Autophagy activation by Jiang Zhi Granule protects against metabolic stress-induced hepatocyte injury

Abstract

AIMTo elucidate the potential role of autophagy and the protective effects of Jiang Zhi Granule (JZG) in metabolic stress-induced hepatocyte injury.METHODSAn in vitro and in vivo approach was used in this study. HepG2 cells were incubated in culture medium containing palmitate (PA; 0, 0.1, 0.2, 0.3, 0.4 or 0.5 mmol/L) and treated with or without JZG (100 μg/mL) for 24 h or 48 h, and the progression of autophagy was visualized by stable fluorescence-expressing cell lines LC3 and p62. Western blot analyses were performed to examine the expression of LC3-II/LC3-I, p62, mTOR and PI3K, while mitochondrial integrity and oxidative stress were observed by fluorescence staining of JC-1 and reactive oxygen species. C57BL/6 mice were divided into three groups: control group (n = 10), high fat (HF) group (n = 13) and JZG group (n = 13); and, histological staining was carried out to detect inflammation and lipid content in the liver.RESULTSThe cell trauma induced by PA was aggravated in a dose- and time-dependent manner, and hepatic function was improved by JZG. PA had dual effects on autophagy by activating autophagy induction and blocking autophagic flux. The PI3K-AKT-mTOR signaling pathway and the fusion of isolated hepatic autophagosomes and lysosomes were critically involved in this process. JZG activated autophagy progression by either induction of autophagosomes or co-localization of autophagosomes and lysosomes as well as degradation of autolysosomes to protect against PA-induced hepatocyte injury, and protected mitochondrial integrity against oxidative stress in PA-induced mitochondrial dysfunction. In addition, JZG ameliorated lipid droplets and inflammation induced by HF diet in vivo, leading to improved metabolic disorder and associated liver injury in a mouse model of non-alcoholic fatty liver disease (NAFLD).CONCLUSIONMetabolic stress-induced hepatocyte injury exhibited dual effects on autophagy and JZG activated the entire process, resulting in beneficial effects in NAFLD.

Systems pharmacology-based exploration reveals mechanisms of anti-steatotic effects of Jiang Zhi Granule on non-alcoholic fatty liver disease

“…Hepatocytes were incubated with various concentrations of palmitic acid (PA) as well as in the presence or absence of JZG. Biochemical analyses confirmed the findings of our previous study 21 , in which the levels of alanine aminotransferase (ALT) in the cell supernatants were increased in a dose- and time-dependent manner, and JZG could mitigate the elevated ALT levels (Fig. 1A,B ).…”

“…A great number of signalling pathways have been discovered during the past decade, and we postulated that the mTOR signalling was critically involved in the observed activities of JZG-treated cells. Unlike our previous findings 21 , we confirmed that JZG could inhibit the mTOR activation to up-regulate the IRS-1/PI3K/AKT signalling conversely as well as to activate autophagy. Fluorescence staining with BODIPY and Lyso-Tracker indicated that JZG could promote the co-localization of lipid droplets and lysosomes for degradation, and we also measured the MTP and ROS generation to demonstrate that JZG could protect mitochondrial integrity against oxidative stress.…”

“…At three weeks after diet and drug administration, sodium pentobarbital was given as anaesthesia to the mice. Blood samples were drawn via cardiac puncture, while livers were excised as described previously 21 .…”

“…Density gradient centrifugation with Nycodenz was performed to purify the hepatocytes, and 6-well plates were processed with gelatine to promote cell attachment. Hepatocytes were cultured following established protocols and infected with lentivirus as described previously 21 .…”

Five Constituents in Psoralea corylifolia L. Attenuate Palmitic Acid-Induced Hepatocyte Injury via Inhibiting the Protein Kinase C-α/Nicotinamide-Adenine Dinucleotide Phosphate Oxidase Pathway

“…Nicotinamideadenine dinucleotide phosphate oxidase (NOX) is considered the major cellular ROS source (Loffredo et al, 2016;Long et al, 2017;Rabelo et al, 2018). Its activation has been associated with hepatic injury (Matsumoto et al, 2018;Zheng et al, 2018) and also plays a very plausible role as the starting point of extrahepatic damage, leading to inflammation and fibrosis through activation of Kupffer cells and hepatic stellate cells (HSCs) (Das et al, 2015;, thus causing a self-perpetuating circle of ROS formation and ROS-mediated damage (Masarone et al, 2018;Zhong and Liu, 2018). Therefore, NOX-specific antioxidant therapies may be a promising intervention to prevent or to treat NASH.…”