Lipotoxicity causes hepatic cell death and therefore plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metformin, a first-line anti-diabetic drug, has shown a potential protective effect against NAFLD. However, the underlying mechanism is still not clear. In this study, we aim to understand the molecular mechanism of the protective effect of metformin in NAFLD, focusing on lipotoxicity. Cell death was studied in HepG2 cells and primary rat hepatocytes exposed to palmitate and metformin. Metformin ameliorated palmitate-induced necrosis and apoptosis (decreased caspase-3/7 activity by 52% and 57% respectively) in HepG2 cells. Metformin also reduced palmitate-induced necrosis in primary rat hepatocytes (P < 0.05). The protective effect of metformin is not due to reducing intracellular lipid content or activation of AMPK signaling pathways. Metformin and a low concentration (0.1 μmol/L) of rotenone showed moderate inhibition on mitochondrial respiration indicated by reduced basal and maximal mitochondrial respiration and proton leak in HepG2 cells. Moreover, metformin and rotenone (0.1 μmol/L) preserved mitochondrial membrane potential in both HepG2 cells and primary rat hepatocytes. In addition, metformin and rotenone (0.1 μmol/L) also reduces reactive oxygen species (ROS) production and increase superoxide dismutase 2 (SOD2) expression. Our results establish that metformin AMPK-independently protects against palmitate-induced hepatic cell death by moderate inhibition of the mitochondrial respiratory chain, recovering mitochondrial function, decreasing cellular ROS production, and inducing SOD2 expression, indicating that metformin may have beneficial actions beyond its glucose-lowering effect and also suggests that mitochondrial complex І may be a therapeutic target in NAFLD.

The number of patients with non-alcoholic fatty liver disease (NAFLD) is rapidly increasing due to the growing epidemic of obesity. Non-alcoholic steatohepatitis (NASH), the inflammatory stage of NAFLD, is characterized by lipid accumulation in hepatocytes, chronic inflammation and hepatocyte cell death. Scopoletin and umbelliferone are coumarin-like molecules and have antioxidant, anti-cancer and anti-inflammatory effects. Cytoprotective effects of these compounds have not been described in hepatocytes and the mechanisms of the beneficial effects of scopoletin and umbelliferone are unknown. To investigate whether scopoletin and/or umbelliferone protect hepatocytes against palmitate-induced cell death. For comparison, we also tested the cytoprotective effect of scopoletin and umbelliferone against bile acid-induced cell death. Primary rat hepatocytes were exposed to palmitate (1 mmol/L) or the hydrophobic bile acid glycochenodeoxycholic acid (GCDCA; 50 μmol/L). Apoptosis was assessed by caspase-3 activity assay, necrosis by Sytox green assay, mRNA levels by qPCR, protein levels by Western blot and production of reactive oxygen species (ROS) by fluorescence assay. Both scopoletin and umbelliferone protected against palmitate and GCDCA-induced cell death. Both palmitate and GCDCA induced the expression of ER stress markers. Scopoletin and umbelliferone decreased palmitate- and GCDCA-induced expression of ER stress markers, phosphorylation of the cell death signaling intermediate JNK as well as ROS production. Scopoletin and umbelliferone protect against palmitate and bile acid-induced cell death of hepatocytes by inhibition of ER stress and ROS generation and decreasing phosphorylation of JNK. Scopoletin and umbelliferone may hold promise as a therapeutic modality for the treatment of NAFLD.

Acetaminophen (APAP)-induced acute liver failure (ALF) is a serious health problem in developed countries. N-acetyl-L-cysteine (NAC), the current therapy for APAP-induced ALF, is not always effective, and liver transplantation is often needed. Opuntia spp. fruits are an important source of nutrients and contain high levels of bioactive compounds, including antioxidants. The aim of this study was to evaluate the hepatoprotective effect of Opuntia robusta and Opuntia streptacantha extracts against APAP-induced ALF. In addition, we analyzed the antioxidant activities of these extracts. Fruit extracts (800mg/kg/day, orally) were given prophylactically to male Wistar rats before intoxication with APAP (500 mg/kg, intraperitoneally). Rat hepatocyte cultures were exposed to 20mmol/LAPAP, and necrosis was assessed by LDH leakage. Opuntia robusta had significantly higher levels of antioxidants than Opuntia streptacantha. Both extracts significantly attenuated APAP-induced injury markers AST, ALT and ALP and improved liver histology. The Opuntia extracts reversed APAP-induced depletion of liver GSH and glycogen stores. In cultured hepatocytes, Opuntia extracts significantly reduced leakage of LDH and cell necrosis, both prophylactically and therapeutically. Both extracts appeared to be superior to NAC when used therapeutically. We conclude that Opuntia extracts are hepatoprotective and can be used as a nutraceutical to prevent ALF.

Acetaminophen (APAP) misuse or overdose is the most important cause of drug-induced acute liver failure. Overdoses of acetaminophen induce oxidative stress and liver injury by the electrophilic metabolite N-acetyl-p-benzoquinone imine (NAPQI). Plant-based medicine has been used for centuries against diseases or intoxications due to their biological activities. The aim of this study was to evaluate the therapeutic value of Opuntia robusta and Opuntia streptacantha fruit extracts against acetaminophen-induced liver damage and to identify the major biocomponents on them. Opuntia fruit extracts were obtained by peeling and squeezing each specie, followed by lyophilization. HPLC was used to characterize the extracts. The effect of the extracts against acetaminophen-induced acute liver injury was evaluated both in vivo and in vitro using biochemical, molecular and histological determinations. The results showed that betacyanins are the main components in the analyzed Opuntia fruit extracts, with betanin as the highest concentration. Therapeutic treatments with Opuntia extracts reduced biochemical, molecular and histological markers of liver (in vivo) and hepatocyte (in vitro) injury. Opuntia extracts reduced the APAP-increased expression of the stress-related gene Gadd45b. Furthermore, Opuntia extracts exerted diverse effects on the antioxidant related genes Sod2, Gclc and Hmox1, independent of their ROS-scavenging ability. Therefore, betacyanins as betanin from Opuntia robusta and Opuntia streptacantha fruits are promising nutraceutical compounds against oxidative liver damage.

Coumarin derivates have been proposed as a potential treatment for metabolic-dysfunction-associated fatty liver disease (MAFLD). However, the mechanisms underlying their beneficial effects remain unclear. In the present study, we explored the potential of the coumarin derivate esculetin in MAFLD, focusing on hepatocyte lipotoxicity and lipid accumulation. Primary cultures of rat hepatocytes were exposed to palmitic acid (PA) and palmitic acid plus oleic acid (OA/PA) as models of lipotoxicity and lipid accumulation, respectively. Esculetin significantly reduced oxidative stress in PA-treated hepatocytes, as shown by decreased total reactive oxygen species (ROS) and mitochondrial superoxide production and elevated expression of antioxidant genes, including Nrf2 and Gpx1. In addition, esculetin protects against PA-induced necrosis. Esculetin also improved lipid metabolism in primary hepatocytes exposed to nonlipotoxic OA/PA by decreasing the expression of the lipogenesis-related gene Srebp1c and increasing the expression of the fatty acid β-oxidation-related gene Ppar-α. Moreover, esculetin attenuated lipid accumulation in OA/PA-treated hepatocytes. The protective effects of esculetin against lipotoxicity and lipid accumulation were shown to be dependent on the inhibition of JNK and the activation of AMPK, respectively. We conclude that esculetin is a promising compound to target lipotoxicity and lipid accumulation in the treatment of MAFLD.

Lipotoxicity plays a critical role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Hesperetin, a flavonoid derivative, has anti-oxidant, anti-inflammatory and cytoprotective properties. In the present study, we aim to examine whether hesperetin protects against palmitate-induced lipotoxic cell death and to investigate the underlying mechanisms in hepatocytes. Primary rat hepatocytes and HepG2 cells were pretreated with hesperetin for 30 min and then exposed to palmitate (1.0 mmol/L in primary rat hepatocytes; 0.5 mmol/L in HepG2 cells) in the presence or absence of hesperetin. Necrotic cell death was measured via Sytox green nuclei staining and quantified by LDH release assay. Apoptotic cell death was determined by caspase 3/7 activity and the protein level of cleaved-PARP. The unfolded protein response (UPR) was assessed by measuring the expression of GRP78, sXBP1, ATF4 and CHOP. Results show that hesperetin (50 μmol/L and 100 μmol/L) protected against palmitate-induced cell death and inhibited palmitate-induced endoplasmic reticulum (ER) stress in both primary rat hepatocytes and HepG2 cells. Hesperetin (100 μmol/L) significantly activated sXBP1/GRP78 signaling, whereas a high concentration of hesperetin (200 μmol/L) activated p-eIF2α and caused hepatic cell death. Importantly, GRP78 knockdown via siRNA abolished the protective effects of hesperetin in HepG2 cells. In conclusion, hesperetin protected against palmitate-induced hepatic cell death via activation of the sXBP1/GRP78 signaling pathway, thus inhibiting palmitate-induced ER stress. Moreover, high concentrations of hesperetin induce ER stress and subsequently cause cell death in hepatocytes.