Leucodin Attenuates Inflammatory Response Inmacrophages And Lipid Accumulation In Steatotic Hepatocytes Via P2x7 Receptor Pathway:Apotential Role In Alcoholic Liver Disease

Scope:Leucodin is a sesquiterpene lactone isolated from the aerial parts of Artemisia capillaris Thunberg.The study found that Artemisia capillaris has certain anti-inflammatory and hepatoprotective activities,but it is still unknown whether the bioactive component leucodin from Artemisia capillaris can improve ethanol-induced hepatocyte injury with inflammation.This study used a combination of lipopolysaccharide(LPS)and adenosine triphosphate(ATP)to stimulate several types of macrophages,as well as a model of hepatocyte exposure to ethanol,to investigate how leucodin inhibited inflammatory responses in macrophages through the Purinergic receptor P2Xligand-gated ion channel 7(P2x7R)pathway and alleviated ethanol-induced lipid accumulation in hepatocytes.Methods:Several types of macrophages including mouse peritoneal macrophages,mouse bone marrow-derived macrophages and human macrophages THP-1 cells were pretreated with different concentrations of leucodin(1?M and 5?M)for 1h and then stimulated with LPS and ATP.Protein expression levels of IL-1?,P2x7R and NLRP3 were analyzed by Western blot.HepG2 cells in the logarithmic growth phase were exposed to ethanol to induce lipid deposition.Prior to this,logarithmic growth phase HepG2 cells were pretreated with different concentrations of leucodin(1?M and 5?M).Oil red O staining assay was used to analyze the morphological changes of HepG2 cells,and the fluorescence intensity changes of Sterol Regulatory Element-binding Protein-1(SREBP1)immunofluorescence staining,an important factor related to lipid synthesis,were observed.Protein expression of AMP-activated Kinase a(AMPK),P-AMPK,Acetyl-CoA carboxylase(ACC),P-ACC was detected by Western blot to determine whether or not leucodin inhibited lipid deposition in HepG2 cells.The toxicity test of leucodin was performed,and the survival rate of mouse peritoneal macrophages was detected by in vitro MTT assay.The nitrite accumulated in the medium was measured based on the Griess reaction as an indicator of NO production.Results:LPS plus ATP initiated IL-1? cleavage and release in mouse peritoneal macrophages and peaked at 4 h.Leucodin did not show significant toxicity within 200?M and effectively inhibited pro-IL-1? cleavage and release of mature-IL-1? in macrophages.Also,P2x7R antagonist and caspase-1 inhibitor also decreased IL-1?release and cleavage.Additionally,leucodin suppressed P2x7R,Toll like receptor-4(TLR4)and NOD-like receptor pyrin domain containing 3(NLRP3)expression in LPS/ATP-stimulated macrophages.HepG2 cells were pretreated with different concentrations of leucodin for 1h and then exposed to ethanol for 24 h.Leucodin suppressed lipid accumulation and enhanced phosphorylation of AMPK and ACC in HepG2 cells exposed to ethanol.In addition,leucodin inhibited the expression of SREBP1 and ACC in ethanol-treated HepG2 cells.Conclusion:Leucodin possessed the capacity for inhibiting inflammatory response in macrophages and suppressing lipid accumulation in hepatocytes,suggesting a promising therapeutic potential targeting inflammation and lipid metabolism in alcoholic liver disease.