Betulinic Acid Down-regulates Expression Of Oxidative Stress-induced LPL Via PKC/AMPK/C-FOS Pathway In Macrophages

Background：Atherosclerosis is a major cause of coronary artery disease, whichcharacterized by cellular lipid accumulation, represents a state of heightened oxidativestress. Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism.Studies showedthat macrophage derived LPL exhibit proatherogenic properties, which plays a majorrole in macrophage lipid accumulation. Evidents suggested oxidative stress wereeffective enhancer of macrophage LPL production. Betulinic acid (BA) is apentacyclic lupane triterpene, which has potent antioxidant activity. In this study, weinvestigated whether BA affects expression of LPL and the potential subsequent effecton cellular lipid accumulation, and discussed the major mechanism involved.Methods: RAW264.7macropage cells were treated18h with various concentrationsH2O2（0,50,100,200μM）or were incubated200μM H2O2for different time periods（0,6,12,18,24h). Cellular ROS were assessed as described, and the levels of LPLmRNA and protein were measured by real-time PCR and Western blotting assays,respectively, LPL activity was determined as described use kits. Macrophage cellswere incubated in medium containing with or without H2O2(200μM) for18h. H2O2groups were the cells pretreated with BA (0,0.5,1,2μg/ml) for24h or with BA(1μg/ml) for0,12,24and48h. The levels of LPL mRNA, protein and LPL activitywere measured respectively. The content of TG and TC was analyzed quantitativelyby standard enzymatic produce, and cellular lipid deposition was visualized by oilRed O staining. Then, RAW264.7macrophage cells were pretreated with BA (0,0.5,1,2μg/ml) for24h, or10mmol/L the antioxidant N-acetyl-l-cysteine (NAC) for1h, and then exposed to H2O2(200μM) for18h. Cellular ROS were assessed as described.Next, RAW264.7macrophage cells were were pretreated with BA (1μg/ml) for24hprior to incubation with H2O2(200μM) for18h, the levels of protein kinase C(PKC)protein，extracellular signal-regulated protein kinase1/2(ERK1/2) phosphorylationprotein and c-Fos phosphorylation protein were measured by Western blotting assays.The levels of LPL expression and activity were determined when add to BA, inhibitorof PKC and MAPK, and depletion c-Fos, respectively. Furthermore, measure theexpression of H2O2-induced ERK1/2and c-Fos phosphorylation protein wheninhibition of PKC, as well as measure the expression of H2O2-induced c-Fosphosphorylation protein when inhibition of MAPK.Results：H2O2led to a dose-dependent and time-dependent augmentation of ROSproduction, as well as LPL protein, mRNA expression and its activity. BAdownregulated H2O2simulated macrophage LPL protein, mRNA level and its activityin a concentration and time-dependent manner. Furthermore, BA decreased LPLinvolved total cholesterol (TC) and triglyceride (TG) level in macrophages，andcellular lipid staining by Oil Red O also observed that BA decreased H2O2induced-LPL involved cellular lipid droplet deposition. Next, we confirmed that BApretreatment decreased the H2O2-induced production of intracellular ROS in adose-dependent manner. Further studies suggested that BA inhibited H2O2-inducedPKC membrane protein expression, ERK1/2phosphorylation protein expression andc-Fos phosphorylation protein expression. Furthermore, the induction of H2O2on LPLproduction and activity were abolished by BA, as well as inhibition of PKC andERK1/2, depletion c-Fos, respectively. In addition, we have observed inhibition ofPKC inhibited H2O2induced expression of ERK1/2and c-Fos phosphorylationprotein, and inhibition of MAPK abolished H2O2induced expression of c-Fosphosphorylation protein.Conclusions：BA performed the role of antioxidant activity, attenuated oxidativestress-induced macrophage-derived LPL expression and activity, effectively reduced cellular lipid accumulation, through the inhibition of ROS mediated PKC/MAPK/c-Fos signaling pathways.