NOX4 promotes Kupffer cell inflammatory response via ROS-NLRP3 to aggravate liver inflammatory injury in acute liver injury

Abstract

Aim: This work aimed to investigate the mechanism of NOX4 in promoting Kupffer cells (KCs) activation and tissue inflammatory response in acute liver injury. Methods: Initially, the mouse KCs were cultured in vitro. Thereafter, the NOX4 overexpression plasmid was transfected into KCs to construct the overexpression cell line. Then, KCs inflammatory response was induced by LPS + Nigericin treatment. CCK-8 assay was performed to detect cell viability, flow cytometry (FCM) was conducted to measure cell apoptosis, enzyme-linked immunosorbent assay (ELISA) was performed to detect inflammatory factor levels in the culture medium, NLRP3 and ASC expression in cells was detected by immunofluorescence (IF) staining, and ROS expression was detected by the DCFH-DA probe. Furthermore, the expression levels of NLRP3, ASC and Caspase-1 proteins were detected by Western-Blot (WB) assay. Furthermore, cells were pre-treated with NOX inhibitor or NAC to suppress NOX4 expression or ROS production, aiming to further investigate the effect on KCs inflammatory response. In mouse experiments, the NOX4 knockdown mice and wild-type (WT) mice were adopted for carrying out experiments. The mouse model of ALI was constructed with LPS and D-GalN treatment. Thereafter, the changes in tissue samples were detected by H&E staining, NLRP3 expression was measured by histochemical staining, inflammatory factors in tissues were analyzed by ELISA, and the levels of NLRP3, ASC and Caspase-1 proteins in tissues were detected by WB assay. Results: LPS induced KCs inflammatory response. NOX4 overexpression decreased the mouse viability and increased the apoptosis rate. The levels of inflammatory factors were up-regulated in the culture medium. In addition, ROS were activated, and the positive cell number increased. Moreover, NOX4 promoted NLRP3 activation and significantly increased the expression of NLRP3 and ASC. Pretreatment with NOX4 inhibitor or NAC antagonized the effects of NOX4 and suppressed the KCs inflammatory response. In the mouse model, NOX4 knockdown significantly suppressed the activation and inflammatory response of microglial cells in tissues, reducing the NLRP3 expression in tissues. Conclusion: NOX4 activates the NLRP3 inflammasome via ROS to promote inflammatory response in KCs and the release of inflammatory factors, suppressing NOX4 can improve ALI in mice, and NOX4 is promising as a new target for ALI treatment.

Mitochondrial dysfunction: A promising therapeutic target for liver diseases

“…Some ROS-producing oxidases, such as NADPH oxidase 4 and the 66-kDa isoform of Shc (p66Shc) promote the activation of liver Kupffer cells and HSCs by activating NLRP3, finally leading to liver inflammatory damage and fibrosis. 95 , 96 Pharmacological induction of HSC apoptosis is a feasible strategy to promote fibrosis regression, Li et al reported that forsythiaside A has anti-fibrosis potential by remolding extracellular matrix and improving oxidation stress to promote apoptosis of HSCs in vitro . Forsythiaside A down-regulates the NADPH oxidase 4/ROS signaling pathway to improve oxidation imbalance, decreases collagen-1 and α -SMA expression, and increases the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2.…”

Investigating the Link between Ketogenic Diet, NAFLD, Mitochondria, and Oxidative Stress: A Narrative Review

“…The expression of NOX4 and related ROS generation was significantly increased during development of steatohepatitis in mice [156]. As recently reported by Zhai and colleagues in mouse model of liver injury, NOX4 activates the NLRP3 inflammasome and promotes inflammatory response in KCs by releasing of inflammatory factors, such as IL-6, IL-1β and TNF-α, speculating that NOX4 could be considered as a key factor in inflammatory response [157].…”

Redox Regulation of Microglial Inflammatory Response: Fine Control of NLRP3 Inflammasome through Nrf2 and NOX4

“…It has been demonstrated by different authors that there is a link between NOX4 activity and NLRP3 inflammasome activation. In Kupffer cells, NOX4-derived ROS promoted NLRP3 activation and significantly increased the expression of inflammasome components both in vitro and in vivo, aggravating liver inflammatory injury [ 27 ]. This mechanism has also been observed in various models of inflammation, such as acute pancreatitis [ 28 ], in high-glucose-induced endothelial dysfunction [ 29 ], in osteoarthritis [ 30 ], and in myocardial ischemia-reperfusion injury [ 31 ].…”