Apigenin Alleviated High-Fat-Diet-Induced Hepatic Pyroptosis by Mitophagy-ROS-CTSB-NLRP3 Pathway in Mice and AML12 Cells

Abstract

Apigenin is considered the most-known natural flavonoid and is abundant in a wide variety of fruits and vegetables. A high fat diet (HFD) can induce liver injury and hepatocyte death in multiple ways. Pyroptosis is an innovative type of programmed cell death. Moreover, excessive pyroptosis of hepatocytes leads to liver injury. We used HFD to induce liver cell pyroptosis in C57BL/6J mice in this work. After gavage of apigenin, apigenin can significantly reduce the level of lactate dehydrogenase (LDH) in liver tissue ignited by HFD and reduce the levels of NLRP3 (NOD-like receptor family pyrin domain containing 3), the N-terminal domain of GSDMD (GSDMD-N), cleaved-caspase 1, cathepsin B (CTSB), interleukin-1β (IL-1β) and interleukin-18 (IL-18) protein expression and the colocalization of NLRP3 and CTSB and increase the level of lysosomal associated membrane protein-1 (LAMP-1) protein expression, thus alleviating cell pyroptosis. In a further in vitro mechanism study, we find that palmitic acid (PA) can induce pyroptosis in AML12 cells. After adding apigenin, apigenin can clear the damaged mitochondria through mitophagy and reduce the generation of intracellular reactive oxygen species (ROS), thus alleviating CTSB release caused by lysosomal membrane permeabilization (LMP), reducing the LDH release caused by PA and reducing the levels of NLRP3, GSDMD-N, cleaved-caspase 1, CTSB, IL-1β, and IL-18 protein expression. By adding the mitophagy inhibitor cyclosporin A (CsA), LC3-siRNA, the CTSB inhibitor CA-074 methyl ester (CA-074 Me), and the NLRP3 inhibitor MCC950, the aforementioned results were further confirmed. Therefore, our results show that HFD-fed and PA can damage mitochondria, promote the production of intracellular ROS, enhance the lysosomal membrane permeabilization (LMP), and cause the leakage of CTSB, thus activating the NLRP3 inflammatory body and inducing pyroptosis in C57BL/6J mice and AML12 cells, while apigenin alleviates this phenomenon through the mitophagy-ROS-CTSB-NLRP3 pathway.

Elevated PINK1/Parkin-Dependent Mitophagy and Boosted Mitochondrial Function Mediate Protection of HepG2 Cells from Excess Palmitic Acid by Hesperetin

“…In addition to energy and metabolism, mitochondria play a crucial role in regulating some key cellular processes, such as apoptosis and inflammation. A growing body of literature suggests that excessive ROS generated by defective mitochondria can result in the activation of NLRP3 inflammasome. , Recent studies have indicated the associations of PA-induced hepatocyte death with dysregulated inflammasome. NLRP3 inflammasome comprising multiple proteins could be activated by a wide variety of danger signals and provoke innate immune responses. , Assembly of the NLRP3 inflammasome activates caspase-1 via initiating cleavage of procaspase 1 into caspase-1, which subsequently converts the cytokine precursors pro-IL-1β into mature and biologically active IL-1β .…”

“…PINK1 acting as a molecular sensor detects signals of abnormal mitochondria for the recruitment and activation of Parkin. Through its E3 ubiquitin ligase activity, Parkin binds to the outer mitochondrial membrane, initiating multiple signaling events culminating in the engulfment of damaged mitochondria within lysosomes. , It has been shown that loss of PINK1 impairs mitophagy and that disabled mitophagy is one of the potential contributors to the onset of NAFLD. , In support of this, some studies reported inhibited PINK1/Parkin-mediated mitophagy in the livers of mice with NAFLD and PA-treated hepatocytes. , Conversely, restoring PINK1/Parkin-driven mitophagy rescued the mitochondrial dysfunction and preserved cellular integrity and homeostasis in hepatocytes. Here, our results connect the hepatoprotective effects of Hst to PINK1/Parkin-mediated mitophagy upregulation.…”

“…Constant exposure to high concentrations of ROS makes mitochondria particularly susceptible to oxidative damage, causing their structural and functional failure. , Therefore, cells employ a specific form of macroautophagy to selectively target and degrade abnormal mitochondria, which is known as mitophagy . Efficient mitophagy contributes to mitochondrial quality control and plays a fundamental role in tissues with a high need for ATP and dependence on mitochondrial energy production, such as the brain, heart, liver, and kidney. − Several previous studies have shown that mitophagy induction in hepatocytes enables the maintenance of adequate functional mitochondria and meets cellular energy demands, prevents FFAs-induced hepatocyte injury, and eventually alleviates the development of NAFLD. , As such, targeting mitochondrial function appears to be a viable approach to prevent liver injury and relieve the progression of NAFLD.…”

Screening of an FDA-approved compound library identifies apigenin for the treatment of myocardial injury

“…Indeed, previous studies had uncovered important mitoprotective actions of apigenin in several disease models 37 . For example, apigenin was reported to reduce palmitic acid-induced mitochondrial dysfunction through mitophagy activation, resulting in decreased hepatic pyroptosis 38 . During LPS-caused neurotoxicity, apigenin was shown to activate mitochondrial fusion, an important mechanism safeguarding mitochondrial homeostasis, through a mechanism involving the Sirt3/PINK1/Parkin pathway 39 .…”

The Intricate Balance between Life and Death: ROS, Cathepsins, and Their Interplay in Cell Death and Autophagy

“…Experiments on mice that were fed either a special diet or acid showed an increase in the level of reactive oxygen species. This led to the release of cathepsin B, which activated the NLRP3 inflammasome [154].…”