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

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.

Microbe-Derived Antioxidants Reduce Lipopolysaccharide-Induced Inflammatory Responses by Activating the Nrf2 Pathway to Inhibit the ROS/NLRP3/IL-1β Signaling Pathway

“…Cells (1 × 10 4 cells/well) were planted in 96-well plate overnight and then treated with different concentrations of MA (0-500 mg/L) for 12 h, followed by stimulation with or without LPS for another 24 h. Inflammation model was constructed using 1 mg/L LPS to stimulate RAW264.7 cells [5]. After discarding supernatant, the treated cells were rinsed three times with DPBS, followed by addition of 100 µL medium and 10 µL CCK-8, and incubated for two hours at 37 • C. An absorbance measurement at 490 nm was conducted, and the results were presented as a fold change compared with the untreated group.…”

“…Lipopolysaccharide (LPS) is an exogenous endotoxin found in Gram-negative bacteria’s outer cell membrane, which causes inflammation. It has been extensively used to stimulate inflammation in previous studies [ 5 ]. Among the significant immune cells involved in the inflammatory response, macrophages play an important role in initiating, maintaining, and resolving inflammation [ 1 ].…”

“…It is widely accepted that inflammation is associated with oxidative stress and that elevated levels of intracellular ROS are considered to be the most potent mediators of inflammation [ 8 ]. LPS-activated macrophages lead to excessive production of ROS [ 5 ]. ROS overproduction also functions as a signal to activate the nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome [ 9 ].…”

“…When activated, caspase-1 creates precursors of the pro-inflammatory cytokines IL-18 and IL-1β, which are then matured and secreted to participate in the resulting inflammatory response [ 9 , 10 ]. Studies have shown that reducing ROS inhibits the activation of NLRP3, indicating that oxidative stress plays a significant role in inflammation [ 5 , 11 ]. Nuclear factor (erythroid-derived-2)-like 2 (Nrf2) translocates to the nucleus and regulates the expression of antioxidant and anti-inflammatory factors in cells [ 11 , 12 ].…”

“…There is growing evidence that activation of Nrf2 blocks ROS overproduction and thus inhibits NLRP3 inflammasome activation. Inflammatory conditions can be effectively treated by targeting Nrf2 signaling [ 5 , 6 , 14 ].…”