated control cells, and ###p < 0.001 compared with LPS/ATP-stimulated cells which was significant.
Figure 3
Figure 3
CoQ0 induces autophagy in LPS/ATP-stimulated RAW264.7 macrophages. Cells were first treated with 2.5-10 μM of CoQ0 and/or 2.5 mM 3-MA (autophagy inhibitor) for 1 h and then followed by stimulation of LPS (1 μg/mL) for 5 h and ATP (5 mM) for 1 h. (a) The expression of LC3-I/LC3-II and p62/SQSTM1 protein was estimated by Western blot. (b) The modifications in LC3B expression were observed by immunofluorescence staining. Cells were incubated with anti-LC3B antibody followed by secondary antibody labelled with FITC. 630x magnification of a confocal microscope was used to visualize the subcellular localization of LC3B. (c) Fold changes in LC3B were determined. (d) CoQ0 affects Beclin-1 and Bcl-2 expression in a dose-dependent manner shown by Western blot analysis. (e) Beclin-1/Bcl-2 ratio relative changes were measured by commercial software representing control as 1-fold. The results were calculated as the mean ± SD of three independent experiments. ∗p < 0.05;  ∗∗p < 0.01;  ∗∗∗p < 0.001, compared with untreated control cells, and #p < 0.05; ##p < 0.01; ###p < 0.001 compared with 3-MA- or LPS/ATP-stimulated cells assigned statistically significant.
Figure 4
Figure 4
CoQ0 induces mitophagy in LPS/ATP-stimulated RAW264.7 macrophages. (a) Time-dependent expression of p-PI3K, PI3K, p-AKT, AKT, p-p70 S6 kinase, p70 S6 kinase, p-mTOR, and mTOR was determined by Western blot. Cells were first treated with CoQ0 (10 μM) for 0-60 min and then stimulated with LPS (1 μg/mL) for 5 h followed by ATP (5 mM) for 1 h. (b) Dose-dependent expression of p-p70 S6 kinase and p-AKT was determined by Western blot. Cells were pretreated with CoQ0 (2.5-10 μM) for 60 min and then LPS (1 μg/mL) stimulation for 5 h followed by ATP (5 mM) for 1 h, and the changes in the intensities of protein bands were measured by commercial quantitative software. (c) The cells were first treated with CoQ0 (2.5-10 μM) for 60 min and then stimulated with LPS (1 μg/mL) for 5 h followed by ATP (5 mM) for 1 h, and lastly, expression of Parkin and PINK1 was determined by Western blot.
Figure 5
Figure 5
CoQ0 inhibits NLRP3 inflammasome activation through autophagy induction in LPS/ATP-stimulated RAW264.7 macrophages. (a) Cells were pretreated with CoQ0 (10 μM) and/or autophagy inhibitor 3-MA (2.5 mM) for 1 h and then stimulated with LPS (1 μg/mL) for 5 h followed by ATP (5 mM) for 1 h. NLRP3, procaspase-1, pro-IL1β, and LC3-I/II were determined by Western blot. (b) LC3 knockdown attenuated the protective effects of CoQ0. Cells were first transfected with siRNA that is specific to either LC3B or a nonsilencing control then pretreated with CoQ0 (10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 5 h followed by ATP (5 mM) for 1 h, and the expression of LC3-I/II or pro-IL1β proteins in both control and siLC3B was determined using Western blot analysis.
Figure 6
Figure 6
CoQ0 attenuates LPS/ATP-stimulated ROS generation in RAW264.7 macrophages. (a) Cells were pretreated with CoQ0 (10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 5 h followed by ATP (5 mM) for 1 h. The level of intracellular ROS was measured by DCF fluorescence using fluorescence microscopy (200x magnification). (b) Data are presented as fold change, and the results were calculated as the mean ± SD of three experiments, where ∗∗∗p < 0.001, compared with untreated control cells, and ###p < 0.001 compared with LPS/ATP-stimulated cells.
Figure 7
Figure 7
CoQ0 inhibits ROS-mediated NLRP3 inflammasome activation through autophagy induction and Nrf2 activation in LPS/ATP-stimulated RAW264.7 macrophages. Cells were pretreated with CoQ0 (2.5-10 μM), Mito-TEMPO (0.5 mM), or NAC (2 mM) for 1 h and then stimulated with LPS (1 μg/mL) for 5 h followed by ATP (5 mM) for 1 h. (a) Immunofluorescence staining of RAW264.7 cells and the nuclear localization of NLRP3 were visualized by the immunofluorescence method. Cells were stained with DAPI (1 μg/mL) for 5 min and examined by fluorescence microscopy. (b) The expression of pro-IL1β or LC3-I/II proteins was measured by Western blot analysis. (c) Nrf2 knockdown attenuated the protective effects of CoQ0. Cells were transfected with siRNA that is specific to either Nrf2 or a nonsilencing control. Transfected cells were pretreated with CoQ0 (2.5-10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 5 h followed by ATP (5 mM) for 1 h, and the expression of Nrf2 or pro-IL1β proteins in both control and siNrf2 was measured by Western blot analysis.
Figure 8
Figure 8
Graphical summary. Collectively, our results showed that subcytotoxic treatments of macrophages with CoQ0 displayed antioxidant and anti-inflammatory properties. CoQ0 inhibited the NLRP3 inflammasome and procaspase-1 activation which in turn suppressed pro-IL1β expression levels. On the other hand, CoQ0 exposure in LPS/ATP-stimulated macrophages incited autophagy which was evident by the accumulation of LC3-II and p62/SQSTM1 as well as dysregulation of Beclin1/Bcl-2. This was accompanied by reduced phosphorylation of PI3K/AKT, p70 S6 kinase, and mTOR. Besides, CoQ0 inhibited ROS-mediated NLRP3 inflammasome activation through mitophagy induction and Nrf2 activation in LPS/ATP-stimulated macrophages. Altogether, we propose that CoQ0 might be a promising candidate for the therapeutics of inflammatory abnormalities due to its effective anti-inflammatory as well as antioxidant properties.
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