Reduction of the Oxidative Injury to the Rabbits with Established Atherosclerosis by Protein Bound Polysaccharide from Coriolus Vesicolor

American Journal of Chinese Medicine, Spring, 2000 by Mei Zhou, Yuan Chen, Qian Ouyang, Shang-Xi Liu, Zhan-Jun Pang

Abstract: Recent evidence has emerged that macrophage glutathione (GSH) content and selenium dependent glutathione peroxidase (SeGSHPx) activity are inversely related to cell-mediated oxidation of LDL, and intervention means to enhance the macrophage GSH-SeGSHPx status may contribute to attenuation of the atherosclerotic process. Our previous works showed that protein bound polysaccharide (PSK) injected intraperitoneally could enhance SeGSHPx activity and mRNA content of mouse macrophages. The aim of the present study is to demonstrate whether PSK can reduce the oxidative injury to the established atherosclerotic rabbits. Using the established atherosclerotic rabbit model, we studied the effect of PSK on oxidatively modified LDL (Ox-LDL), lipoperoxide (LPO) cholesterol contents and SeGSHPx activities of plasma and tissues (aorta, heart and liver) in the established atherosclerotic rabbits. As compared with the control group, Ox-LDL, LPO and cholesterol contents were much lower; SeGSHPx activities and SeGSHPx/LPO ratios were much higher in plasma and tissues (aorta, heart and liver); and the lesion area of aortae was reduced in the PSK group. Through the increment of SeGSHPx activity in macrophages and aortae, PSK enhances their antioxidation potentiality and improves the antioxidant/prooxidant imbalance in them, and thus decreases Ox-LDL, LPO and cholesterol contents of plasma and tissues, and regresses lesion area of aortae in the established atherosclerotic rabbits.

An imbalance between antioxidant and oxidant-generating systems leading to an oxidative stress has already been proposed in the pathogenesis of atherosclerosis (Simon et al., 1998). Many studies have indicated that there is a close relationship between the development of atherosclerosis and oxidative modification of low density lipoprotein (Steinberg et al., 1989; Steinberg et al., 1990; Witztum et al., 1991; Ross, 1993; Steinberg, 1997; Steinberg, 1997). Antioxidants have been proposed to have antioatherogenic potential by their inhibition of low density lipoprotein (LDL) oxidation. Thus inhibition of LDL oxidation has been suggested as an important means of preventing and reducing atherosclerotic lesions. Evidence has increased to show that prevention of LDL oxidation by antioxidants could inhibit and alleviate atherogenesis in the experimental animals and clinical investigation (Frei, 1995; Rice-Evan et al., 1993; Meyers et al., 1993; William et al., 1992; Hertog et al., 1993; Bjorkhem et al., 1991; Steinberg et al., 1988; Kita et al., 1998; Nagano et al., 1992; Sparrow et al., 1992). Steinberg (1997) summarized the antioxidants, probucol, Vitamin E (VE), DPPD (N,N'-dephenyl-1,4-phenylenediamine) and BHT (butylated hydroxytoluene) in animal models of athorosclerosis: of 23 published studies 16 have been strongly positive, 2 have been borderline positive, and 5 have been negative. It seems fair to say that LDL oxidative modification hypothesis is strongly supported by data in experimental animal models. The result of the Cambridge Heart Antioxidant Study with adequate dose of VE for effects on coronary events also supports the hypothesis. VE can significantly decrease nonfatal myocardial infarctions (77%), and the sum of nonfatal myocardial and cardiovascular deaths (47%; p=0.005) (Slephens et al., 1996).

Based on macrophage-mediated oxidation of LDL playing a key role during early atherogenesis, Rosenblat et al. recently examined the effect of the macrophage GSH and SeGSHPx (GSH-SeGSHPx) status on the ability of the cells to oxidize LDL and found that the macrophage GSH content and SeGSHPx activity are inversely related to cell-mediated oxidation of LDL and that intervention means to enhance the macrophage GSH-SeGSHPx status may contribute to attenuation of the atherosclerotic process (Aviram et al., 1997; Rosenblat et al., 1998). In our previous studies we found that a protein bound polysaccharide (PSK) injected intraperitoneally could enhance SeGSHPx activity and mRNA content of mouse macrophages (Li et al., 1993; Chen et al., 1994; Zhou et al., 1996; Chen et al., 1996). The aim of this study is to further determine whether it has an important role in reducing the oxidative injury to rabbits with established atherosclerosis.

Materials and Methods

Preparation of PSK and Reagents

PSK was extracted from the sporophores of Coriolus vesicolor in our laboratory according to the method of Xi et al. (1993). The highest molecular weight was about 1,200 kD measured by high performance chromatography analysis; the percentage of carbohydrate was about 65.9% as determined by the method of phenol sulfuric acid and using glucose as standard. Endotoxin was not detectable at the concentration used by the Limulus amoebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA, USA), which had a sensitivity level of 50 pg/ml. Monoclonal antibody (mAb) against Ox-LDL (HOL5) was prepared in our laboratory (Mei et al., 1997).

DTNB [5, 5'-dithiobis (2-Nitrobenzoic acid)], 1,1,3,3-tetramethoxypropane, reduced glutathione, cholesterol and RPMI-1640 were purchased from the Sigma Chemical Company (St. Louis, MO, USA). Pentobarbital sodium was purchased from Shanghai Chemical Company (Shanghai, China). All other chemicals were of analytical grade.