Production of nerve growth factor enhanced in cultured mouse astrocytes by glycerophospholipids, sphingolipids, and their related compounds

The presence of cysteine and methionine groups together with an ability to bind long-chain fatty acid (LCFA) oxidation products makes liver fatty acid binding protein (L-FABP) an attractive candidate against hepatocellular oxidative stress. In this report, we show that pharmacological treatment directed at modulating L-FABP level affected hepatocellular oxidant status. L-FABP expressing 1548-hepatoma cells, treated with dexamethasone or clofibrate, decreased and increased intracellular L-FABP levels, respectively. Oxidative stress was induced by H₂O₂ incubation or hypoxia–reoxygenation. The fluorescent marker, dichlorofluorescein (DCF), was employed to measure intracellular reactive oxygen species (ROS). Hepatocellular damage was assessed by lactate dehydrogenase (LDH) level. Dexamethasone treatment resulted in a significant increase in DCF fluorescence with higher LDH release compared to control cells. Clofibrate treatment, however, resulted in a significant decrease in both parameters (p < 0.05). Drug treatments did not affect cytosolic activites of glutathione peroxidase (GPx), superoxide dismutase (SOD), or catalase suggesting that the differences between treated and control cells may likely be associated with varying L-FABP levels. We conclude that L-FABP may act as an effective endogenous cytoprotectant against hepatocellular oxidative stress.     

The expression of PPAR-associated genes is modulated through postnatal development of PPAR subtypes in the small intestine

In this study, we found that the mRNA level of peroxisome proliferator-activated receptor (PPAR) alpha, but not of PPARdelta, was elevated in the jejunum during the postnatal development of the rat. Moreover, we found that the expressions of PPAR-dependent genes, such as acyl-CoA oxidase, L-FABP, and I-FABP, were also increased during the postnatal development of the small intestine. Electrophoretic mobility shift assay revealed that both the PPARalpha-9-cis-retinoic acid receptor alpha (RXRalpha) heterodimer and the PPARdelta-RXRalpha heterodimer bound to the peroxisome proliferator response element (PPRE) of acyl-CoA oxidase and L-FABP genes. The binding of the PPARalpha-RXRalpha heterodimer to the PPREs of the various genes was enhanced by the addition of PPARalpha, with a concomitant reduction of the binding of PPARdelta-RXRalpha to the PPREs. Furthermore, the binding activity of PPARalpha-RXRalpha, but not PPARdelta-RXRalpha, to the PPREs was enhanced by the addition of a PPAR ligand, WY14,643. The GAL4-PPAR-chimera reporter assay showed that WY14,643 transactivated the reporter gene through action of PPARalpha, but not through PPARdelta, in Caco-2 cells. Furthermore, oral administration of a PPAR ligand, clofibrate, during 3 consecutive days of the weanling period caused a parallel increase in the mRNA levels of these PPAR-dependent genes. These results suggest that acyl-CoA oxidase, L-FABP and the other PPAR-dependent genes in the small intestine may be coordinately modulated during postnatal development by the disproportional expression of PPARalpha over PPARdelta.     

Antioxidant activity of different dihydropyridines

Lacidipine, a dihydropyridine-based calcium antagonist (DHP), has already been demonstrated to possess antioxidant activity and to reduce the intracellular production of reactive oxygen species (ROS). To verify if this effect is a peculiarity of this molecule, or belongs to other DHPs, the activity of lacidipine was compared with those of amlodipine, lercanidipine, nimodipine, and nifedipine. The DHPs were incorporated in bovine aortic endothelial cells (BAECs). Cu(2+)-oxidized LDL (ox-LDL, 5 microM) was incubated with BAECs for 5 min. 2',7'-Dichlorofluorescein (DCF) as expression of intracellular ROS production was measured by flow cytometry. Ox-LDL induced a strong increase in intracellular ROS formation (p<0.001) that was significantly reduced only with lacidipine and lercanidipine (p from <0.05 to <0.01); the effect of lacidipine, however, resulted in being much more evident than lercanidipine (p<0.01); amlodipine, nimodopine, and nifedipine had no effect on ROS formation. The lowest IC50s, i.e. the concentrations determining the 50% reduction of ROS, were obtained with lacidipine (p<0.01). The inhibitory effect of lacidipine on ox-LDL-induced ROS production in endothelial cells is a peculiarity of this molecule through its antioxidant activity.     

Induction of hepatic cytosolic fatty acid binding protein with clofibrate accelerates both membrane and cytoplasmic transport of palmitate

The role of liver cytosolic fatty acid binding protein (L-FABP) in fatty acid transport and metabolism is unclear. Female liver contains substantially more L-FABP than male liver. Female liver also has a different fatty acid transport phenotype, including more rapid uptake, efflux and cytoplasmic transport. However, it is not known if the greater levels of L-FABP are responsible for these differences. We therefore determined whether increasing L-FABP using clofibrate causes male liver to acquire a female transport phenotype. The multiple indicator dilution (MID) method was used to estimate the rate constants for influx, efflux and cytoplasmic diffusion of palmitate in isolated perfused rat livers. Clofibrate treatment increased cytosolic concentrations of L-FABP 4.2+/-0.8-fold, the rate of cytoplasmic diffusion of palmitate 4.3+/-1.7-fold, and the steady-state palmitate extraction 1.5+/-0.3-fold (mean+/-S.E.). Influx and efflux constants were both increased (by 44% and 79%, respectively) to levels typical of female livers. These data suggest that clofibrate-induced elevation of cytosolic L-FABP not only stimulates intracellular diffusion but also influx and efflux of fatty acids. Possible mechanisms include reducing fatty acid binding to cytoplasmic membranes, induction of membrane fatty acid carriers, and catalyzing fatty acid exchange between aqueous cytoplasm and the plasma membrane.     

BRCA1 down-regulates cellular levels of reactive oxygen species

Previous studies have shown that the breast cancer suppressor BRCA1 stimulates antioxidant gene expression and protects cells against oxidative stress. To further examine this important function, we tested whether BRCA1 could modulate intracellular levels of reactive oxygen species (ROS). Wild-type BRCA1 (but not a cancer-associated mutant) significantly reduced ROS levels, determined by DCF fluorescence assays by flow cytometry and confocal microscopy. The BRCA1 and REF1 pathways for reduction of ROS levels appear to exhibit cross-talk. BRCA1 also reduced the levels of protein nitration and H₂O₂-induced oxidative damage to DNA. Thus, BRCA1 may protect cellular macromolecules by reducing intracellular ROS levels.     

Peroxisome proliferator-activated receptor-α activation protects against endoplasmic reticulum stress-induced HepG2 cell apoptosis

Live ischemia–reperfusion injury is associated with endoplasmic reticulum (ER) stress-induced apoptosis. Activation of peroxisome proliferator-activated receptor-α (PPARα) may inhibit hepatocyte apoptosis induced by oxidative stress and protect against liver injury. This study aimed to investigate the effects of PPARα activation, through a specific agonist, on ER stress-induced apoptosis in human liver hepatocellular carcinoma (HepG2) cells. HepG2 cells were challenged with H₂O₂ and treated with WY14643, a selective PPARα agonist, in the presence or absence of the PPARα antagonist of MK886. Cell viable assay (MTT) and immunostaining were used to evaluate cell viability. The level of apoptotic cell death was quantified through Annexin V/PI staining. Alanine aminotransferase, asparatate aminotransferase, and malondialdehyde levels were measured to determine the presence of cellular injury and oxidative stress. RT-PCR and Western blot analysis were used to detect mRNA and protein expression of PPARα, BiP, and CHOP. Immunofluorescence was utilized to determine the intracellular localization of CHOP. H₂O₂ and MK886 both reduced the viability of HepG2 cells, increased oxidative stress and apoptosis, up-regulated the BiP and CHOP expression, and induced CHOP translocation from the cytoplasm to the nucleus. Compared with cells treated with H₂O₂ alone, pre-administration of WY14643 increased cell viability, attenuated apoptosis, improved cell function, down-regulated BiP and CHOP expression and inhibited CHOP translocation. The effects of WY14643 were completely abolished using the MK886 antagonist. PPARα activation protects against H₂O₂-induced HepG2 cell apoptosis. The underlying mechanisms may be associated with its activation to suppress excessive ER stress.     

Mechanism for antioxidative effects of thiazolidinediones in pancreatic β-cells

Thiazolidinediones (TZDs) are synthetic ligands of peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear receptor superfamily. TZDs are known to increase insulin sensitivity and also to have an antioxidative effect. In this study, we tested whether TZDs protect pancreatic β-cells from oxidative stress, and we investigated the mechanism involved in this process. To generate oxidative stress in pancreatic β-cells (INS-1 and βTC3) or isolated islets, glucose oxidase was added to the media. The extracellular and intracellular reactive oxygen species (ROS) were measured to directly determine the antioxidant effect of TZDs. The phosphorylation of JNK/MAPK after oxidative stress was detected by Western blot analysis, and glucose-stimulated insulin secretion and cell viability were also measured. TZDs significantly reduced the ROS levels that were increased by glucose oxidase, and they effectively prevented β-cell dysfunction. The antioxidative effect of TZDs was abolished in the presence of a PPARγ antagonist, GW9662. Real-time PCR was used to investigate the expression levels of antioxidant genes. The expression of catalase, an antioxidant enzyme, was increased by TZDs in pancreatic β-cells, and the knockdown of catalase significantly inhibited the antioxidant effect of TZDs. These results suggest that TZDs effectively protect pancreatic β-cells from oxidative stress, and this effect is dependent largely on PPARγ. In addition, the expression of catalase is increased by TZDs, and catalase, at least in part, mediates the antioxidant effect of TZDs in pancreatic β-cells.     

The liver-enriched transcription factor CREBH is nutritionally regulated and activated by fatty acids and PPARα

To elucidate the physiological role of CREBH, the hepatic mRNA and protein levels of CREBH were estimated in various feeding states of wild and obesity mice. In the fast state, the expression of CREBH mRNA and nuclear protein were high and profoundly suppressed by refeeding in the wild-type mice. In ob/ob mice, the refeeding suppression was impaired. The diet studies suggested that CREBH expression was activated by fatty acids. CREBH mRNA levels in the mouse primary hepatocytes were elevated by addition of the palmitate, oleate and eicosapenonate. It was also induced by PPARα agonist and repressed by PPARα antagonist. Luciferase reporter gene assays indicated that the CREBH promoter activity was induced by fatty acids and co-expression of PPARα. Deletion studies identified the PPRE for PPARα activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay confirmed that PPARα directly binds to the PPRE. Activation of CREBH at fasting through fatty acids and PPARα suggest that CREBH is involved in nutritional regulation.     

抑制核转录因子PPARα的反义寡核苷酸的抗乙型肝炎病毒活性研究

目的:研究靶向过氧化物酶体增殖活化受体α(PPARα)的寡核苷酸是否具有抑制乙型肝炎病毒(HBV)复制的活性作用。方法:反义寡核苷酸作用于能稳定表达HBV丹氏颗粒的HepG2.2.15细胞,ELISA检测细胞上清中HBV表面抗原(HBsAg)及e抗原(HBeAg)的分泌;实时荧光定量PCR考察反义寡核苷酸对HBV DNA复制水平的影响;通过反转录PCR和Western印迹考察反义寡核苷酸作用于细胞后靶基因及靶蛋白的差异表达情况。结果:抑制PPARα表达的反义序列PPARα-2可剂量依赖且特异性地抑制肝癌细胞中HBsAg和HBeAg的表达,且显著降低细胞中PPARαmRNA水平和蛋白水平。结论:通过筛选初步确定了基于PPARα设计的反义寡核苷酸有较好的抗HBV活性,同时也验证确定了PPARα可能成为抗HBV药物的新型作用靶点。     

木香烃内酯抑制乙醇诱导的肝细胞损伤与脂肪变性

探究木香烃内酯体外对乙醇诱导肝细胞损伤及脂肪变性的影响。建立乙醇导致人LO2肝细胞损伤模型,检测木香烃内酯对细胞活力、ALT和AST释放、脂质生成、脂质调控因子表达及AMPK活性的影响。发现乙醇在高于100 mM浓度时显著抑制肝细胞活力,据此将100 mM浓度的乙醇作为体外刺激肝细胞的实验浓度。木香烃内酯能够逆转乙醇对肝细胞活力的抑制作用,并降低乙醇导致的肝细胞ALT、AST的释放。木香烃内酯能够降低乙醇诱导的肝细胞脂质成分集聚,降低细胞内TG、TC水平。此外,乙醇导致肝细胞中重要的脂质调控转录因子SREBP-1c的表达显著上调,使PPARα的表达显著下调;而木香烃内酯能够减少SREBP-1c的表达并增加PPARα的表达。进一步发现,木香烃内酯显著促进肝细胞中AMPK的磷酸化,且AMPK抑制剂BML-275能够显著削弱木香烃内脂对SREBP-1c和PPARα的调控作用。综上,木香烃内酯体外显著改善乙醇诱导的肝细胞损伤与脂肪变性,该作用与激活AMPK进而调控SREBP-1c与PPARα的表达有关。本研究为将木香烃内酯作为抗酒精性脂肪肝候选药物研究提供实验依据。