Abstract
BackgroundN-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA; 22:6n-3), has clinical significance in the prevention and reversal of nonalcoholic steatohepatitis (NASH). However, the precious mechanism underlying remains unclear. The inflammasome, a multiprotein complex formed by NOD-like receptor (NLR) family members, has been recently shown to be activated in NASH and promote the cleavage of the pro-inflammatory cytokines to their maturation forms.MethodsHepG2 cells were exposed to different dose of PA for 24 h with or without the preincubation of 50 μM DHA for another 24 h and then lipid deposition was assessed with Oil red O staining and intracellular triglyceride (TG) determination. Secretory levels of inflammatory cytokines and Caspase-1 activity were determined by ELISA assays. Gene expression and protein levels were determined by quantitative RCR and western blotting, respectively.ResultsPalmitate (PA) dose-dependently increased lipid accumulation, TG content and induced the secretion of interleukin-1β (IL-1β), IL-18, TNF-α and MCP-1 from HepG2 cells. Preincubation with DHA significantly alleviated PA-induced lipid accumulation and inflammatory agents. DHA was also found to attenuate PA-induced NOD-like receptor protein 4 (NLRC4) mRNA expression. Furthermore, PA induced caspase-1 activation in a dose-dependent manner, resulting in exacerbating of procaspase-1 and pro-IL-1β processing. Knockdown of NLRC4 partially abrogated PA-induced caspase-1 activation and IL-1β maturation and completely abolished these events in the presence of DHA.ConclusionsOur findings indicate DHA attenuates PA-induced lipid accumulation and inflammation through suppressing NLRC4 inflammasome activation, caspase-1 activation and IL-1β cleavage.
Highlights
N-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA; 22:6n-3), has clinical significance in the prevention and reversal of nonalcoholic steatohepatitis (NASH)
Our findings demonstrated that NODlike receptor protein 4 (NLRC4) inflammasome is activated in PA-induced lipid accumulation and inflammation; and that DHA alleviates these events by suppressing the activity of caspase-1 and the cleavage of procaspase-1 and pro-IL-1β through inhibiting NLRC4 inflammasome activation
The membranes were blocked with 5% bovine serum albumin (BSA) in TBS for 1 h at room temperature and incubated overnight at 4°C using the following primary antibodies: 1:1000 rabbit anti β-actin, 1:500 NLRC4 (Santa Cruz, CA, USA), 1:1000 caspase-1 and 1:500 IL-1β, followed by 1:2000 dilution of goat anti-rabbit horseradish peroxidaselabeled antibody
Summary
N-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA; 22:6n-3), has clinical significance in the prevention and reversal of nonalcoholic steatohepatitis (NASH). The inflammasome, a multiprotein complex formed by NOD-like receptor (NLR) family members, has been recently shown to be activated in NASH and promote the cleavage of the pro-inflammatory cytokines to their maturation forms. The role of another inflammasome NODlike receptor protein 4 (NLRC4) on the generation of inflammatory mediators in NASH has not been elucidated. Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), have been found to decrease liver fat content in children with NAFLD and possess anti-inflammatory effects [15,16]. The effects of DHA on NLRC4 inflammasome in PA-induced inflammatory responses remain unknown. Our findings demonstrated that NLRC4 inflammasome is activated in PA-induced lipid accumulation and inflammation; and that DHA alleviates these events by suppressing the activity of caspase-1 and the cleavage of procaspase-1 and pro-IL-1β through inhibiting NLRC4 inflammasome activation
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