ALIMENTAZIONE e PLEUROTUS ostreatus:

Fibre. La prima misura terapeutica è senz’altro costituita dall’introduzione nella alimentazione di sostanze che aumentano la massa intestinale assorbendo acqua, e riducono l’assorbimento dei grassi dal lume intestinale: psillio, crusca, glucomannano, guar e Plantago ovata, per le mucillagini contenute nel tegumento del seme. Queste sostanze devono essere obbligatoriamente assunte con un abbondante quantitativo di acqua. In particolare, le fibre rallentano la velocità di assorbimento dei lipidi e la formazione delle molecole lipidiche finali, aumentano il transito intestinale, riducono il riassorbimento degli acidi biliari (blocco del loro circolo entero-epatico). Tra le fibre, sono quelle solubili contenute nella frutta, nella verdura e nei legumi (pectine, beta glucano ecc.) a ridurre maggiormente il colesterolo totale, e selettivamente il colesterolo LDL.

Olio di pesce. Un elemento non fitoterapico, ma pur sempre considerato naturale e come tale spesso assimilato ai prodotti a base di piante medicinali è rappresentato dall’olio di pesce (PUFA n-3), ricco di acidi grassi polinsaturi Omega 3, EPA e DHA (acido eicosapentaenoico e acido docosaesaenoico in rapporto 0,9:1,5) le cui principali azioni farmacologiche consistono in una riduzione di numerose funzioni:
– livello di trigliceridi
– sintesi di acido arachidonico
– aggregazione piastrinica
– chemiotassi dei monociti
– tendenza alla trombosi
– aritmie
Il dato maggiormente interessante è quello emerso dallo studio italiano Gissi-prevenzione condotto su 11.324 pazienti e pubblicato su Lancet nel 1999, cioè la dimostrazione di efficacia nella prevenzione secondaria nel paziente con pregresso infarto del miocardio e nella riduzione del rischio di mortalità. Per la riduzione dei trigliceridi si utilizza 1 g di olio di pesce due-tre volte al giorno, mentre nella prevenzione del reinfarto 1 g/die. Può aumentare il rischio di sanguinamento nei soggetti che assumono anticoagulanti.

Nell’alimentazione quotidiana, è inoltre raccomandato l’uso di olio di oliva: anche un recente studio pubblicato su Annals of Internal Medicine ha confermato l’importanza della dieta mediterranea nella riduzione del colesterolo totale, del livello di LDL. La dieta ricca di acidi grassi poliinsaturi favorisce anche la vasodilatazione. L’unica avvertenza rimane nei confronti del contenuto calorico.

Un recente lavoro clinico (Kurowska EM, 2000) ha dimostrato come un’alta dose di succo d’arancia (750 ml/giorno) abbia aumentato del 21 % la concentrazione di HDL colesterolo nel sangue di 25 volontari con ipercolesterolemia, riducendo del 16 % il rapporto LDL/HDL (p < 0.005). Il risultato non era invece significativo per l’assunzione di quantitativi più limitati (250 o 500 ml/die ). L’assunzione di succo d’arancia, ma anche il consumo alimentare dei frutti, potrebbe pertanto diventare un consiglio abituale per l’alimentazione del soggetto dislipidemico.

La soia invece può contribuire alla riduzione del tasso di colesterolo attraverso numerosi suoi costituenti:
– Proteine
– Isoflavoni
– Lecitina
– Saponine
– Fibre
Diventa fondamentale il ricorso alla soia come alimento, utilizzata ad esempio all’interno di zuppe di legumi e cereali integrali, ma anche come lecitina assunta pura alla dose di alcuni grammi al giorno (5-10) ed anche di isoflavoni qualora si sia di fronte ad una ipercolesterolemia in menopausa.

 

In particolare è stato dimostrato un aumento del colesterolo HDL, una riduzione delle LDL ed una attività antiossidante. I fitoestrogeni sarebbero anche in grado di inibire l’attività dell’enzima 7 alfa reduttasi, che rappresenta una delle tappe iniziali della biosintesi del colesterolo. Gli isoflavoni inoltre sperimentalmente prevengono le lesioni intimali, inibendo la formazione di neointima. E’ stato pure registrato un aumento dei recettori per le LDL.
L’integrazione dietetica con soia può consentire una riduzione tra il 15 ed il 25 % del colesterolo.

 

2008 Jul-Sep;26(3):256-99. doi: 10.1080/10590500802350086.

Selenium in edible mushrooms.

Falandysz J.

Source

Department of Environmental Chemistry, Ecotoxicology & Food Toxicology, University of Gdańsk, Gdańsk, Poland. jfalandy@chem.univ.gda.pl

Abstract

Selenium is vital to human health. This article is a compendium of virtually all the published data on total selenium concentrations, its distribution in fruitbody, bioconcentration factors, and chemical forms in wild-grown, cultivated, and selenium-enriched mushrooms worldwide. Of the 190 species reviewed (belonging to 21 families and 56 genera), most are considered edible, and a few selected data relate to inedible mushrooms. Most of edible mushroom species examined until now are selenium-poor (< 1 microg Se/g dry weight). The fruitbody of some species of wild-grown edible mushrooms is naturally rich in selenium; their occurrence data are reviewed, along with information on their suitability as a dietary source of selenium for humans, the impact of cooking and possible leaching out, the significance of traditional mushroom dishes, and the element’s absorption rates and co-occurrence with some potentially problematic elements. The Goat’s Foot (Albatrellus pes-caprae) with approximately 200 microg Se/g dw on average (maximum up to 370 microg/g dw) is the richest one in this element among the species surveyed. Several other representatives of the genus Albatrellus are also abundant in selenium. Of the most popular edible wild-grown mushrooms, the King Bolete (Boletus edulis) is considered abundant in selenium as well; on average, it contains approximately 20 microg Se/g dw (maximum up to 70 microg/g dw). Some species of the genus Boletus, such as B. pinicola, B. aereus, B. aestivalis, B. erythropus, and B. appendiculus, can also accumulate considerable amounts of selenium. Some other relatively rich sources of selenium include the European Pine Cone Lepidella (Amanita strobiliformis), which contains, on average, approximately 20 microg Se/g dw (up to 37 microg/g dw); the Macrolepiota spp., with an average range of approximately 5 to < 10 microg/g dw (an exception is M. rhacodes with < 10 microg/g dw); and the Lycoperdon spp., with an average of approximately 5 microg Se/g dw. For several wild-grown species of the genus Agaricus, the selenium content ( approximately 5 microg/g dw) is much greater than that from cultivated Champignon Mushroom; these include A. bisporus, A. bitorquis, A. campestris, A. cesarea, A. campestris, A. edulis, A. macrosporus, and A. silvaticus. A particularly rich source of selenium could be obtained from selenium-enriched mushrooms that are cultivated on a substrate fortified with selenium (as inorganic salt or selenized-yeast). The Se-enriched Champignon Mushroom could contain up to 30 or 110 microg Se/g dw, while the Varnished Polypore (Ganoderma lucidum) could contain up to 72 microg Se/g dw. An increasingly growing database on chemical forms of selenium of mushrooms indicates that the seleno-compounds identified in carpophore include selenocysteine, selenomethionine, Se-methylselenocysteine, selenite, and several unidentified seleno-compounds; their proportions vary widely. Some aspects of environmental selenium occurrence and human body pharmacokinetics and nutritional needs will also be briefly discussed in this review.

PMID:

 

18781538

 

[PubMed – indexed for MEDLINE]

 

1996 Sep;35(3):249-52.

Oyster mushroom (Pleurotus ostreatus) reduces the production and secretion of very low density lipoproteins in hypercholesterolemic rats.

Bobek P, Ozdin L.

Source

Research Institute of Nutrition, Bratislava, Slovak Republich.

Abstract

Oyster mushroom (Pleurotus ostreatus) reduced the production and secretion of nascent very low density lipoproteins in hypercholesterolemic rats. In male Wistar rats (initial body weight about 70 g) fed a semisynthetic diet with 0.3% of cholesterol, the addition of 5% of powdered oyster mushroom (Pleurotus ostreatus) to the diet reduced after 8 weeks the level of serum cholesterol (by 36%) and accumulation of cholesterol and triglycerides in liver (by 51 and 32%, respectively). The decreased levels of serum cholesterol were caused to the same extent by reduction of cholesterol content in very low density lipoproteins (VLDL) and in low density lipoproteins (LDL) (by 53 and 47%, respectively). Biosynthesis of all structural lipids of VLDL (phospholipids, cholesterol, triglycerides) in liver and incorporation of de novo synthesized lipids into secreted nascent VLDL (measured by simultaneous application of Na-acetate-1-14 C and Triton WR 1339 which inhibits peripheral lipolysis) was reduced by application of diet with oyster mushroom.

2012 Oct 27. [Epub ahead of print]

Antihypercholesterolemic and antioxidative effects of an extract of the oyster mushroom, Pleurotus ostreatus, and its major constituent, chrysin, in Triton WR-1339-induced hypercholesterolemic rats.

Anandhi R, Annadurai T, Anitha TS, Muralidharan AR, Najmunnisha K, Nachiappan V, Thomas PA, Geraldine P.

Source

Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India.

Abstract

Hypercholesterolemia and oxidative stress are known to accelerate coronary artery disease and progression of atherosclerotic lesions. In the present study, an attempt was made to evaluate the putative antihypercholesterolemic and antioxidative effects of an ethanolic extract of the oyster mushroom (Pleurotus ostreatus) and chrysin, one of its major components, in hypercholesterolemic rats. Hypercholesterolemia was induced in rats by a single intraperitoneal injection of Triton WR-1339 (300 mg/kg body weight (b.wt.)), which resulted in persistently elevated blood/serum levels of glucose, lipid profile parameters (total cholesterol, triglycerides, low-density lipoprotein-, and very low-density lipoprotein-cholesterol), and of hepatic marker enzymes (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase). In addition, lowered mean activities of hepatic antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) and lowered mean levels of nonenzymatic antioxidants (reduced glutathione, vitamin C, and vitamin E) were observed. Oral administration of the mushroom extract (500 mg/kg b.wt.) and chrysin (200 mg/kg b.wt.) to hypercholesterolemic rats for 7 days resulted in a significant decrease in mean blood/serum levels of glucose, lipid profile parameters, and hepatic marker enzymes and a concomitant increase in enzymatic and nonenzymatic antioxidant parameters. The hypercholesterolemia-ameliorating effect was more pronounced in chrysin-treated rats than in extract-treated rats, being almost as effective as that of the standard lipid-lowering drug, lovastatin (10 mg/kg b.wt.). These results suggest that chrysin, a major component of the oyster mushroom extract, may protect against the hypercholesterolemia and elevated serum hepatic marker enzyme levels induced in rats injected with Triton WR-1339.

PMID:

 

23104078

1997 Dec;54(4):240-3.

evidence for the anti-hyperlipidaemic activity of the edible fungus Pleurotus ostreatus.

Opletal L, Jahodár L, Chobot V, Zdanský P, Lukes J, Brátová M, Solichová D, Blunden G, Dacke CG, Patel AV.

Source

Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy, Charles University, Heyrovského, Czech Republic.

Abstract

The effects are described of adding either the dried fruiting bodies of the oyster fungus Pleurotus ostreatus, or an ethanolic extract of it, to the diet of normal Wistar male rats and a strain with hereditary hypercholesterolaemia. Addition of the dry oyster fungus to the diet significantly increased, by more than two-fold, the triacylglycerol (TAG) level in the plasma of both groups of rats compared with their respective controls. In contrast, the ethanolic extract did not significantly change TAG levels. Values for total cholesterol and its high- and low-density lipoprotein fractions in the plasma, as well as the calculated atherogenic index, did not show any significant change. Levels of liver cholesterol were significantly lowered by the dried oyster fungus in both hypercholesterolaemic and normal groups of rats, and by the ethanolic extract in normal rats. A significantly increased phospholipid-to-cholesterol ratio in the aortas of both groups of rats, after the administration of either dried oyster fungus or the ethanolic extract of it, suggests a favourable anti-atherogenic effect for both.

2003 Jul;30(7):470-5.

Dietary mushroom (Pleurotus ostreatus) ameliorates atherogenic lipid in hypercholesterolaemic rats.

Hossain S, Hashimoto M, Choudhury EK, Alam N, Hussain S, Hasan M, Choudhury SK, Mahmud I.

Source

Department of Physiology, Shimane Medical University, Shimane, Japan.

Abstract

1. The effects of edible oyster mushroom Pleurotus ostreatus on plasma and liver lipid profiles and on the plasma total anti-oxidant status were estimated in hyper- and normocholesterolaemic Long Evans rats. 2. The feeding of 5% powder of the fruiting bodies of P. ostreatus mushrooms to hypercholesterolaemic rats reduced their plasma total cholesterol by approximately 28%, low-density lipoprotein-cholesterol by approximately 55%, triglyceride by approximately 34%, non-esterified fatty acid by approximately 30% and total liver cholesterol levels by > 34%, with a concurrent increase in plasma high-density lipoprotein-cholesterol concentration of > 21%. However, these effects were not observed in mushroom-fed normocholesterolaemic rats. 3. Mushroom feeding significantly increased plasma fatty acid unsaturation in both normo- and hypercholesterolaemic rats. 4. Plasma total anti-oxidant status, as estimated by the oxidation of 2,2′-azino-bis-[3-ethylbenz-thiazoline-6-sulphonic-acid], was significantly decreased in mushroom-fed hypercholesterolaemic rats, concomitant with a decrease in plasma total cholesterol. 5. The present study suggests that 5% P. ostreatus supplementation provides health benefits, at least partially, by acting on the atherogenic lipid profile in the hypercholesterolaemic condition.

Mycobiology. 2011 March; 39(1): 45–51.

Published online 2011 March 23. doi: 10.4489/MYCO.2011.39.1.045

PMCID: PMC3385090

Ostreolysin, a pore-forming protein from the oyster mushroom, interacts specifically with membrane cholesterol-rich lipid domains

Edited by Gerrit van Meer

· Kristina Sepčića,

· Sabina Bernea,

· Katja Rebolja,

· Urška Batistab,

· Ana Plemenitašc,

· Marjeta Šentjurcd,

· Peter Mačeka, ,

· a Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia

· b Institute of Biophysics, Medical Faculty, University of Ljubljana, Lipičeva 2, 1000 Ljubljana, Slovenia

· c Institute of Biochemistry, Medical Faculty, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia

· d Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia

· http://dx.doi.org/10.1016/j.febslet.2004.07.093, How to Cite or Link Using DOI

· Permissions & Reprints

Abstract

Ostreolysin, a 15 kDa pore-forming protein from the edible oyster mushroom (Pleurotus ostreatus), is lytic to membranes containing both cholesterol and sphingomyelin. Its cytotoxicity to Chinese hamster ovary cells correlates with their cholesterol contents and with the occurrence of ostreolysin in the cells detergent resistant membranes. Moreover, ostreolysin binds to supported monolayers and efficiently permeabilizes sonicated lipid vesicles, only if cholesterol is combined with either sphingomyelin or dipalmitoylphosphatidylcholine. Addition of mono- or di-unsaturated phosphatidylcholine to the cholesterol/sphingomyelin vesicles dramatically reduces the ostreolysin’s activity. It appears that the protein recognizes specifically a cholesterol-rich lipid phase, probably the liquid-ordered phase.

 

1. Introduction

Ostreolysin (Oly) [1] and [2] is a representative of the aegerolysin protein family (Pfam Accession No. PF06355). So far, these proteins have been found in the bacteria Clostridium bifermentans[3] andPseudomonas aeruginosa (TrEMBL Accession No. Q9I710), moulds Aspergillus fumigatus[4] andNeurospora crassa (TrEMBL Accession No. Q8WZT0), and mushrooms Pleurotus ostreatus and Agrocybe aegerita [1] and [5]. It has been suggested that they may have a role in processes such as modulation of bacterial sporulation [3], virulence of A. fumigatus[4], and mushroom fruiting [6].

Oly interacts with lipids and forms pores in eukaryotic cell membranes and lipid vesicles prepared from total membrane lipids [2]. So far, searching for a specific lipid acceptor of the proteins belonging to the aegerolysin protein family has not been successful. Lysophospholipids have been reported to be inhibitors of Asp-hemolysin from A. fumigatus[7] and Oly, but no direct interaction with lysophospholipids could be proved for Oly [2]. Very recently, an Oly isoform from P. ostreatus, pleurotolysin A, was suggested to be an SM-inhibited hemolysin [5].

Here, we addressed the question of membrane lipid acceptor(s) for ostreolysin. We provide evidence that ostreolysin is partitioned in both cell and artificial lipid membrane detergent resistant membranes (DRMs). Moreover, its binding to lipid mono- and bilayers, and lipid vesicle permeabilization, is specifically dependent on the presence of both cholesterol (Chol) and sphingomyelin (SM). SM can be replaced by saturated dipalmitoylphosphatidylcholine (DPPC); however, this results in the decreased activity of Oly.

2. Materials and methods

2.1. Ostreolysin and lipids

Ostreolysin was isolated from immature mushroom fruit bodies and N-sequenced to check purity as described [1]. In addition, its homogeneity was confirmed by ESI-mass spectroscopy, which gave an Mr for ostreolysin of 14975.4. Dioleoylphosphatidylcholine (DOPC), DPPC, palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylethanolamine, wool grease Chol, and porcine brain SM were obtained from Avanti Polar Lipids (USA). Ergosterol was from Fluka (Switzerland) and ganglioside GM1 from Serva (Germany).

2.2. Cytotoxicity assay

To determine the influence of cell membrane Chol on the Oly cytotoxicity, we used Chinese hamster ovary wild-type CHO-K1 and CHO-215 cells with deficient Chol synthesis (impaired 4-carboxysterol decarboxylase) [8], grown in a McCoy’s 5A medium (Sigma, Germany) with 10% fetal calf serum (FCS) at 37 °C and 5% CO2. Cells were plated overnight in 96-well microtiter plates (Costar, USA) at a concentration of 5 × 103 cells/well in the growth medium. After washing them with a Dulbecco’s PBS buffer, fresh growth medium, with either 10% FCS or a lipoprotein poor serum (LPPS) with diminished Chol contents [8] and [9], was added. After 24 h, the cells were treated with 1 μg/ml Oly for 0–25 min. Cells were then washed three times with the growth medium and cytotoxicity was determined with an MTT assay [2]. The treated cells were inspected under a phase-contrast microscope.

2.3. Isolation of DRMs and Oly Western immunoblotting

DRMs were isolated from the cells or sonicated vesicles (SV) by Triton X-100 (0.5% v/v) extraction as described in detail before [9] and [10]. In the growth media, 2–5 × 107 cells were pre-treated with 2.5 μg/ml Oly for 15 min at 23 °C before the detergent extraction. The pelleted cells, lysed for 30 min at 0 °C by 0.5% Triton X-100 in buffered saline (25 mM 1-piperazineethane sulfonic acid, 4-(2-hydroxyethyl)-monosodium salt (HEPES), 150 mM NaCl, 1 mM ethylene-diaminotetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride, and 10 μg/ml aprotonin, pH 6.5), were mixed (1/1, v/v) with 85% sucrose (w/v) in the same buffer mixture. In a centrifuge tube, the resulting 42.5% sucrose mixture was overlaid successively with 5 ml of a 35 and 5% (w/v) sucrose gradient in the buffered saline supplemented with 1 mM EDTA and 1 mM Na3VO4. Following 18 h centrifugation at 4 °C (200 000×g, SW41 rotor, Beckman L8-M preparative centrifuge), centrifuged sucrose fractions were collected and analyzed for Oly using Western immunoblotting. Proteins were stained with rabbit anti-Oly polyclonal and secondary goat anti-rabbit horseradish peroxidase-conjugated antibodies [1]. Chol contents/mg of cell DRMs protein were determined as reported [8] and [9]. Using the same procedure as for the cell DRMs, SV DRMs were isolated after treatment of 10 mg/ml SV with 100 μg/ml Oly in 140 mM NaCl, 20 mM Tris–HCl, and 1 mM EDTA, pH 8.0, for 15 min at room temperature. In that case, only fractions containing visible Triton X-100-insoluble lipids were analyzed for Oly as described above.

2.4. Lipid vesicles and measurement of calcein release

Sonicated vesicles, either without or loaded with 80 mM calcein (Sigma), were prepared by sonication of 4–20 mg/ml lipids in 140 mM NaCl, 20 mM Tris–HCl, and 1 mM EDTA, pH 8.0. Non-encapsulated 80 mM calcein was removed on a Sephadex G-50 column. After sonication and centrifugation, the lipid concentration of SV suspensions (without calcein) was determined colorimetrically with Waco Free Cholesterol C and Wako test Phospholipids B (990-54009) kits (Waco Chemicals GmbH, Germany) in order to quantify total lipids in suspension and to check the cholesterol/phospholipid ratio. Before use, the sonicated vesicles were kept overnight at room temperature to avoid their instability due to fusion [11]. Size of SV was estimated by photon correlation spectroscopy on a Zeta Sizer instrument (Malvern Instruments, UK) as reported before [2].

Permeabilization of SV was measured as the increase in fluorescence intensity of calcein (exc./em. wave length, 485/535 nm), dequenched on release from SV, in a fluorescence microplate reader Tecan 02A (Tecan, Austria). The time-course of increasing fluorescence intensity, Ft, was fitted to a stretched exponential equation used in various chemical and physical rate processes in disordered media [12]:

equation(1)

where Fmin and Fmax are the starting and final fluorescence intensities, k is a rate constant, and b an exponential scaling factor. Cooperativity of the permeabilization process was estimated by determining the slope of the log–log dependence of the relative rate of permeabilization on concentration of Chol or Oly.

2.5. Hemolysis assay and inhibition of ostreolysin by lipids

Binding of Oly to SV (without encapsulated calcein) was estimated by measuring the hemolytic activity of the free Oly fraction after pre-incubation of 0.2 μg/ml Oly with varied concentration of SV lipids for 30 min at 37 °C. The lipid concentration necessary for reducing the rate of hemolysis of bovine erythrocytes by 50%, IC50, was determined in a MRX microplate reader (Dynex Technologies, Germany) as described [2].

2.6. EPR spectroscopy

EPR spectroscopy was used to evaluate potential effect of ostreolysin on the domain structure of lipid vesicles and their fluidity characteristics [13]. SV (20 mg/ml lipids, without calcein) were spin-labeled with MeFASL (10.3) (N-oxyl-2-undecyl-2-(3′-methoxycarbonyl)-propyl-4,4-dimethyloxazolidine, provided by Prof. S. Pečar, University of Ljubljana) using a molar ratio of 200:1. Spectra were taken on a X-band EPR spectrometer ESP 300 (Bruker, Germany) at temperatures ranging between 20 and 40 °C as described [14]. EPR spectra were analyzed with an EPRSIM Ver 4.9 software package for simulation of the spectra of nitroxide spin labels [13]. The simulation method takes into account the fact that the spectra are composed of several components that belong to the spin labels in different types of coexisting membrane regions with different ordering and dynamics of the alkyl chains. From the best fit to the experimental spectra, a number of the coexisting domains, their order parameter, rotational correlation time, and the corresponding portion of each region in the membrane of SV were derived. The effect of Oly on the parameters p and S was studied at a lipid/protein molar ratio of 100:1.

2.7. SPR determination of binding of ostreolysin to lipid monolayers

Oly binding to lipid monolayers was determined in a Biacore X surface plasmon resonance (SPR) apparatus (Biacore AB, Sweden). Monolayers were deposited on a HPA Chip (Biacore AB) using SV (∼0.5 mM lipids in 140 mM NaCl, 20 mM Tris–HCl, and 1 mM EDTA, pH 8.0) as recommended by the producer. Dissolved in the same buffer, Oly (0.081, 0.162, 0.325, 0.75, and 1.2 mg/ml) was injected at a flow rate of 40 μl/min and sensorgrams were recorded at 25 °C. Bulk controls were run without protein.

3. Results and discussion

In membranes, dynamic complexion of cholesterol with sphingomyelin and saturated glycerophosphatides results in liquid-ordered, Lo, and disordered, Ld, lipid phases [15]. This is the basis for the formation of separate lipid domains, lipid rafts (operationally DRMs). In cell membranes, rafts are enriched in cholesterol, sphingolipids, and specific proteins. They have roles in membrane trafficking and signaling [16],[17] and [18], and are attachment/entry sites for cell pathogens, toxins and other ligands [19] and [20].

Our preliminary screening for lipid acceptor(s) had demonstrated that only vesicles reconstituted from erythrocyte neutral lipids (mainly Chol) and phospholipids (SM and glycerophosphatides) could be permeabilized by Oly. This observation has suggested a specific cholesterol-induced lipid microdomain, such as Lo phase, which is characteristic of lipid rafts, to be a possible binding site. To check this hypothesis, we first employed wild-type CHO-K1 and mutant CHO-215 cells, with impaired Chol synthesis, to assess dependence of Oly cytotoxicity on the cell membrane contents of Chol and occurrence of Oly in DRMs. Fig. 1 demonstrates that (i) Oly is more cytotoxic to cells, both wild-type and mutant, grown in the presence of serum Chol and (ii) the higher the cellular Chol level, the more abundant is Oly in the cells’ DRMs. Despite the lower content of Chol, the mutant cells appear more sensitive to Oly, which could be the consequence of the lower viability of the cells with deficient Chol-synthesis as observed before [9] and [10]. Microscopy revealed that the extent of cytotoxicity correlated well with changes of cell shape, cell membrane blebbing, and cell lysis (not shown), similar to that observed for some tumor cells exposed to Oly[2].

 

So far, screening of dispersions of pure Chol, SM, DPPC, and some other phospholipids, or binary mixtures of phospholipids for inhibition of hemolysis produced by Oly has failed to reveal specific acceptor lipid(s)[1] and [2]. Following this fact and the results in Fig. 1, we used artificial lipid membranes to study Oly preference for lipid mixtures typical of DRMs. Only SV composed of Chol combined with either SM or DPPC exerted inhibition of hemolysis, as indicated by IC50 values in Table 1. None of the other combinations tested (up to 20 mg/ml lipids), such as those including POPC, DOPC or egg phosphatidylethanolamine, was inhibitory. The striking finding that, while inhibition is essentially dependent on Chol, this is only in combination with either SM or the saturated phosphatidylcholine species, strongly suggested that the protein might recognize a specific Chol-induced lipid phase.

Table 1. Lipid composition of sonicated vesicles, dimensions, and inhibition of ostreolysin-induced hemolysis

 

Composition (molar ratio)	Diameter (nm)	IC50 (mg/ml)
Chol:SM 1:9	104 ± 1	∼17
Chol:SM 2:8	123 ± 2	∼15
Chol:SM 3:7	88 ± 1	∼11
Chol:SM 4:6	94 ± 1	6.6
Chol:SM 5:5	91 ± 1	1.0
Chol:SM 6:4	97 ± 3	0.1
Chol:DOPC 1:1	119 ± 2	≫20
Chol:POPC 1:1	71 ± 6	≫20
Chol:DPPC 1:1	98 ± 3	∼3

 

Mean diameter D ± S.D. (standard deviation) was estimated by photon correlation spectroscopy. IC50 is the concentration of lipids that decreases the rate of hemolysis of bovine erythrocytes by 50% [2].

Table options

As shown in Fig. 2, we studied in more detail the dependence of SV permeabilization on specific Chol binary or ternary lipid mixtures allowing formation of the Lo phase and lipid rafts [21], [22] and [23]. Time courses of calcein release, quantifying permeabilization activity as exemplified in Fig. 2A, were used to derive a rate constant k and the percentage of maximal calcein release at different final Oly concentrations (Fig. 2B and C). The highest rate and extent of calcein release is pertinent to the Chol/SM SV, while those obtained with Chol/DPPC are approx. 20 times lower. POPC, 20 mol%, in SV containing Chol/SM (1:1) reduces both k and the percentage of release by approx. 60-fold, while at 33%, it results in a decrease of k by ∼1000-fold. Inclusion of 1% ganglioside GM1, well-known to be preferentially associated into the Lo lipid phase [24], has no effect on Oly permeabilizing activity. Replacement of Chol by the fungal sterol ergosterol in SV has a similar effect, but at a very much lower efficiency (Fig. 2B, C). In this respect, it is known that sterols other than Chol, typical for fungi or plants, and Chol precursors, have a similar function in lipid raft maintenance[25] and [26].

Detergent extraction of cell and artificial lipid membranes may produce artifact DRMs [27] and [28] and, therefore, inclusion of Oly in the cell and lipid DRMs might be an artifact. However, this seems unlikely due to the SPR results in Fig. 3, where direct binding, without intervention of the detergent, is shown. Further, incubation of spin labeled Chol/SM (1: 1) SV with Oly had no effect on their EPR spectra (not shown). This evidence thus supports the conclusion that the permeabilizing effect of Oly results from its direct recognition of an existing specific lipid complex, rather than any effect on recruiting and ordering of lipids.

Formation of a transmembrane pore by pore-forming proteins is a multi-step process that includes binding to the membrane and in-plane protein oligomerization [29] and [30]. Certain of such proteins may exploit cholesterol or SM for binding or protein insertion. Chol is crucial for permeabilization of bacterial cholesterol-dependent cytolysins but not for binding and pre-pore formation on the membrane [30]. An exception is perfringolysin O and its C-terminal domain, of which binding step is cholesterol-specific [31]. Another cytolysin, lysenin, uses SM as a binding acceptor [32], as also suggested for pleurotolysin A [5]. To discriminate between the possible effects of the specific Chol/lipid composition on either binding or pore-formation by Oly, we used SPR to detect directly its binding to lipid monolayers [29] and [33]. In Fig. 3, only sensorgrams obtained at the highest Oly concentration of 1.2 mg/ml are shown because no Oly binding to Chol/POPC could be observed below this concentration. On the Chol/SM monolayer, saturation with ostreolysin was observed by increasing the protein concentration (not shown); a half-saturation concentration was ∼90 μg/ml. The efficiency of Oly binding, in the order Chol/SM≫Chol/DPPC > Chol/POPC and no binding to the Chol/DOPC monolayers (Fig. 3A), strongly suggests that the binding step is essentially dependent on the specific Chol/lipid composition. This is also supported by partition of Oly into SV DRMs (Fig. 3B). There is evidence that the coexistence of Lo, Ld, and some other lipid domains, together with their contents, is dependent on the Chol/lipid(s) ratio [23], [34] and [35]. We, therefore, analyzed in more detail the effect of the Chol/SM ratio on the permeabilizing activity of Oly in SV (Fig. 4). The rate constants and extent of calcein release coincide with increased Chol. The concentrations of Chol (above 30 mol%) that produce detectable permeabilization are similar to those inducing the Lo lipid phase in mono- and bilayers[23] and [34]. If the marked activity is dependent on a Chol-rich phase, then one should expect that Oly interacts with certain Chol complexes. We used the method of initial rate estimates of the permeabilization curves to derive the coefficient n with respect to Chol concentration (Fig. 4C). The slope reveals unusual ever-increasing values for n, from ∼2 to about 45. In contrast, n determined for Oly from the plot of logvrel vs. log conc. of Oly was uniformly equal to ∼ 2.3 over its whole concentration range (not shown), in agreement with the previously reported Oly oligomerization on vesicles made of erythrocyte lipids [2]. The increasing Chol cooperativity suggests that Oly interacts with specific Chol-containing complexes, which may grow with increasing Chol concentration. On the other hand, the opposing effect of POPC (see Fig. 2) implies that mono- and di-unsaturated lipids promote dissociation of productive Chol-rich complexes and thus prevent interaction with Oly.

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4. Conclusions and perspectives

So far, the majority of studies of lipid domains were performed on monolayers or giant unilamellar vesicles. Our study suggests, however, that the specific cholesterol-rich phases could be studied just as well in vesicles of a smaller diameter (see Table 1). It is evident that Oly does not interact with either pure Chol or SM but that it does react, specifically, with a Chol-rich lipid phase, the integrity of which is highly sensitive to unsaturated glycerophosphatides. The underlying mechanism could be dissociation of Chol-complexes by unsaturated phospholipids and, in turn, increase of chemical activity of Chol [24]. According to recent data, we suggest that the Chol-induced phase may be the Lo phase, however, some other unprecedented Chol-rich lipid phase, dependent on the presence of saturated phosphatidylcholines in the membrane, cannot be excluded. In fact, the diversity of Chol-rich lipid phases in membranes in terms of the stoichiometry of Chol/Chol and/or Chol/other lipid complexes is not obvious [15] and [24]. The properties of Oly described here thus make Oly and its engineered mutants – for example, fused with fluorescent proteins or labeled with fluorescent, as reported for perfringolysin O [36], or spin probes – a new tool in further studies of Chol-rich lipid phases and Chol-complexes in natural and artificial lipid membranes.

Acknowledgements

The study was supported by Ministry of Education, Science, and Sports of Slovenia. We thank Prof. B.Kralj (Jožef Stefan Institute, Ljubljana) for mass spectroscopy measurements, Prof. R. Pain for critical reading of the manuscript, and I. Pavešić for skillful technical assistance.

Hypolipidemic Activities of Dietary Pleurotus ostreatus in Hypercholesterolemic Rats

Nuhu Alam, Ki Nam Yoon, Tae Soo Lee, and U Youn Lee

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This work was conducted to investigate dietary supplementation of oyster mushroom fruiting bodies on biochemical and histological changes in hyper and normocholesterolemic rats. Six-week old female Sprague-Dawley albino rats were divided into three groups of 10 rats each. Feeding a diet containing a 5% powder of Pleurotus ostreatus fruiting bodies to hypercholesterolemic rats reduced plasma total cholesterol, triglyceride, low-density lipoprotein (LDL), total lipid, phospholipids, and LDL/high-density lipoprotein ratio by 30.18, 52.75, 59.62, 34.15, 23.89, and 50%, respectively. Feeding oyster mushrooms also significantly reduced body weight in hypercholesterolemic rats. However, it had no adverse effects on plasma albumin, total bilirubin, direct bilirubin, creatinin, blood urea nitrogen, uric acid, glucose, total protein, calcium, sodium, potassium, chloride, inorganic phosphate, magnesium, or enzyme profiles. Feeding mushroom increased total lipid and cholesterol excretion in feces. The plasma lipoprotein fraction, separated by agarose gel electrophoresis, indicated that P. ostreatus significantly reduced plasma β and pre-β-lipoprotein but increased α-lipoprotein. A histological study of hepatic cells by conventional hematoxylin-eosin and oil red O staining revealed normal findings for mushroom-fed hypercholesterolemic rats. These results suggest that a 5% P. ostreatus diet supplement provided health benefits by acting on the atherogenic lipid profile in hypercholesterolemic rats.

Keywords: Agarose gel electrophoresis, Atherogenic lipid profile, Histopathology, Hypercholesterolemic rats, Hypolipidemic, Pleurotus ostreatus

Pleurotus ostreatus, the oyster mushroom, is increasingly being recognized as an important food product with a significant role in human health and nutrition [1]. It is generally accepted that lowering high plasma cholesterol levels plays a significant role in preventing atherosclerosis. Oyster mushrooms are an ideal dietary substance for the prevention and treatment of hypercholesterolemia due to high content of dietary fiber, sterol, proteins, and microelements [2].

The fact that lovastatin is present in high proportions in this mushroom, is an important food supplement for patients suffering from hypercholesterolemia [3]. Besides lovastatin, P. ostreatuscontains various biologically active phenolic compounds such as gallic acid, protocatechuic acid, chlorogenic acid, naringenin, hesperetin, and biochanin-A [4]. The general idea that controlling blood cholesterol is an important for reducing the risk of developing atherosclerosis [5] has stimulated the investigation and study of natural substances with hypocholesterolemic activity.

Considering the widely accepted concept about the key role of reactive oxygen species in the pathogenesis of atherosclerosis [6], reduced lipid peroxidation in blood is an additional positive effect of oyster mushrooms. Oyster mushrooms and other related mushrooms are used in traditional oriental medicine as components of natural diets with an antisclerotic effect [7].

There is considerable data supporting the hypothesis that the health benefit obtained through lowering blood cholesterol may be derived from the effects of eicosapentaenoic acid and docosahexaenoic acid [8]. In addition to their roles in the development and functioning of the central nervous system, these two fatty acids play an important role in the physiological functions of the cardiovascular system [9].

A hypolipidemic activity study is pertinent because the hypolipidemic activity of P. ostreatus is essential for its antiatherosclerotic function. Moreover, P. ostreatus has the potential to serve as an effective therapeutic agent for hyperlipidemic diseases, especially cardiovascular disease. Despite the medicinal importance of P. ostreatus and its therapeutic potential, no detailed studies on the biochemical and histological function of hypercholesterolemia have been performed, and comprehensive studies on the antihyperlepidemic effects of this mushroom are not available. Therefore we examined the potential hypolipidemic activity of P. ostreatus to generate awareness of its health benefit.

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Mushroom

Fresh fruiting bodies of P. ostreatus (cultivar Chun-chu 2) were obtained from Hanultari mushroom farm, Korea. A pure culture was deposited in the Culture Collection and DNA Bank of Mushroom, Division of Life Sciences, University of Incheon, Korea with the acquired accession number, IUM-4143. Fresh fruiting bodies were dried with hot air at 40℃ for 48 hr and pulverized.

Animals

Thirty female Sprague-Dawley albino rats (101 ± 4.2 g, 6-week old, purchased from Central Lab. Animal Inc., Seoul, Korea) were used. All animals were acclimated to the animal room for 1-week. The rats were housed in an animal room at 23 ± 2℃ under a 12 hr darklight cycle (17:00~5:00 hr) and relative humidity of 50~60%. Rats were divided into three feed groups: a basal diet (normocholesterolemic control rats; NC rats), basal diet with 1% cholesterol (hypercholesterolemic rats; HC rats), and a basal diet with 1% cholesterol and 5% P. ostreatus powder (oyster mushroom-fed hypercholesterolemic rats; HC + PO rats). The basal diet compositions are presented in Table 1, and the rats were feed for 42 days.

Plasma chemical analysis

At the end of the experimental period, overnight-fasted animals were sacrificed under injectable anesthetic (Zoletil 50; VIRBAC Laboratories, Carros, France). Blood samples were collected with a disposable plastic syringe into heparinized tubes. Plasma was prepared by centrifugation at 2,493 ×g for 10 min. Plasma triglyceride (TG) concentration was measured enzymatically using the glycerophosphate oxidase assay. Plasma total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C), total lipid (TL), and phospholipid (PL) levels were measured enzymatically by the cholesterol oxidase assay [10] using commercially available assay kits (Sekisui Medical Co., Ltd., Tokyo, Japan). Plasma albumin, total bilirubin, direct bilirubin, creatinin, blood urea nitrogen, uric acid, glucose, total protein, and electrolyte parameters, including calcium, sodium, potassium, chloride, inorganic phosphate, and magnesium were measured by standard methods using an auto analyzer (Hitachi 7600-210; Hitachi, Tokyo, Japan).

Very low density lipoprotein cholesterol was calculated as follows:

VLDL-C = [TC – (HDL-C + LDL-C)]

Plasma enzyme analysis

The activity of the plasma transaminases, glutamate pyruvate transaminase (GPT), and glutamate oxaloacetate transaminase (GOT) were determined using the kinetic method [10]. The oxoacids formed in the transaminase reactions were measured indirectly by enzymatic reduction to their corresponding hydroxyacids. The accompanying change in NADH concentration was measured at 340 nm using a spectrophotometer (Optizen POP; Mecasys Co. Ltd., Daejeon, Korea). Plasma alkaline phosphatase (ALP) activity was determined using 4-nitrophenyl phosphate. ALP catalyzes the hydrolysis of 4-nitrophenyl phosphate, forming phosphate and free 4-nitrophenol, which is colorless in dilute acid solutions. But, under alkaline conditions 4-nitrophenol is converted to the 4-nitrophenoxide ion, which is an intense yellow color. The absorbance of this color compound was measured spectrophotometrically at 420 nm to determine plasma ALP activity.

Fecal cholesterol and TL analysis

Feces were collected for 7 days before and at the end of 42 days, lyophilized, and then milled into powder. Total lipids were extracted with chloroform/methanol (2 : 1 v/v) according to the method of Folch et al. [11]. One gram of fecal powder was mixed with 10 mL of chloroform and 5 mL of methanol solution and stirred at 150 rpm for 3 days at room temperature. The suspension was filtered through Whatman No. 2 filter paper (Whatman, Maidstone, UK), the methanol was aspirated, and the chloroform was evaporated. The extracted lipids were then weighed. Two mL of H2O was added, and a suspension was created using a bath sonicator. This suspension was used to estimate fecal cholesterol content, which was estimated by the enzymatic method using the cholesterol oxidase assay.

Plasma lipoprotein separation by agarose gel electrophoresis

Plasma lipoprotein fractions were determined by agarose gel electrophoresis [12]. Three lipoprotein fractions were detected by electrophoresis, which will henceforth be referred to as β-lipoprotein (LDL), pre-β-lipoprotein (VLDL), and α-lipoprotein (HDL). Sample application (2 µL), electrophoresis (80 V, 30min), staining (Fat Red 7B), drying, and densitometric scanning (525 nm) were performed automatically by the Helena TITAN GEL Lipoprotein Electrophoresis System (Helena Laboratories, Beaumont, TX, USA). After electrophoresis, lipoprotein fractions were visualized with enzymatic staining reagents. The visualized gel plate was scanned on a densitometer, and the lipoprotein scanning patterns were identified using analytical software (electrophoresis data bank, K.K. Helena Laboratories, Saitama, Japan). The scanned patterns were divided into lipoprotein fractions using the nadirs of the lipoprotein sequential curve. Lipoprotein levels were estimated from the area percentages and total concentrations.

Histological analysis of liver

Liver tissues were rapidly dissected, fixed in liquid nitrogen and 10% formalin solution, and stored until use at -80℃. A representative part of the frozen tissues was processed with a cryo microtome (Cryotome FSE Cryostat; Thermo Electron Corp., Cambridge, MA, USA) using sections 5-µm thick and stained with oil red-O [13]. A representative part of the formalin fixative liver tissues was processed for 4-µm thick paraffin embedded sections using a microtome (Microtome HM 450; Thermo Electron Corp.) and then stained with hematoxylin and eosin. Both stained tissue samples were then examined and photographed under a light microscope to assess the presence of lipid. Digital images were obtained using an Olympus BX51 microscope equipped with a Camedia C3040ZOOM digital camera (Olympus America Inc., Melville, NY, USA). All images were taken under 40× magnification.

Statistical analysis

Results are expressed as means ± SDs. Intergroup differences were analyzed by a one-way analysis of variance followed by post-hoc tests. We used the SPSS ver. 11.5 (SPSS Inc., Chicago, IL, USA). A p ≤ 0.05 was considered statistically significant.

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Effects of feeding oyster mushroom on bodyweight

Feeding P. ostreatus reduced body weight significantly in hypercholesterolemic and normocholesterolemic rats by 16.89 and 13.38%, respectively (Table 2). This finding is of special significance because obesity is associated with many diseases including diabetes, atherosclerosis, coronary heart disease, and others [14].

Effects of feeding oyster mushroom on plasma lipid profile

Plasma lipid profile concentrations in NC, HC, and HC + PO rats after P. ostreatus feeding for 6-wk are presented in Table 3. Plasma TC, TG, HDL-C, LDL-C, VLDL-C, TL, and PL in HC rats increased by 17.09, 36.68, 12.23, 22.35, 19.01, 19.82, and 16.14%, respectively, compared with levels in NC rats, whereas these parameters decreased significantly by 30.18, 52.75, 19.91, 59.62, 27.08, 34.15, and 23.89%, respectively, in HC + PO rats compared with HC rats. The ratio of plasma LDL and HDL is shown in Fig. 1. In HC rats, this ratio increased by 11%, compared with NC rats, whereas this ratio was reduced significantly by 50% in HC + PO compared with HC rats. The results show that feeding 5% P. ostreatus to rats significantly ameliorated the plasma atherogenic lipid profiles in experimentally induced HC rats.

Effects of Pleurotus ostreatus on plasma low density lipoprotein (LDL)/high density lipoprotein (HDL) ratio in hypercholesterolemic rats. Results are mean ± SDs. Different symbols indicate significant differences at p ≤ 0.05. NC, normocholesterolemic …

Rats are particularly resistant to the development of hypercholesterolemia and atherosclerosis [15] and have a strong ability to maintain their plasma cholesterol levels [16, 17]. Therefore, to induce hypercholesterolemia or atherosclerosis in rats, cholesterol feeding is used with other additives, including bile acids and propylthiouracil (an anti-thyroid drug), which increase intestinal absorption of cholesterol [18]. However, in the present study, the addition of 1% cholesterol to the basal diet without bile acids and/or anti-thyroid drugs produced hypercholesterolemia in the rats, because cholesterol feeding itself increases bile acid secretion by approximately three to four-fold in rats [19]. The 30.18% increase in plasma cholesterol in the HC rats in the present study was comparable with that reported by Bobek et al. [20], who feed rats cholesterol (0.3%) diet with added bile acids (0.5%) and showed a 1.7-fold higher cholesterolemia in their cholesterol-feed rats than normal rats. In this experiment, feeding 5% P. ostreatus to HC rats significantly repressed the increase in plasma cholesterol. The mechanism by which mushrooms reduce plasma lipoprotein levels in HC rats is not clearly understood. Mushrooms contain the hypocholesterolemic agent mevnolin (monacolin K, lovastatin) [21], which may be involved in decreasing the activity of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase enzyme [20], the rate-limiting enzyme of cholesterol biosynthesis. Thus, feeding mushrooms may involve suppression of endogenous cholesterol biosynthesis by inhibiting HMG-CoA reductase activity.

Effects of feeding oyster mushroom on plasma biochemical and electrolyte function

The results of the plasma biochemical and electrolytes concentrations indicated that albumin, uric acid, glucose, total protein, potassium, inorganic phosphate, and magnesium decreased significantly in HC rats by 20.59, 70.83, 27.92, 19.18, 37.33, 40.52, and 36.11%, respectively, compared with levels in oyster mushroom-fed rats. In contrast, no significant difference was found for plasma total bilirubin, direct bilirubin, creatinin, blood urea nitrogen, calcium, sodium, and chloride levels among the normocholesterolemic, hypercholesterolemic, and oyster mushroom-fed HC rats (Table 4). The glucose-lowering effect of propionate is associated with gluconeogenesis and the regulation of serum lipid levels [22]. Reduction in plasma potassium, sodium, and chloride concentrations is one of the mechanisms of action of antihypertensive drugs, particularly diuretics [23]. Diuretics act by diminishing sodium chloride reabsorption at different sites in the nephrons, thereby increasing urinary sodium chloride and water losses and consequently leading to decreased plasma levels of these electrolytes. Antonov et al. [24] reported that plasma electrolyte contents increased significantly in hypertensive rats. Impaired function of Na, K-ATPase and the Na-H antiport, which is typical of arterial hypertension, may promote an increase in plasma electrolytes.

Effects of feeding oyster mushroom on plasma enzyme profile

Lower plasma GOT, GPT, and ALP concentrations were observed in oyster mushroom-fed HC rats than normocholesterolemic rats (Table 5). No significant difference was observed in the activities of plasma ALP in the NC, HC, or HC + PO rats groups. Plasma GOT and GPT activities were significantly higher in HC rats than in NC rats, whereas 5% HC + PO rats revealed significantly decreased plasma GOT and GPT activities by 13.60 and 11.28%, respectively.

Due to the increasing frequency of antihyperlipidemic drug use and their common side effects, there is a need to identify natural products with few or no side effects. Thus, development continues for highly effective natural ingredients from food, such as mushrooms, which decrease hyperlipidemia [3, 20]. Previous studies have shown that GOT and GPT are typically elevated following cellular damage as a result of enzyme leakage from the cells into blood [25]. Therefore, the increased enzyme activities resulting from oyster mushroom treatment may prevent oxidative damage by detoxifying reactive oxygen species; thus, reducing hyperlipidemia.

Effects of feeding oyster mushroom on fecal TL and cholesterol

The fecal TL and cholesterol of the 5% P. ostreatus-fed HC rats significantly increased by 2.7 and 3.2-fold, respectively, compared with NC rats (Table 6). Thus, the decreased plasma cholesterol may have attributed to such a mechanism. The higher level of plasma HDL-C indicates that more cholesterol from peripheral tissues was returning to the liver for catabolism and subsequent excretion. Plasma VLDL-C and TG contents in HC + PO rats were lower compared with hypercholesterolemic control rats. VLDL-C is the major transport vehicle for TG from the liver to extrahepatic tissues, whereas LDL-C is not secreted as such in the liver but seems to be formed from VLDL-C after partial removal of TG by lipoprotein lipase [26]. LDL-C became the prime carrier for cholesterol after feeding cholesterol to the rats, leading to decreased VLDL-C and HDL-C content in HC + PO rats.

Effects of feeding oyster mushroom on the plasma lipoprotein fraction by agarose gel electrophoresis

The α-lipoprotein band was the fast-moving fraction and was located nearest the anode. The β-lipoprotein band was usually the most prominent fraction and was near the origin, migrating only slightly anodic to the point of application. The pre-β lipoprotein band migrated between the α and β-lipoprotein (Fig. 2). The effects of feeding P. ostreatus on the plasma lipoprotein fraction are presented inFig. 3. The results indicated no significant difference in the lipoprotein fractions between NC and HC + PO rats and HC rats. The results revealed that feeding 5% oyster mushrooms significantly reduced plasma β-lipoprotein and pre-β-lipoprotein but increased α-lipoprotein.

Separation of plasma lipoproteins by agarose gel electrophoresis. Lanes 1~5 represent the plasma lipoprotein fraction of five different rats from each group. NC, normocholesterolemic control rats; HC, hypercholesterolemic rats; HC + PO, Pleurotus ostreatus …

The hypocholesterolemic effect of oyster mushrooms is mediated by the interplay of a complex mixture of substances [27]. Water-soluble gel-forming components of the fiber substance (β-1,3-D-glucan with a low degree of polymerization, forming 15~20% of dry matter) interacts with bile acids and affects micelle formation. Such substances might be interfering with the absorption of cholesterol in this manner.

Effects of feeding oyster mushroom on rat liver histopathology

The effect of P. ostreatus on hepatocyte cells of HC rats is presented in Fig. 4. Liver tissues were stained with hematoxylin-eosin and oil red O. The hepatic cords were typically arranged and located in liver tissue near the central vein in the NC, HC, and HC + PO groups. Lipid droplets were observed only in the liver tissue of HC rats. This could be attributed to lipid accumulation in the hepatocyte cell cytoplasm. Oxidized LDL induces the expression of scavenger receptors on the macrophage surface. These scavenger receptors promote the accumulation of modified lipoproteins, forming an early atheroma. The histological results indicated that the liver tissues of 5% HC + PO rats were almost similar to NC rats and that the hepatic biosynthesis of cholesterol was suppressed, which might be due to a reduction in the activity of HMG-CoA [28]. Hyperlipidemia is the leading risk factor for atherosclerosis, but the atherosclerotic pathological process could be slowed or reversed by reducing serum LDL, TGs, and PLs and increasing serum HDL. Several studies have demonstrated a protective effect of HDL in atherosclerosis and cardiovascular disease, whereas high levels of LDL constitute a risk factor. Excess LDL in the blood is deposited on the blood vessel walls and becomes a major component of atherosclerotic plaque lesions, whereas HDL facilitates translocation of cholesterol from peripheral tissues, such as arterial walls, to the liver for catabolism [29]. Bobek and Galbavý [7] observed that oyster mushrooms prevented the formation of atheromatous plaques and reduced the incidence and extent of atherosclerotic lesions in the aorta and coronary arteries as well as focal fibrosis in the myocardium of rabbits.

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This research was supported by a grant from the University of Incheon in 2009.