1-2-linoleoylphosphatidylcholine and 1-palmitoyl-2-linoleoylphosphatidylcholine

Polyenylphosphatidylcholine (PPC), a mixture of polyunsaturated phosphatidylcholines, protects against alcoholic and nonalcoholic liver fibrosis in baboons and rats, respectively. In this study, we assessed the antifibrogenic action of dilinoleoylphosphatidylcholine (DLPC), the main phosphatidylcholine species of PPC, against transforming growth factor-beta1-mediated expression of alpha1(I) procollagen, tissue inhibitor of metallopreoteinase-1 (TIMP-1) and matrix metalloproteinase-13 (MMP-13) in cultured rat hepatic stellate cells (HSCs). In primary culture-activated HSCs, TGF-beta1 up-regulated the alpha1(I) procollagen mRNA level with a concomitant increase in type I collagen accumulation in culture media. Whereas TIMP-1 mRNA levels and TIMP-1 accumulation in media were also increased by TGF-beta1, MMP-13 mRNA expression and MMP-13 concentration in media were not altered. DLPC fully blocked TGF-beta1-induced increase in alpha1(I) procollagen mRNA expression and decreased collagen accumulation in media. Whereas TIMP-1 mRNA level and TIMP-1 accumulation in media were decreased by DLPC, MMP-13 mRNA expression and MMP-13 concentration in media were not changed by this treatment. Palmitoyl-linoleoylphosphatidylcholine (PLPC), the second most abundant component of PPC, had no effect on the concentrations of collagen, TIMP-1, and MMP-13 in HSC culture. We conclude that DLPC prevents TGF-beta1-mediated HSC fibrogenesis through down-regulation of alpha1(I) procollagen and TIMP-1 mRNA expression. The latter effect leads to a decreased accumulation of TIMP-1 that, in the presence of unchanged MMP-13 mRNA expression and MMP-13 concentration, results in a larger ratio of MMP-13/TIMP-1 concentrations in the culture media, favoring collagen degradation and lesser collagen accumulation. This effect of DLPC may explain, at least in part, the antifibrogenic action of PPC against alcoholic and other fibrotic disorders of the liver.

Topics: Actins; Animals; Cells, Cultured; Collagen Type I; Collagenases; Desmin; Fluorescent Antibody Technique; Gene Expression; Hepatocytes; Male; Matrix Metalloproteinase 13; Phosphatidylcholines; Procollagen; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vimentin

The prevention of cirrhosis in alcohol-fed baboons by the administration of a soybean extract-43% to 50% of which was dilinoleoyl-phosphatidylcholine (DLPC) and 24% of which was 1,palmitoyl 2,linoleoyl-phosphatidylcholine (PLPC)-was associated with a significant reduction in the number of stellate cells transformed to myofibroblast-like cells. To study whether these two major phospholipids affect the similar transformation that occurs by culturing stellate cells on uncoated plastic, we assessed their effects on proliferation (by (methyl-3H)-thymidine incorporation into DNA), expression of alpha-smooth muscle actin and type I procollagen (by densitometry of Western blots), and collagen synthesis (by incorporation of tritiated proline into collagenase-digestible proteins). These manifestations of stellate cell activation were decreased by 10 micromol/L DLPC but not by 10 micromol/L PLPC when compared with controls incubated either with 17 mmol/L ethanol (used as solvent for the phospholipids) or without addition. These agents did not affect cell viability, contamination with other cells, or the capacity of stellate cells to synthesize protein. Thus DLPC specifically decreases the in vitro activation of stellate cells, as judged by the decreases in proliferative activity, alpha-smooth muscle actin and procollagen I expressions, and collagen synthesis, whereas PLPC did not show such effects. alpha-Procollagen (type I) mRNA was not affected by DLPC, suggesting a post-translational effect. The reduction in the activation of hepatic stellate cells by DLPC may be responsible for, or at least contribute to, the prevention of fibrosis by the polyenylphosphatidylcholine mixture administered in vivo.

Topics: Actins; Animals; Blotting, Northern; Cell Division; Cell Survival; Collagen; DNA; Gene Expression; Liver; Male; Phosphatidylcholines; Procollagen; Rats; Rats, Sprague-Dawley

Polyenylphosphatidylcholine (PPC), a polyunsaturated phospholipid extract from soy beans, prevents the development of liver cirrhosis in animal models. Its mechanism of action is unknown. Based on the hypothesis that PPC might act by decreasing hepatic stellate cell proliferation, we studied the effect of PPC and its main components, dilinoleoylphosphatidylcholine (DLPC) and palmitoyl-linoleoylphosphatidylcholine (PLPC), on PDGF-induced stellate cell proliferation and intracellular signal transduction. Normal rat hepatic stellate cells in tissue culture were serumstarved, and incubated with 10ng/ml PDGF in the absence or presence of phospholipids. Cell proliferation was measured by 3H-thymidine incorporation. P44MAPK activation was determined by kinase assay, and AP-1 binding by electrophoretic mobility shift assay. PPC (200 ng/ml) significantly inhibited PDGF-induced proliferation (p < 0.05; ANOVA, n = 3) and antagonized PDGF-induced P44MAPK activation and AP-1 binding. This effect was mimicked by DLPC but not by PLPC. Neither DLPC nor PLPC prevented PDGF receptor activation. We conclude that PPC exerts a previously unrecognized effect on mitogen-induced stellate cell proliferation which may be mediated by DLPC. Inhibition of this cascade represents a potential mechanism for the inhibitory effect of PPC on hepatic fibrogenesis.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cell Nucleus; Cells, Cultured; Enzyme Activation; Female; Liver; Phosphatidylcholines; Phosphorylation; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Transcription Factor AP-1

Phenolic antioxidants of the hydroxychroman class, alpha-tocopherol (alpha-TOC) and 2,2,5,6,7-pentamethyl-6-hydroxychroman (PMHC), and the hindered phenols 2,3-dihydro-5-hydroxy-2,2,4-trimethylnaphtho[1,2-b]furan (NFUR), 2,6-di-tert-butyl-4-methoxyphenol (DBHA), and 2,6-di-tert-butyl-4-methyl phenol (BHT), were delivered into oxidizable (ACCEPTOR) liposomes of dilinoleoylphosphatidylcholine (DLPC) or 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) from saturated DONOR liposomes of dimyristoylphosphatidylcholine (DMPC) by liposomal transfer. The antioxidant activities, k(inh), by the inhibited oxygen uptake method were compared with the k(inh)s determined when the antioxidants were introduced into the liposomes by coevaporation from organic solvents. The peroxidations were initiated using either thermal initiators, water-soluble azo-bis-amidinopropane hydrochloride (ABAP), lipid-soluble azo-bis-2,4-dimethylvaleronitrile (ADVN) and di-tert-butylhyponitrite (DBHN), or the photoinitiator benzophenone. The antioxidants PMHC, NFUR, DBHA, and BHT transferred rapidly between liposomes, but several hours of incubation were needed to transfer alpha-TOC. The average k(inh)s in liposomes, in the relative order NFUR approximately DBHA > PMHC > BHT approximately alpha-TOC, were markedly lower than known values in organic solvent. k(inh) values in liposomes appear to be controlled by effects of hydrogen bonding with water and by restricted diffusion of antioxidants, especially in the case of alpha-TOC. Product studies of the hydroperoxides formed during inhibited oxygen consumption were carried out. The cis,trans/trans,trans (c,t/t,t) product ratios of the 9- and 13-hydroperoxides formed from PLPC during inhibited peroxidation by PMHC were similar for both the coevaporated and liposomal transfer procedures. The c,t/t,t ratio for the same concentration of alpha-TOC, 1.52, compares to a value of 1.69 for PMHC at the start of the inhibition period. The higher c,t/t,t ratio observed for NFUR in DLPC, which varied between values of 7.0 at the start of the inhibition to about 1.8 after the break in the induction period, is a reflection of the increased hydrogen atom donating ability of the antioxidant plus the increased concentration of oxidizable lipid provided by DLPC.

Topics: Amidines; Antioxidants; Azo Compounds; Butylated Hydroxytoluene; Chromans; Dimyristoylphosphatidylcholine; Furans; Lipid Peroxidation; Liposomes; Models, Chemical; Nitriles; Oxygen Consumption; Phosphatidylcholines; Vitamin E

Phospholipid hydroperoxides and phospholipid alcohols are two of the major forms of oxidatively modified phospholipids produced during oxidant stress and lipid peroxidation. The process of lipid peroxidation is known to affect the physiological function of membranes. We, therefore, investigated the effects of lipid peroxidation products on the molecular interactions in membranes. Our study was specifically focused on the effects of lipid peroxidation products on static membrane structure (molecular orientational order) and on the reorientational dynamics of the probe molecules in lipid bilayers. The study was done by performing angle-resolved fluorescence depolarization measurements (AFD) on the fluorescent probe diphenylhexatriene (DPH) and by performing angle-resolved electron spin resonance (A-ESR) measurements on cholestane (CSL) nitroxide spin probes embedded in macroscopically oriented planar bilayers consisting of 2-10% 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)phosphatidylcholine (PLPC-OOH) or 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) in 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC) or dilinoleoylphosphatidylcholine (DLPC). Both probe molecules have rigid cylindrical geometries and report on the overall molecular order and dynamics. However, being more polar, the nitroxide spin probe CSL is preferentially located near the surface of the membrane, while the less polar fluorescent probe DPH reports preferentially near the central hydrophobic region of the lipid bilayers. The results show that the presence of relatively small amounts of oxidatively modified phospholipids within the PLPC or DLPC membranes causes pronounced structural effects as the molecular orientational order of the probe molecules is strongly decreased. In contrast, the effect on membrane reorientational dynamics is minimal.

Topics: Diphenylhexatriene; Electron Spin Resonance Spectroscopy; Fluorescence Polarization; Lipid Bilayers; Lipid Peroxidation; Membrane Lipids; Oxidation-Reduction; Phosphatidylcholines; Phospholipids; Spin Labels