The Effects Of FAT/CD36on Hepatic Lipid Accumulation And Injury Under Inflammatory Stress

Objective: Inflammatory stress was induced by adding cytokines tohuman hepatoma cell HepG2and primary hepatocytes from C57BL/6Jmice in vitro and by subcutaneous casein injection in C57BL/6J mice invivo. In this study, we investigated whether CD36plays an important rolein hepatic lipid accumulation and injury under inflammatory stress.Methods: CD36was knocked down by transfection with humanCD36small interfering RNA (CD36i) or overexpressed by transfectionwith human CD36cDNA (O/E CD36) in HepG2. A two-step collagenaseperfusion technique was used to isolate the hepatocytes fromWT andCD36KO mice. These cells were treated with three different treatments:control (0.2%BSA+0.04μmol/ml palmitic PA), inflammation I (tumornecrosis factor TNF-α,20ng/ml+palmitic acid PA,0.04μmol/ml+0.2%BSA), inflammation II (interleukin6IL-6,20ng/ml+palmitic acid PA,0.04μmol/ml+0.2%BSA). Protein expreesions of CD36were detectedby western blotting. A realtime uptaking fatty acid fluorescence detectiontechnology was used to observe process of uptaking fatty acid.Intracellular and hepatic free fatty acids (FFA) and triglycerides (TG)were detected using ELISA kit and enzymic assay. The mRNA expressionof ER stress signs including activating transcription factor6(ATF6),glucose regulated protein78(GRP78) and Inositol requiring protein1(IRE1) and fibrotic genes containing collagen I (col I), collagen Ⅳ (col Ⅳ) and α-smooth muscle actin (α-SMA) were detected by real-time PCR.Levels of reactive oxygen species (ROS) and H2O2in hepatocytes weremeasured by enzymic assay.Male C57BL/6J and CD36KO mice were injected with casein onalternate days for14weeks to induce inflammation. At termination, bloodsamples were taken for cytokines and lipid assays, and tissue sampleswere collected for further detection. Real-time PCR (RT-PCR) wasapplied to detect mRNA expression of tumor necrosis factor (TNF-α) andmonocyte chemoattractant protein-1(MCP-1) and interleukin6(IL-6) inliver. Lipid droplet accumulation was observed by oil red O staining.Results: These inflammatory mediators increased CD36proteinexpression in hepatocytes (P<0.05). The rates of upktaking FFA in WTprimary hepatocytes and normal HepG2cells were accelerated underinflammatory stress (P<0.05). Detection of intracellular TG and FFAshowed that inflammation aggravated lipid accumulation in hepatocytes(P<0.05). Meanwhile, enzymic assay indicates that inflammationincreases intracellular levels of ROS and H2O2(P<0.05).The data ofreal-time PCR showed that the mRNA expressions of ER stress signs andfibrotic genes in hepatocytes were increasd under inflammatory stress(P<0.05). To test the specific roles of CD36on hepatic lipid accumulationand injury under inflammatory stress in cells, CD36was knocked downby transfection with human CD36small interfering RNA (CD36i) oroverexpressed by transfection with human CD36cDNA (O/E CD36) inHepG2cells. The increased lipid accumulation and injury induced byinflammatory stress were blocked by knocking down CD36(CD36i) incomparison with negative control, whereas O/E CD36enhanced lipidaccumulation and injury in HepG2cells.Casein injection increasd levels of SAA and IL-6in serum and liver. Western blotting showed that inflammation increased hepatic CD36protein expression (P<0.05). Oil red O staining showed that inflammationaggravated lipid accumulation in livers of C57BL/6J mice. Quantitativeassay of FFA and TG consisted with the Oil red O staining (P<0.05).Hepatic injury were markedly increased with casein injection inC57BL/6J mice, however, the hepatic lipid accumulation and injuryinduced by inflammatory stress were decreased in knocking out CD36mice (CD36KO) mice comparing with wild type (WT) mice.Conclusions: Inflammation increased hepatic CD36proteinexpression. CD36plays an important role in hepatic lipid accumulationand damage under inflammatory stress.