ShenFu Preparation Protects AML12 Cells Against Palmitic Acid-Induced Injury Through Inhibition of Both JNK/Nox4 and JNK/NFκB Pathways

Abstract

Background/Aims: Nonalcoholic steatohepatitis includes steatosis along with liver inflammation, hepatocyte injury and fibrosis. In this study, we investigated the protective role and the potential mechanisms of a traditional Chinese medicine ShenFu (SF) preparation in an in vitro hepatic steatosis model. Methods: In palmitic acid (PA)-induced murine hepatic AML12 cell injury, effects of SF preparation on cellular apoptosis and intracellular triglyceride (iTG) level were assessed using TUNEL and TG Colorimetric Assay. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels were measured using DCF and JC-1 assay. Cytokine levels were evaluated using ELISA assay. Immunoblot was used to compare the activation level of c-Jun N terminal kinase (JNK), NADPH oxidase (Nox4), and NFκB pathways. Results: Addition of SF preparation prevented PA-mediated increase of apoptosis and iTG as well as IL-8 and IL-6. In PA-treated cell, SF preparation reduced the level of Nox4 and ROS, while increasing the level of MMP and the expression of manganese superoxide dismutase (MnSOD) and catalase, indicating emendation of mitochondrial dysfunction. Nox4 inhibitor GKT137381 prevented PA-induced increase of ROS and apoptosis, while decreasing iTG slightly and not influencing the level of IL-8 and IL-6. SF preparation prevented PA-induced upregulation of phospho-JNK. JNK inhibitor SP600125 prevented PA-mediated increase of Nox4, IL-8, IL-6 and iTG. Nuclear translocation of NFκB/p65 was detected in PA-treated cells, which was prevented by SF preparation. An IκB degradation inhibitor, BAY11-7082, prevented PA-induced increase of IL-8 and IL-6 as well as iTG, whereas it only decreased ROS levels slightly and showed no influence on cellular apoptosis. Conclusion: SF preparation shows a beneficial role in prevention of hepatocyte injury by attenuating oxidative stress and cytokines production at least partially through inhibition of JNK/Nox4 and JNK/NFκB pathway, respectively.

microRNA-363-3p reduces endothelial cell inflammatory responses in coronary heart disease via inactivation of the NOX4-dependent p38 MAPK axis

“…siRNA-mediated NOX4 silencing reduces ROS generation, p38 expression and ERK1/2 phosphorylation level in HUVECs, thus retarding TNF-α-induced oxidative stress and inflammation in AS [ 44 ]. In palmitic acid (PA)-induced murine hepatic AML12 cells, treatment with the JNK inhibitor SP600125 decreases NOX4 expression [ 45 ]. Inhibition of NOX4 and inactivation of JNK helped to mitigate the inflammatory response in fibroblast-like synoviocytes, exerting protective effects against the progression of rheumatoid arthritis [ 46 ].…”

Berberine Inhibits Nod-Like Receptor Family Pyrin Domain Containing 3 Inflammasome Activation and Pyroptosis in Nonalcoholic Steatohepatitis via the ROS/TXNIP Axis

“…In the current study, we established two distinct-mechanical NASH in vitro models by exposing AML12 cells to PA or MCD medium plus LPS and one in vivo model by feeding MCD diet to C57BL/6J mouse for 5 weeks. PA, accounting for 20-30% of total fatty acids in the human body (Carta et al, 2017) and the cause of lipotoxicity in hepatocytes, is widely used to establish NASH in vitro models (Ji et al, 2018). Deprivation of methionine and choline is known to cause many disturbances in hepatic lipid metabolism due to the decreased availability of methionine and choline for phospholipid synthesis (Rinella et al, 2008;Pickens et al, 2009).…”

SBT (Composed of Panax ginseng and Aconitum carmichaeli) and Stigmasterol Enhances Nitric Oxide Production and Exerts Curative Properties as a Potential Anti-Oxidant and Immunity-Enhancing Agent

“…The treatment with SBT or Stig did not affect the pH values of the culture medium ( Supplementary Figure S2 ), indicating that there were no changes in conditions of the culture medium or stable conditions of the SBT or Stig-added medium. Furthermore, several reports have suggested that SBT and Stig exert beneficial effects in various long-term experiments [ 22 , 78 , 79 , 80 , 81 , 82 , 83 ]. Thus, we presuppose that SBT or Stig may be stably maintained in the culture medium.…”