CHAPTER Ⅱ Ferroptosis affects the progression of non-alcoholic steatohepatitis via the modulation of lipid peroxidation-mediated cell death in miceABSTRACT BACKGROUND&AIMS: Oxidative stress and its-associated lipid peroxidation play a key role in non-alcoholic steatohepatitis (NASH). Ferroptosis is a recently recognized type of cell death characterized by an iron dependent and lipid peroxidation-mediated non-apoptotic cell death. METHODS: We herein try to demonstrate the impact of ferroptosis on the progression of NASH induced by methionine/choline-deficient diet (MCD) feeding for 10 days. RESULTS: RSL-3 (a ferroptosis inducer) treatment showed decreased hepatic expression of GPX4, and conversely increased 12/15-lipoxygenase, and apoptosis inducing factor, indicating that ferroptosis plays a key role in NASH-related lipid peroxidation and its-associated cell death. Consistently, levels of serum biochemical, hepatic steatosis, inflammation and apoptosis in MCD-fed mice were exacerbated with RSL-3 treatment. However, MCD-fed mice treated with sodium selenite (a GPX4 activator) showed increase of hepatic GPX4, accompanied by reduced NASH severity. To chelate iron, we used deferoxamine mesylate salt (DFO). DFO administration significantly reduced NASH severity, and abolished the harmful effects of RSL-3 in MCD-fed mice. Finally, Liproxstatin-1 (a ferroptosis inhibitor) treatment showed repressed hepatic lipid peroxidation, and its associated cell death resulting in decreased NASH severity. Consistent with the in vivo findings, modulation of ferroptosis/GPX4 affected hepatocellular death in palmitic acid-induced in vitro NASH milieu. CONCLUSIONS: We conclude that GPX4 and its-related ferroptosis might play a major role in the development of NASH.
CHAPTER Ⅲ Amlexanox ameliorates acetaminophen-induced acute liver injury by reducing oxidative stress in mice ABSTRACTBACKGROUND&AIMS: Amlexanox, a clinically approved small-molecule therapeutic presently used to treat allergic rhinitis, ulcer, and asthma, is an inhibitor of the non-canonical IkB kinase-ε (IKKε) and TANK-binding kinase 1 (TBK1). This study was to investigate the protective mechanism of amlexanox in acetaminophen (APAP)-induced acute liver injury (ALI). METHODS: Mice were intraperitoneally injected with APAP (300 mg/kg, 12 hours) to induce ALI and were orally administrated with amlexanox (25, 50, and 100 mg/kg) one hour after APAP treatment. RESULTS: Inhibition of IKKε and TBK1 by treatment of amlexanox attenuated APAP-induced ALI as confirmed by decreased serum levels of aspartate aminotransferase and alanine aminotransferase. Furthermore, amlexanox significantly decreased hepatocellular apoptosis in injured livers of mice as evidenced by histopathologic observation. Consistently, reduced oxidative stress by amlexanox was observed by increased hepatic glutathione concomitant with decreased levels of malondialdehyde. Amlexanox also enhanced expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) target genes including heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1, and glutamate-cysteine ligase in injured livers of mice. Mechanistic insights into the mode of action of amlexanox against APAP-induced hepatotoxicity were involved in increasing phosphorylation of AMP-activated protein kinase (AMPK) and nuclear translocation of Nrf2, both in vivo and in vitro. Furthermore, the protective effects of amlexanox on APAP-induced hepatotoxicity were abolished by compound C, an AMPK inhibitor. CONCLUSIONS: Taken together, our findings suggest that amlexanox exerts antioxidative activities against APAP-mediated hepatotoxicity via AMPK/Nrf2 pathway.
CHAPTER Ⅳ Modulation of PPARγ activity affects carbon tetrachloride-induced acute liver injury in mice 1ABSTRACTBACKGROUND&AIMS: The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor that regulates glucose and lipid metabolism. The present study aimed to investigate the effect of PPARγ activation on carbon tetrachloride (CCl4)-induced acute liver injury (ALI) in vivo and in vitro and the underlying molecular mechanisms.METHODS: To induce ALI in mice, a single intraperitoneal injection of CCl4 was administered. Before CCl4 treatment, mice were treated with PPARγ agonist rosiglitazone (RSG) and/or PPARγ antagonist SR202. Mice were sacrificed 6, 12, and 24 h post CCl4 administration. An in vitro system using mouse primary hepatocytes was also applied.RESULTS: Significantly increased protein levels of PPARγ were observed in injured livers. PPARγ activation markedly attenuated liver injury, which was confirmed by histopathologic analysis, decreased serum levels of aspartate aminotransferase and alanine aminotransferase. Consistently, reduced oxidative stress by PPARγ activation was shown by increased hepatic glutathione concomitant with decreased levels of malondialdehyde. Mechanistically, activated PPARγ suppresses oxidative stress in injured livers by inducing autophagy, as evidenced by increased conversion of microtubule-associated protein 1 light chain 3B (LC3B)-II from LC3B-I and decreased protein expression of p62. Additionally, PPARγ activation inhibited the phosphorylation of c-Jun amino-terminal kinase (JNK) in livers of mice treated with CCl4. However, PPARγ antagonist reversed the beneficial effects of PPARγ agonist in CCl4-induecd ALI in mice. Furthermore, similar results were found in mouse primary hepatocytes. Finally, treatment with 3-methyladenine, an autophagy inhibitor, partially abolished the protection of PPARγ activation in the injured liver.CONCLUSIONS: The present findings demonstrated that activation of PPARγ reduces oxidative stress induced by CCl4 hepatotoxicity in mice, which may be closely associated with induction of autophagy. Also, PPARγ activity could become an important target to attenuate ALI.