Quercetin Protects Ethanol-Induced Hepatocyte Pyroptosis via Scavenging Mitochondrial ROS and Promoting PGC-1α-Regulated Mitochondrial Homeostasis in L02 Cells

Abstract

Alcoholic liver disease (ALD) is a multifaceted process that involves excessive lipid, reactive oxygen species (ROS) production, unbalanced mitochondrial homeostasis, and ultimate cell death. Quercetin is a dietary phytochemical presented in various fruits and vegetables, which has anti-inflammatory and antioxidant effects. According to recent advances in pharmanutritional management, the effects of quercetin on various mitochondrial processes have attracted attention. In the study, we explored whether quercetin could attenuate ethanol-induced hepatocyte pyroptosis by maintaining mitochondrial homeostasis and studied its hepatoprotective effect and the underlying mechanism. We chose L02 cells to establish an in vitro model with ethanol-induced hepatocyte pyroptosis. Then, the cells at approximately 80% confluence were treated with quercetin (80, 40, and 20 μM). The cell viability (CCK-8) was used to investigate the effect of quercetin on ethanol-induced L02 cell proliferation. Relative assay kits were used to measure oxidative stress index ( OSI = TOS / TAS ), lipid peroxidation (LPO) release, and mitochondrial membrane potential ( δ Ψ m ). The morphology of mitochondria was characterized by transmission electron microscopy- (TEM-) based analysis. Mitochondrial dynamics (Mito Tracker Green), mitROS (MitoSOX Red Mitochondrial Superoxide) production, and nuclear DNA (nDNA) damage (γH2AX) markers were detected by immunofluorescence. The mRNA levels of mitochondrial function (including mitochondrial DNA (mtDNA) transcription genes TWNK, MTCO1, and MFND) and pyroptosis-related genes were detected by RT-qPCR, and the protein levels of NLRP3, ASC, caspase1, cleaved-caspase1, IL-18, IL-1β, and GSDMD-N were detected by western blot. The results showed that quercetin treatment downregulated redox status, lipid droplets, and LPO release, restored damaged mitochondrial membrane potential, and repaired mtDNA damage, PGC-1α nuclear transfer, and mitochondrial dynamics. The gene and protein expressions of NLRP3, ASC, cleaved-caspase1, IL-18, IL-1β, and GSDMD-N were decreased, which effectively inhibited cell pyroptosis. Therefore, the results indicated that quercetin protected ethanol-induced hepatocyte pyroptosis via scavenging mitROS and promoting PGC-1α-mediated mitochondrial homeostasis in L02 cells.

Kaempferol prevents acetaminophen-induced liver injury by suppressing hepatocyte ferroptosis via Nrf2 pathway activation

“…The CCK8 assay was performed as described previously. 48 The L02 cells were plated into 96-well plates with a complete medium and treated with different concentrations of KA (0, 5, 10 and 20 μM) and APAP (5 mM) for 24 h. After treatments, a CCK8 reagent was added into each well, followed by incubation for another 2 h in a 37 °C incubator with 5% CO 2 . Then, the absorbance was measured using a microplate reader at 450 nm (Bio-Rad, Hercules, CA, USA), and the cell viability was expressed as percentage values, as compared with the control group.…”

Remodeling the hepatic fibrotic microenvironment with emerging nanotherapeutics: a comprehensive review

“…Whereas collagen fibers between adjacent hepatic blood sinusoids contact each other and encircle hepatocytes in a grid pattern, which weakens oxygen exchange between hepatic sinusoids and hepatocytes, therefore leads to hypoxia in the microenvironment of liver fibrosis [75]. In addition, abnormal immune inflammation leads to the release of inflammatory factors, chemokines, ROS, adipokines, and pro-angiogenic mediators [76]. Among them, intracellular NO and ROS reactions are significantly increased in extracellular matrix (ECM) subjected to peroxynitrite (ONOO-)-mediated oxidization, and MMP-2 activity, directly mediated by S-glutathionylation of its cysteine residues in the presence of ONOO(-) and by phosphorylation of its serine and threonine residues [77], as well as increased fibrosis [78].…”

Alcohol and the mechanisms of liver disease

“…78,79 Several mitochondrial-targeted antioxidants, such as MitoQ, have been shown to reduce oxidative stress and liver injury in preclinical studies. 80 Also, focusing on upstream of ROS generating pathways has been thought as options for drug development. Enzymes such as NADPH oxidases and xanthine oxidases are major sources of ROS in ALD.…”

“…Antioxidants such as vitamin E, N‐acetylcysteine, and S‐adenosylmethionine have been shown to reduce oxidative stress and improve liver function in patients with ALD 78,79 . Several mitochondrial‐targeted antioxidants, such as MitoQ, have been shown to reduce oxidative stress and liver injury in preclinical studies 80 . Also, focusing on upstream of ROS generating pathways has been thought as options for drug development.…”