Hypolipidemic Effect of Mannan in Mice with Acute Lipemia Induced by Poloxamer 407.

We studied biological effects of mannan, a polysaccharide immunomodulator from C. albicans, that interacts with mannose receptor in vivo. It is shown that preliminary administration of mannan (5 times in a dose of 50 mg/kg or 2 times in a dose of 100 mg/kg) to mice with acute lipemia induced by lipase inhibitor poloxamer 407 (300 mg/kg) reduces the serum concentrations of atherogenic LDL, cholesterol, and triglycerides. Administration of mannan to intact mice and animals with acute lipemia reduces triglyceride concentration and causes labilization of lysosomal membranes in the liver. Serum activity of chitotriosidase, a marker of macrophage activation, was elevated in mice with acute lipemia treated with mannan. Thus, mannan from C. albicans is a promising hypolipidemic polysaccharide compound, similar by its activity to ß-glycan, a component of LPS.

Depression of Macrophages Modifies Serum Lipid Profile in Hyperlipidemia.

Suppression of functional activity of macrophages by gadolinium chloride, suppressing the macrophage population and the endocytosis velocity, was studied in vivo. Injection of GdCl3 led to an increase in serum cholesterol concentration. Preliminary injection of GdCl3 to mice with lipidemia 24 h before poloxamer 407 reduced the concentrations of triglycerides and LDL during marked depression of macrophages (in 24 h). Macrophage repopulation (days 5, 7) was associated with development of a trend to an increase of triglyceride and LDL levels. lectron microscopic study of Kupffer cells after injection of poloxamer 407 and its combination with gadoliniun chloride detected the intralysosomal accumulation syndrome in these cells (formation of auto- and heterophagolysosomes). Activity of cathepsin B, characteristic of macrophages, reduced 24 h after injections of GdCl3 and poloxamer 407 alone and restored in response to their combination.

Effects of Anti-CD206 Antibodies on Macrophage Functions in Male CBF1 Mice with Lipidemia.

The effects of anti-CD208 antibodies (mannose receptor) on functional characteristics of peritoneal macrophages were studied in intact mice and mice with lipidemia induced by poloxamer-407. Lipidemia was associated with suppression of phagocytosis and increase in spontaneous proliferative potential and NO production by macrophages. Anti-CD206 antibodies suppressed NO production by macrophages in mice with lipidemia.

Cellular Composition of the Spleen and Changes in Splenic Lysosomes in the Dynamics of Dyslipidemia in Mice Caused by Repeated Administration of Poloxamer 407.

We studied the effect of dyslipidemia induced by poloxamer 407 (300 mg/kg twice a week for 30 days) on cellular composition of the spleen and splenocyte lysosomes in mice. Changes in blood lipid profile included elevated concentrations of total cholesterol, aterogenic LDL, and triglycerides most pronounced in 24 h after the last poloxamer 407 injection; gradual normalization of lipid profile was observed in 4 days (except triglycerides) and 10 days. The most pronounced changes in the spleen (increase in organ weight and number of cells, inhibition in apoptosis, and reduced accumulation of vital dye acridine orange in lysosomes) were detected on day 4; on day 10, the indices returned to normal. Cathepsin D activity in the spleen also increased at these terms. The relationship between changes in the cellular composition of the spleen and dynamics of serum lipid profile in mice in dyslipidemia caused by repeated administrations of relatively low doses of poloxamer 407 is discussed.

Comparative characteristics of lipemia models induced by injections of Triton WR-1339 and poloxamer 407 in mice.

Lipemia modeled by injections of Triton WR-1339 (500 mg/kg) and poloxamer 407 (500 mg/kg) to mice were compared. LP fraction and subfraction compositions were compared by small-angle X-ray scattering on a diffractometer. Both compounds in the same dose caused a sharp increase in serum concentrations of total cholesterol (CH) and triglycerides (TG), the increases in response to poloxamer 407 being more pronounced. The differences in the models consisted in the levels of atherogenic fractions: CH-VLDL (subfractions CH-VLDL1-2) and CH-LDL, which were higher under the effect of poloxamer 407. Similar increases were observed for atherogenic fractions: TG-VLDL (subfractions TG-VLDL1-2) and TG-LDL (subfractions TG-LDL1-3). A specific feature of the model induced by poloxamer 407 was elevation of the concentrations of antiatherogenic CH-HDL and TG-HDL (subfractions CH-HDL2 and TG-HDL2). Both models exhibited high similarity, but changes in atherogenic fractions were more pronounced under the effect of poloxamer 407.

Fractional composition of blood serum lipoproteins in mice and rats with Triton WR 1339-induced lipemia.

We compared fractional composition of blood serum lipoproteins (LP) in female ICR mice and Wistar rats induced by single administration of a nonionic detergent Triton WR 1339 in doses of 300 and 500 mg/kg. Lipemia in animals of both species was characterized by a sharp increase in the concentration of cholesterol and, particularly, of triglycerides in blood serum lipoproteins by the 24th hour after administration of the detergent. We revealed a significant increase in the concentrations of atherogenic VLDL cholesterol (due to VLDL2), intermediate density lipoproteins, and LDL. These changes were more pronounced in rats. The model of lipemia can be used to study the role of fractional composition of lipoproteins and, particularly, of triglycerides in the pathogenesis of atherosclerosis. Moreover, this model holds much promise for evaluation of the efficiency of hypolipidemic drugs (statins and fibrates) in normalizing the increased level of atherogenic cholesterol of VLDL and LDL.

[The role of changes in matrix metalloproteases and chitotriosidase in the mechanism of protective effect of atorvastatin in experimental murine lipemia].

The effect of atorvastatin on the activity of chitotriosidase (CTO) and total matrix metalloproteases (MMPs)-new markers of cardiovascular disorder-was studied on the model of murine lipemia induced by single administration of Triton WR 1339 in two doses, 500 mg/kg (mild lipemia) and 850 mg/kg (severe lipemia). A hypolipidemic effect of atorvastatin was observed in mice with mild lipemia, but not in those with severe lipemia. In both mild and severe lipemia cases, the serum CTO activity was increased upon the combined administration of atorvastatin and Triton WR 1339, correlating with cholesterol and triglyceride concentration. The total serum MMP activity decreased only in experiments with atorvastatin administration to intact mice. In mice with experimental lipemia induced by Triton WR 1339, the administration of atorvastatin also increased the ALT and AST activity in the blood serum.

Serum cystatin C and chitotriosidase in acute P-407 induced dyslipidemia: Can they serve as potential early biomarkers for atherosclerosis?

In an attempt to better understand potential biomarkers for, and the role of macrophages in, the development of atherosclerosis, the toxicologic, and any therapeutic pharmacologic effects of carboxymethylated ß-glucan, gadolinium chloride, and poloxamer 407 were studied in mice for their capacity to perturb serum lipids, cystatin C, and chitotriosidase-1. Gadolinium and carboxymethylated ß-glucan dosed separately to control mice had no effect on serum lipids, whereas carboxymethylated ß-glucan, but not gadolinium, exerted a significant (p<0.01) and unexpected hypolipidemic effect in poloxamer 407-induced hyperlipidemic mice. An acute hyperlipidemic state (∼4 days), induced with poloxamer 407 administration alone, resulted in a significant (p<0.01) time-dependent decrease and increase in serum cystatin C and chitotriosidase, respectively. Carboxymethylated ß-glucan administration to hyperlipidemic mice significantly (p<0.05) increased the serum concentration of cystatin C, but significantly (p<0.01) decreased chitotriosidase activity, when each was compared to mice treated with poloxamer 407 only. Gadolinium administration caused a significant decrease in serum chitotriosidase activity in both controls (p<0.01) and poloxamer 407-induced hyperlipidemic (p<0.001) mice, but had no effect on the concentration of cystatin C in either controls or poloxamer 407-induced hyperlipidemic mice. Gadolinium administration resulted in both morphological and functional changes to liver macrophages, which included incorporation of excess lipids, especially when simultaneously administered with poloxamer 407. It is suggested that serum cystatin C and chitotriosidase may represent potential early biomarkers for eventual atherosclerosis in the poloxamer 407-induced mouse model of atherogenesis, and that two compounds known to either increase (carboxymethylated ß-glucan) or decrease (gadolinium chloride) the number of macrophages in vivo were able to modulate serum chitotriosidase activity, This, in turn, would appear to support the premise that serum chitotriosidase activity may be a more sensitive indicator of macrophage involvement than cystatin C in the context of future atherosclerosis.

[Effect of triton WR 1339 on the ultrastructure of liver parenchymal and stromal cells in rats with chronic toxic hepatitis]. / Vliianie tritona WR 1339 na ul'trastrukturu kletok parenkhimy i stromy pecheni krys pri khronicheskom toksicheskom gepatite.

A preliminary injection of triton WR 1339 protectively influenced the chronic toxic CCl4-hepatitis. The ultrastructural peculiarities were close to the normal ones in hepatocytes, in addition to the increased triglyceride rejection from cells by means of autophagy. The revealed stimulation signs of pinocytosis in hepatocytes and the Kupfer cells are accompanied by the activation of the Golgi complex and, probably, by the increased protein catabolism in lysosomes. The signs of phagocytosis increase in the Kupfer cells were determined. It is supposed that the marked ultrastructural peculiarities of liver cells reflect a more effective restoration or a lesser injury under the influence of triton WR 1339.

Activity of lysosomal enzymes in the bile and serum of mice with intrahepatic cholestasis.

Lysosomal enzyme activity in the bile and blood serum was compared in mice with experimental intrahepatic cholestasis induced by alpha-naphthyl isothiocyanate and Triton WR 1339. Triton WR 1339 increases the synthesis of cholesterol (fatty acid precursor) in liver cells. The development of intrahepatic cholestasis was confirmed by the increase in activities of alkaline phosphatase and gamma-glutamyltransferase in blood serum. Administration of Triton WR 1339 in a dose of 100 mg/100 g was followed by a 10-fold increase in beta-galactosidase activity (hepatocyte lysosomal enzyme) in the bile, but not in the serum of mice. beta-Galactosidase activity significantly increased in the bile, but decreased in the serum of mice after treatment with a-naphthyl isothiocyanate in a dose of 200 mg/kg. Our results indicate that intrahepatic cholestasis is manifested in increased secretion of lysosomal glycosidases into the bile. Bile components can aggravate damage to liver cells by affecting the processes of hepatocyte apoptosis and necrosis.

Changes in the concentration of tissue inhibitor of type 1 metalloproteinases in blood serum and liver of mice with CCl4-induced hepatitis.

The concentration of tissue inhibitor of type 1 metalloproteinases in blood serum from intact CBA mice measured by enzyme immunoassay is similar to that in healthy humans. The concentration of tissue inhibitor of type 1 metalloproteinases in mouse bile was higher than in blood serum, while its concentration in liver homogenate more than 1000-fold exceeded that in the serum, which attests to its primarily intracellular localization in the liver. Loading of liver cell lysosomes with Triton WR-1339 and development of intrahepatic cholestasis did not affect the concentration of tissue inhibitor of type 1 metalloproteinases in liver homogenate and bile. Administration of CCl4 to mice for 4.5 weeks was accompanied by an increase in the concentration of tissue inhibitor of type 1 metalloproteinases in blood serum, but not in liver homogenate. These changes reflect dysregulation of metalloproteinases, development of inflammation, and progression of the initial stage of connective tissue formation in mouse liver.

[Role of vacuolar apparatus overload in lysosomal changes in liver cells in acute toxic hepatitis]. / Rol' peregruzki vakuoliarnogo apparata v izmeneniiakh lizosom kletok pecheni pri ostrom toksicheskom gepatite.

Physical and chemical properties of the rat liver lysosomes with single Triton WR 1339 overloading were studied during the administration of a detergent to intact rats and those with acute toxic hepatitis. Administration of the latter to intact animals was accompanied by a reduction of the floating density of the particles, solubilization of the lysosome enzymes and by increased fragility of the particles in the hypotonic medium. Lysosomes of the hepatocytes in rats with toxic hepatitis also displayed signs of overloading of the vacuolar apparatus with the preparation administered. The most pronounced solubilization of the lysosomal enzymes beta-galactosidase, acid RNA-ase, cathepsin D--was noted in case of combined action of CCl4 and Triton WR 1339 24, 48, 72 hours and 7 days after the CCl4 poisoning. Possible consequences of overloading of the vacuolar apparatus of the rat hepatocytes are discussed.

[Effect of a lysosome triton WR 1339 load on distribution of C14-albumin in the livers of rats with chronic hepatitis]. / Vliianie nagruzki lizosom tritonom WR 1339 na raspredelenie al'bumina-C14 v pecheni krys s khronicheskom gepatitom

A possible mechanism of the protective effect of Triton WR1339 during chronic CC14-hepatitis was considered. Supposing that the improvement of the process was due to the intensification of the lysosome heterophage function the authors studied the intensity of the C14-albumin uptake by the liver and its subcellular distribution in the liver of rats in the administration of the detergent to the animals with chronic CCI4-hepatitis, Preliminary administration of the detergent failed to influence the intensity of C14-albumin uptake; subsequent administration of triton WR1339 to rats with toxic hepatitis decrease the protein uptake which reached the values in intact rats. In chronic hepatitis C14-albumin was concentrated in the lysosome fraction. Administration of trition WR1339 to CCI4-treated animals was not accompanied by any coincidence in the peaks of labeled protein and lysosome enzymes. The selective participation of lysosomes of the Kupffer cells providing a more rapid restoration of the liver in chronic hepatitis is discussed.

[Changes in rat liver lysosomes following stimulation of recovery processes in the damaged organ by triton WR-1339]. / Osobennosti izmenenii lizosom pecheni krys pri stimuliatsii vosstanovitel'nykh protsessov tritonom WR-1339 v povrezhdennom organe.

The effect of a single injection of lysosomotropic agent--Triton WR-1339 on the properties of the rat liver lysosomes and the course of pathological process during chronic toxic hepatitis were studied. Triton WR-1339 administration was followed by a more rapid restoration of the liver structure and function. A possible mechanism of the favourable effect of Triton WR-1339 is discussed.

Heterophagic function and rate of intralysosomal proteolysis during lysosomotropic agents administration.

Lysosomotropic agents--Triton WR 1339 and suramin--are taken up selectively into lysosomes during in vivo administration and cause specific changes of the particles. Overloading of rat liver lysosomes by Triton WR 1339 was accompanied by the labilization of lysosomes and an increased uptake of [14C]-bovine serum albumin ([14C]-BSA) by the rat liver. The rate of intralysosomal proteolysis was not altered. The capture of 125I-labelled poly(vinylpyrrolidone) (PVP) by the liver was slightly decreased. In the case of suramin administration (250 mg/kg b.w.) the uptake of labelled protein by the liver was not changed. The increased amount of acid-insoluble radioactivity in rat liver was caused by the decrease of intralysosomal protein digestion rate. The lysosomes overloaded by the two kinds of lysosomotropic agents--Triton WR 1339 (with no changes of intralysosomal proteolysis) and suramin (with decreased rate of proteolysis) did not prevent the uptake by liver of substances captured by the adsorptive ([14C]-BSA) or fluid ([125I]-PVP) endocytosis.

[Lysosomotropic properties of polyvinylpyrrolidone (an experimental study]. / Lizosomotropnye svoistva polivinilpirrolidona (éksperimental'noe issledovanie).

Three-fold administration of polyvinylpyrrolidone (PVP), molecular weight 24 000, in a dose of 3.3 ml of 0.6% solution per 100 g/bw to rats was accompanied by disorders of lysosomal integrity and increased vulnerability to damage of the particles in the hypotonic medium (as judged from acid ribonuclease and cathepside D release). Five days after a single administration 125I-PVP was retained primarily by liver cells (7.3% of the dose administered) and to a lesser degree by renal cells (0.43%) and spleen cells (0.93%). Ninety per cent of the labeled PVP was bound to granular fractions of the rat liver (pinosomes, heterolysosomes). In the course of particles destruction by 0.1% Triton X-100 90% of 125I-PVP was released into the supernatant, which confirms the lysosomal localization of the compound.

[The effect of loading liver lysosomes with triton WR 1339 on the development of chronic toxic hepatitis]. / Vliianie nagruzki lizosom pecheni tritonom WR 1339 na razvitie khronicheskogo toksicheskogo gepatita

Preliminary administration of triton WR 1339 produced a favourable effect on the course of chronic toxic hepatitis. This was expressed in a reduction of necrotic zones, a delay in development of connective tissue and in improvement of the functional capacity of the liver. Lysosomes of the liver of animals subjected to the action of CCl-4 under conditions of preliminary administration of a detergent were more stable to the injurious actions in vitro.

[Facilitation of the structural and functional disorders of liver lysosomes in toxic hepatitis due to the suppression of intralysosomal proteolysis]. / Usilenie strukturno-funktsional'nykh narushenii lizosom pecheni pri toksicheskom gepatite, vyzvannoe podavleniem vnutrilizosomnogo proteoliza.

Suramin that accumulates in rat liver Kupffer cell lysosomes and inhibits the intralysosomal proteolysis was used to suppress the functional activity of these particles during liver damage (acute CCl4 hepatitis). Polyvinylpyrrolidone that does not disturb protein catabolism in liver lysosomes was employed for reference. According to the characteristic changes in lysosomes induced by suramin (inhibition of acid phosphatase, decrease of the rate of the intralysosomal proteolysis in the liver) and PVP the damaged liver was able to accumulate the lysosomotropic substances under study. Suramin aggravated liver damage and increased the lysosomal labilization, whereas PVP exhibited the protective action. The unfavourable effect of suramin may be linked with the suppression of catabolism of Kupffer cell lysosomes. The data obtained suggest the lack of safety of using the inhibitors of intralysosomal proteolysis in patients with acute hepatitis.

Endocytosis by liver cells during suppression of intralysosomal proteolysis.

The lysosomotropic agent chloroquine is widely used as a specific inhibitor of intralysosomal proteolysis in isolated hepatocytes. It was shown that in vitro chloroquine reversibly inhibited purified cathepsins H, B, L in concentrations less than those observed inside lysosomes in vivo. However, administration of high doses of chloroquine to rats (30-50 mg/kg i.p. as a single or repeated injections) was followed by increased cathepsin D and cysteine proteinase activities, as well as other lysosomal enzymes. Chloroquine administration did not induce any changes of carbon particles phagocytosis by liver cells (macrophages); modifications of fluid-phase (125I-PVP uptake) and receptor-mediated endocytosis (125I-asialo-fetuin uptake) were noted. Chloroquine administered in vivo reproduced some symptoms of lysosomal storage diseases (especially during repeated drug administration).

Chitotriosidase as a marker of macrophage stimulation.

Serum chitotriosidase activity was determined in different conditions accompanied by macrophage stimulation. Stimulation of macrophages with zymosan, yeast polysaccharide carboxymethylglucan (fraction II), and lysosomotropic preparation Triton WR-1339 1.5-2.0-fold increased enzyme activity. Chitotriosidase activity in intact Wistar rats was similar to that in humans, while in CBA and A/Sn mice this parameter was 5-fold higher. Sharp increase in chitotriosidase activity in the serum from patients with type I Gaucher's disease was probably related to intense secretion of the enzyme by macrophages. Under experimental conditions, stimulation of rat and mouse macrophages (mainly liver cells) caused no increase in chitotriosidase activity typical of patients with Gaucher's disease.