Recent data have revealed that inhibiting autophagy exacerbates lipid accumulation in hepatocytes and nitrite treatment reduces total triglyceride levels in the high-fat diet mice. Therefore, the present study aimed to determine the effects of nitrite on simple hepatic steatosis and the possible role of autophagy. Firstly, steatotic L-02 cells were induced by incubating L-02 cells with 1.2 mmol · L(-1) oleic acid (OA) for 24 h. Secondly, steatotic L-02 cells were treated with 0.2 mmol · L(-1) sodium nitrite (SN) plus 3-methyladenine (3-MA), or chloroquine (CQ) for 24 h, and then lipid accumulation was measured with oil red O staining and triglyceride quantification. The notable steatosis could be observed in L-02 cells following exposure to 1.2 mmol · L(-1) OA for 24 h. Treatment with 0.2 mmol · L(-1) sodium nitrite reduced lipid accumulation in steatotic L-02 cells. 3-MA weakened the ability of sodium nitrite to ameliorate hepatic steatosis. Additionally, the sodium nitrite increased number of LC3-II immunostaining puncta and LC3-II protein expression was confirmed by immunofluorescence or Western blot analysis, and the effects were enhanced by CQ treatment. The number of increased cytoplasm vacuoles and lysosomes increased was confirmed by phase contrast and fluorescence microscope respectively. The increased autolysosome was detected by electron microscopy, this phenomenon could be reversed by CQ treatment. These data demonstrated that sodium nitrite enhanced the autophagic flux and decomposition of triglycerides in steatotic L-02 cells.
Adenine ; analogs & derivatives ; Autophagy ; Blotting, Western ; Cells, Cultured ; Chloroquine ; Cytoplasm ; Fatty Liver ; Hepatocytes ; drug effects ; Humans ; Lipid Metabolism ; drug effects ; Microscopy, Fluorescence ; Microtubule-Associated Proteins ; metabolism ; Oleic Acid ; Sodium Nitrite ; pharmacology ; Triglycerides

The Escherichia coli fadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ-proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensis fabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensis fabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; metabolism ; Base Sequence ; Binding Sites ; DNA, Bacterial ; metabolism ; Escherichia coli ; genetics ; metabolism ; Fatty Acid Synthase, Type II ; genetics ; metabolism ; Fatty Acids ; biosynthesis ; Gene Expression Regulation, Bacterial ; drug effects ; Molecular Sequence Data ; Oleic Acid ; pharmacology ; Protein Binding ; drug effects ; Regulon ; genetics ; Repressor Proteins ; chemistry ; metabolism ; Shewanella ; genetics ; metabolism

BACKGROUNDAcute lung injury (ALI) is a common syndrome associated with high morbidity and mortality in emergency medicine. Cell apoptosis plays a key role in the pathogenesis of ALI. Hydrogen sulfide (H(2)S) plays a protective role during acute lung injury. We designed this study to examine the role of H(2)S in the lung alveolar epithelial cell apoptosis in rats with ALI.

METHODSSixty-nine male Sprague Dawley rats were used. ALI was induced by intra-tail vein injection of oleic acid (OA). NaHS solution was injected intraperitonally 30 minutes before OA injection as the NaHS pretreatment group. Single sodium hydrosulfide pretreatment group and control group were designed. Index of quantitative assessment (IQA), wet/dry weight (W/D) ratio and the percentage of polymorphonuclear leukocyte (PMN) cells in the bronchoalveolar lavage fluid (BALF) were determined. H(2)S level in lung tissue was measured by a sensitive sulphur electrode. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Fas protein was measured by immunohistochemical staining.

RESULTSThe level of endogenous H(2)S in lung tissue decreased with the development of ALI induced by OA injection. Apoptosis and Fas protein in alveolar epithelial cells increased in the ALI of rats but NaHS lessened apoptosis and Fas protein expression in alveolar epithelial cells of rats with ALI.

CONCLUSIONEndogenous H(2)S protects rats from oleic acid-induced ALI, probably by inhibiting cell apoptosis.

Acute Lung Injury ; chemically induced ; drug therapy ; metabolism ; Animals ; Apoptosis ; physiology ; Epithelial Cells ; drug effects ; Hydrogen Sulfide ; metabolism ; In Situ Nick-End Labeling ; Male ; Oleic Acid ; toxicity ; Rats ; Rats, Sprague-Dawley ; Sulfides ; pharmacology ; therapeutic use

OBJECTIVETo investigate the effects of peroxisome proliferator activated receptor-alpha (PPAR-a) activation on oleic acid (OA)-induced steatosis and hepatic expression of heme oxygenase-1 (HO-1) using an in vitro cell model system.

METHODSA steatosis human hepatocyte in vitro model system was established by treating HepG2 cells with 0.2 mmol/L of oleic acid for 24 hours. The steatosis cells were then divided into four groups for an additional 24 hours of treatment with 0.2 mmol/L of oleic acid alone (model control group) or with 5, 10 or 50 pnol/L of fenofibrate (FF, a selective PPAR-a agonist; experimental groups). Untreated HepG2 cells served as non-steatosis controls. Effect of PPAR-a activation on fat accumulation was detected by Oil Red O staining and on intracellular triglyceride (TG) levels by enzymatic assay. mRNA and protein expression of PPAR-alpha and HO-1 were quantified by real-time PCR and immunocytochemistry, respectively. One-way ANOVA and the LSD t-test were used for between-group comparisons, and correlation analysis was performed with the Pearson's correlation coefficient.

RESULTSThe steatosis model control cells showed significantly increased TG deposition (379.98 +/- 23.19 mg/g protein, vs. non-steatosis controls F = 148.56, P< 0.01), significantly decreased mRNA and protein expression of PPAR-alpha (0.42 +/- 0.38,F= 177.64,P< 0.01 and 0.47 +/- 0.14, F= 120.76,P< 0.01) and HO-1 (0.36 +/- 0.66, F= 74.77,P< 0.01 and 0.26 +/- 0.10,F= 119.90,P<0.01). FF (5, 10 and 50 micromol/L) inhibited the steatosis induced by OA in a concentration-dependent manner (294.00 +/- 19.80, 250.33 +/- 9.96, and 196.99 +/- 9.14, F = 148.56, P <0.01) and increased the mRNA and protein expression of PPAR-alpha (0.55 +/- 0.65, 0.85 +/- 0.61, and 1.31 +/- 0.36,F= 177.64,P< 0.01; 0.82 + 0.11, 1.31 +/- 0.16, and 1.75 +/- 0.13, F= 120.76,P <0.01) and HO-1 (0.62 +/- 0.05, 0.84 +/- 0.07, and 1.30 +/- 0.11,F= 74.77,P <0.01; 0.44 +/- 0.08, 0.81 +/- 0.08, 1.20 +/- 0.10,F= 119.90,P< 0.01).

CONCLUSIONActivation of PPAR-a prevents OA-induced steatosis in HepG2 cells, and HO-1 may function as a downstream effector of this mechanism.

Fatty Liver ; chemically induced ; Heme Oxygenase-1 ; metabolism ; Hep G2 Cells ; Humans ; Oleic Acid ; pharmacology ; PPAR alpha ; metabolism ; Triglycerides ; metabolism

OBJECTIVETo study the effect of oleic acid (OA) on expression of aquaglyceroporin genes, AQP3 and AQP9, in hepatocyte steatosis and to investigate the underlying molecular mechanisms using an in vitro system.

METHODSHepG2 cells were treated with OA at different concentration to establish in vitro models of nonalcoholic hepatocyte steatosis. The corresponding extents of hepatic steatosis modeling were assessed by oil red O staining and optical density (OD) measurements of the intracellular fat content. The model lines were then treated with inhibitors of the PI3K/Akt and p38 MAPK signaling pathway factors and effects on AQP3/9 expression was measured by real time RT-PCR and western blotting.

RESULTSThe fat concentration, indicative of hepatic steatosis, increased in conjunction with increased concentrations of OA (0 less than 250 less than 500 mumol/L). OA exposure also down-regulated AQP3 mRNA and up-regulated AQP9 mRNA levels in a concentration-dependent manner. The most robust changes in expression occurred in response to the 500 mumol/L concentration of OA for both AQP3 (0.47+/-0.18; t = 4.5450, P less than 0.05) and AQP9 (1.57+/-0.21; t = 3.0306, P less than 0.05). Treatment with OA + PI3K pathway inhibitor (LY294004) significantly decreased AQP9 mRNA expression (4.55+/-0.62) as compared to the control group (1.00+/-0.10; t = 9.7909, P less than 0.01), that 500 mumol/L OA group (2.43+/-0.53; t = 4.5018, P less than 0.05), and the LY294002 group (1.90+/-0.16; t = 7.1683, P less than 0.01). Treatment with p38 MAPK pathway inhibitor (SB230580) significantly increased the OA-suppressed level of AQP3 mRNA to the level detected in the control group (1.27+/-0.11; t = 5.7455, P less than 0.01) and decreased the OA-stimulated AQP9 mRNA (0.38+/-0.09; t = 6.5727, P less than 0.01). No significant changes in mRNA expression of AQP3/9 were observed with inhibition of the ERK1/2 and JNK signal transduction pathways. The OA-induced changes in protein expression levels of AQR3 and AQP9 followed a similar trend of the genes. Finally, OA suppressed the level of phosphorylated Akt (from 0.21+/-0.02 to 0.13+/-0.03; t = 3.8431, P less than 0.05) but elevated the level of phosphorylated p38 (from 0.58+/-0.06 to 1.02+/-0.10; t = 12.5289, P less than 0.01). Again, OA treatment produced no significant affect on ERK1/2 and JNK phosphorylation.

CONCLUSIONOA down-regulates AQP3 expression by stimulating the p38 MAPK signaling pathway, and up-regulates the AQP9 by blocking the PI3K/Akt pathway and activating the p38 MAPK signaling pathway.

Aquaporin 3 ; metabolism ; Aquaporins ; metabolism ; Fatty Liver ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Hep G2 Cells ; Humans ; Oleic Acid ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; metabolism

BACKGROUNDAnimal models that demonstrate changes of renal function in response to acute lung injury (ALI) and mechanical ventilation (MV) are few. The present study was performed to examine the effect of ALI induced by oleic acid (OA) in combination with conventional MV strategy on renal function in piglets.

METHODSTwelve Chinese mini-piglets were randomly divided into two groups: the OA group (n = 6), animals were ventilated with a conventional MV strategy of 12 ml/kg and suffered an ALI induced by administration of OA, and the control group (n = 6), animals were ventilated with a protective MV strategy of 6 ml/kg and received the same amount of sterile saline.

RESULTSSix hours after OA injection a severe lung injury and a mild-moderate degree of renal histopathological injury were seen, while no apparent histological abnormalities were observed in the control group. Although we observed an increase in the plasma concentrations of creatinine and urea after ALI, there was no significant difference compared with the control group. Plasma concentrations of neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C increased (5.6 ± 1.3) and (7.4 ± 1.5) times in the OA group compared to baseline values, and were significantly higher than the values in the control group. OA injection in combination with conventional MV strategy resulted in a dramatic aggravation of hemodynamic and blood gas exchange parameters, while these parameters remained stable during the experiment in the control group. The plasma expression of TNF-α and IL-6 in the OA group were significantly higher than that in the control group. Compared with high expression in the lung and renal tissue in the OA group, TNF-α and IL-6 were too low to be detected in the lung and renal tissue in the control group.

CONCLUSIONSOA injection in combination with conventional MV strategy not only resulted in a severe lung injury but also an apparent renal injury. The potential mechanisms involved a cytokine response of TNF-α and IL-6 in plasma, lung and renal tissues.

Acute Lung Injury ; chemically induced ; pathology ; physiopathology ; Animals ; Cytokines ; analysis ; Hemodynamics ; Kidney ; pathology ; physiopathology ; Lung ; pathology ; Oleic Acid ; pharmacology ; Respiration, Artificial ; Swine ; Swine, Miniature

Cumulative evidence suggests that renal vascular endothelial injury play an important role in initiating and extending tubular epithelial injury and contribute to the development of ischemic acute renal failure. Our previous studies have demonstrated that iptakalim's endothelium protection is related to activation of SUR2B/Kir6.1 subtype of ATP sensitive potassium channel (K(ATP)) in the endothelium. It has been reported that SUR2B/Kir6.1 channels are widely distributed in the tubular epithelium, glomerular mesangium, and the endothelium and the smooth muscle of blood vessels. Herein, we hypothesized that activating renal K(ATP) channels with iptakalim might have directly neroprotective effects. In this study, glomerular endothelial, mesangial and tubular epithelial cells which are the main cell types to form nephron were exposed to oleic acid (OA) at various concentrations for 24 h. 0.25 microl/ml OA could cause cellular damage of glomerular endothelium and mesangium, while 1.25 microl/ml OA could lead to the injury of three types of renal cells. It was observed that pretreatment with iptakalim at concentrations of 0.1, 1, 10 or 100 micromol/L prevented cellular damage of glomerular endothelium and tubular epithelium, whereas iptakalim from 1 to 100 micromol/L prevented the injury of mesangial cells. Our data showed iptakalim significantly increased survived cell rates in a concentration-dependent manner, significantly antagonized by glibenclamide, a K(ATP) blocker. Iptakalim played a protective role in the main cell types of kidney, which was consistent with natakalim, a highly selective SUR2B/Kir6.1 channel opener. Iptakalim exerted protective effects through activating SUR2B/Kir6.1 channels, suggesting a new strategy for renal injury by its endothelial and renal cell protection.
Cells, Cultured ; Epithelial Cells ; metabolism ; Glyburide ; adverse effects ; Humans ; KATP Channels ; metabolism ; Kidney ; cytology ; metabolism ; physiopathology ; Kidney Diseases ; drug therapy ; metabolism ; Oleic Acid ; adverse effects ; Propylamines ; pharmacology ; Protective Agents ; pharmacology

OBJECTIVETo investigate the effect of oleic acid on the proliferation and secretion of pro-inflammatory mediators of human normal fibroblasts and scar fibroblasts.

METHODSHuman normal fibroblasts and scar fibroblasts were cultured in vitro and respectively divided into seven groups according to the random number table, with 8 samples in each group. Cells in blank control (BC) group were routinely cultured without addition of other agents. Cells in ethanol-control (EC) group were cultured with medium with the addition of 2% absolute ethanol. Cells in oleic acid groups were cultured with the addition of oleic acid in concentration of 0.25, 0.50, 1.00, 2.00, or 4.00 mmol/L in 2% absolute ethanol. The growth of cells in each group was observed with trypan blue staining on post culture day (PCD) 1-5. On PCD 2, structure of cells in BC, EC, and 1.00 mmol/L oleic acid groups was observed under inverted phase contrast microscope and transmission electron microscope; cell cycle of BC, EC, and 1.00 mmol/L oleic acid groups was measured by flow cytometer; cell proliferation activity in each group was measured by MTT assay; the level of NO in supernatant was assayed by Griess assay; the levels of TNF-α, IL-1β, IL-6, and IL-8 in supernatants in each group were determined by enzyme-linked immunosorbent assay. Data were processed with multifactor and repeated measurement design analysis of variance.

RESULTS(1) There was no significant difference in each index of normal fibroblasts and scar fibroblasts between BC group and EC group. (2) The numbers of normal fibroblasts and scar fibroblasts in 2.00 and 4.00 mmol/L oleic acid groups were significantly lower than those in corresponding BC and EC groups on PCD 2-5 (with F values respectively 13.773 and 11.344, P values all below 0.01). (3) On PCD 2, the numbers of normal fibroblasts and scar fibroblasts in 1.00 mmol/L oleic acid groups decreased, and the cells were aggregating, rounding, and easy to drop off. Cellular membrane disruption, vacuolar degeneration of mitochondrion, pyknosis, and lipid droplets were observed. (4) The percentages of G0/G1 and G2/M phases of normal fibroblasts in 1.00 mmol/L oleic acid group [(93.56 ± 9.98)%, (2.01 ± 0.75)%] were significantly higher than those in BC group [(84.23 ± 10.96)%, (0.37 ± 0.16)%, with F values respectively 3.026, 34.751, P < 0.05 or P < 0.01], while the percentage of normal fibroblasts in S phase [(4.42 ± 0.87)%] was markedly lower than that in BC group [(16.06 ± 1.74)%, F = 136.120, P < 0.01]. The percentages of scar fibroblasts of G0/G1 and G2/M phases in 1.00 mmol/L oleic acid group [(93.86 ± 13.90)%, (1.89 ± 0.66)%] were significantly higher than those in BC group [(83.88 ± 10.42)%, (0.41 ± 0.17)%, with F values respectively 3.529, 32.710, P < 0.05 or P < 0.01], and the percentage of scar fibroblasts in S phase [(3.87 ± 0.63)%] was markedly lower than that in BC group [(15.89 ± 2.02)%, F = 116.508, P < 0.01]. (5) The proliferation rates of normal fibroblasts and scar fibroblasts in 0.50-4.00 mmol/L oleic acid groups were significantly lower than those in corresponding BC and EC groups (with F values respectively 215.945 and 194.555, P < 0.05 or P < 0.01). (6) The content of NO in supernatant of normal fibroblasts in all oleic acid groups was obviously higher than that in BC and EC groups (F = 30.240, P < 0.05 or P < 0.01). The contents of NO in supernatants of scar fibroblasts in 1.00-4.00 mmol/L oleic acid groups were significantly higher than that in BC and EC groups (F = 12.495, P < 0.01). The contents of TNF-α and IL-6 in supernatants of normal fibroblasts and scar fibroblasts in 2.00 and 4.00 mmol/L oleic acid groups were obviously higher than those in corresponding BC and EC groups (with F(TNF-α) values respectively 6.911, 3.818, F(IL-6) values respectively 16.939, 11.600,P < 0.05 or P < 0.01). The contents of IL-1β in supernatants of normal fibroblasts and scar fibroblasts in groups of every concentration of oleic acid were significantly higher than those in corresponding BC and EC groups (with F values respectively 25.117, 9.137, P values all below 0.01). The contents of IL-8 in supernatants of normal fibroblasts in 1.00-4.00 mmol/L oleic acid groups were markedly higher than those in BC and EC groups (F = 2.717, P < 0.05 or P < 0.01). The contents of IL-8 in supernatants of scar fibroblasts in 2.00 and 4.00 mmol/L oleic acid groups were significantly higher than those in BC and EC groups (F = 3.338, P < 0.05). There was no statistically significant difference in above indexes between normal fibroblasts and scar fibroblasts in the same concentration of oleic acid group (with F values from 0.120 to 3.766, P values all above 0.05).

CONCLUSIONSAlthough oleic acid in high concentration inhibits the proliferation of scar fibroblasts, it also inhibits the proliferation of normal fibroblasts. Oleic acid in high concentration can cause excessive and continued inflammatory reaction by promoting the secretion of pro-inflammatory mediators of normal fibroblasts and scar fibroblasts, and they are detrimental to wound healing.

Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix ; metabolism ; Fibroblasts ; cytology ; drug effects ; secretion ; Humans ; Inflammation Mediators ; metabolism ; Oleic Acid ; pharmacology

OBJECTIVETo observe the expression of P-selectin (Ps), intercellular adhesion molecule-1 (ICAM-1) and nuclear factor-kappa B (NF-kappaB) in lung tissues of acute lung injury (ALI) rat model induced by oleic acid (OA) and to explore the protective effects of melatonin (MT) in lung tissues in rats.

METHODSAll rats were randomly divided into four groups: control group, OA group, MT + OA group and SB203580 + OA group. Rat model of ALI was established by intravenous injection of oleic acid (OA). Lung coefficient was measured, lung tissues were imbedded by paraffin to observe morphological changes and the expression of Ps, ICAM-1 and NF-kappaB in lung tissues by means of immunohistochemistry staining.

RESULTSCompared with control group, the lung coefficient increased significantly in OA group (P < 0.05). Alveolar septum thickened significantly in OA group, there had many infiltrated inflammatory cells and collapsed alveoli of lung; positive expression of Ps, ICAM-1 and NF-kappaB were very obvious (P < 0.05); the administration of MT and SB203580 mitigated above changes significantly (P < 0.05).

CONCLUSIONMT possesses obviously protective effect on lung tissues during ALI, its protective mechanism might be related to the inhibition of the expression of Ps, ICAM-1 and NF-kappaB.

Acute Lung Injury ; chemically induced ; physiopathology ; prevention & control ; Animals ; Down-Regulation ; drug effects ; Intercellular Adhesion Molecule-1 ; metabolism ; Male ; Melatonin ; pharmacology ; therapeutic use ; NF-kappa B ; metabolism ; Oleic Acid ; adverse effects ; P-Selectin ; metabolism ; Protective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley

This study is to investigate the pharmacological effect and mechanism of action of hydroxysafflor yellow A (HSYA) on acute lung injury (ALI). The rat ALI was induced by oleic acid and lipopolysaccharide (LPS) injection. The incidence of acidosis, PaO2 (arterial blood oxygen pressure), W/D (wet weight/dry weight) and lung index (LI) were measured. Electron microscope and optical microscope were applied to observe lung morphological changes in rat. RT-PCR was used to determine TNF-alpha and ICAM-1 mRNA level. Inhibition effect of HSYA on plasma inflammatory cytokine expression was measured by ELISA. HSYA could alleviate pulmonary edema, reduce acidosis, keep PaO2 from descending, inhibit inflammatory cell infiltration, inhibit rat lung TNF-alpha and ICAM-1 mRNA expression and plasma IL-6 and IL-1beta level elevation. HSYA is an effective ingredient to remit ALI induced by oleic acid and LPS in rat.
Acute Lung Injury ; chemically induced ; metabolism ; pathology ; Animals ; Carthamus tinctorius ; chemistry ; Chalcone ; analogs & derivatives ; isolation & purification ; pharmacology ; Flowers ; chemistry ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Interleukin-1beta ; blood ; Interleukin-6 ; blood ; Lipopolysaccharides ; Lung ; metabolism ; pathology ; ultrastructure ; Male ; Oleic Acid ; Plants, Medicinal ; chemistry ; Quinones ; isolation & purification ; pharmacology ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; genetics ; metabolism