The Investigation Of Effect And Mechanism Of Chitosan-Oligosaccharides On Atherosclerosis Of Apolipoprotein E Knockout Mice

ObjectiveOur previous studies have shown that the degradation products of chitin, chitosan-oligosaccharides (COS), plays a big role in the regulation of plasma lipids and thepromotion of reverse cholesterol transport (RCT). However, the effects of COS onatherosclerosis have not been reported. In this study, we use spontaneous atherogenesismouse, the apolipoprotein E knockout (apoE-/-) mouse fed with high-cholesterol/high-fatdiet, to investigate the effects of COS on the atherosclerosis lesions in vivo. To furtherinvestigate the possible mechanism, we determined the cholesterol metabolism-related geneexpression of HepG2cell lines and RAW264.7cell lines with the treatment of variousconcentrations of COS.Methods1. Thirty-five male12-week-old apoE-/-mice were randomly divided into three groups:(1) the model group (Model group, n=11) were given sterile distilled water0.01L/kg/d;(2)the low-dose COS group (COS-L group, n=12), were gastric gavages administration of COS250mg/kg/d;(3) the high-dose COS group (COS-H group, n=12),were gastric gavagesadministration of COS1000mg/kg/d. The mice accessed diet and water ad libitum and werekept in a temperature and humidity controlled room with a12/12hours light-dark cycle. Allgroups were fed a high-fat high-cholesterol diet (HCHF diet)(15.8%fat and1.25%cholesterol) supplemented with vehicle or COS for12weeks.2. At the end of12weeks COS or vehicle administration, fasting blood was harvestedand plasma was isolated for the measurement of total cholesterol (TC), triglyceride (TG),and high density lipoprotein cholesterol (HDL-C). Pooled plasma from each group wasloaded on fast protein liquid chromatography (FPLC) to separate lipoproteins. Cholesterol or TG content in plasma and in FPLC fractions was analysis by enzymatic method.3. Plaque morphological histomorphometric characters were analyzed. The dissectedaortas and sections of aorta root of mice were stained with Oil red O to highlight theatherosclerotic lesion. Quantitative analysis of plaque area was performed by usingImage-Pro Plus software6.0. Data were evaluated by2observers in a double blinded,randomized fashion.4. Sequential sections were stained with macrophage-specific antibody and mousematrix metalloproteinases-9(MMP-9) specific antibody. Staining areas were quantified.5. The content of serum Tumor necrosis factor α (TNF-α) and interleukin-6(IL-6) levelwere determined by enzyme-linked immunosorbent assay (ELISA) kit.6. Cholesterol metabolism related genes (ATP-binding cassette transporter A1and G1,ABCA1and G1, low-density lipoprotein receptor, LDL-R, and the scavenger receptor BI,SR-BI) in liver and peritoneal macrophages from each group were determined by real-timequantitative PCR and Western Blot.7. To further understand the effects of COS on cholesterol metabolism related genes,HepG2and RAW264.7cell lines were treated with various concentrations of COS. Cellswere harvested and total RNA was extracted for the evaluation of cholesterol metabolismrelated genes expression by real-time quantitative PCR.Results1. COS treatment decreased plasma TC: compared with Model group, COS treatmentreduced plasma TC dose-dependently (11%,P＜0.05,and23%,P＜0.01,in COS-L andCOS-H respectively). COS treatment also decreased plasma triglyceride (TG): comparedwith model group, COS treatments reduced plasma TG significantly (29%,P＜0.05,and31%,P＜0.01,in COS-L and COS-H respectively). HDL-C level was not changed in eachgroup(P＞0.05). FPLC analysis showed that COS treatment significantly decreasedcholesterol and triglyceride in non-HDL fractions in a dose dependent manner, whereas noobvious change was observed in HDL in COS-H group. WB results showed that plasmaapolipoprotein B (apoB)100decreased in COS-H by56%(P＜0.05).2. COS treatment attenuated the atherosclerotic lesions in apoE-/-mice: compared withModel group, the atherosclerotic plaque area of en face aorta in COS-L group and theCOS-H group were reduced by27.6%(P＜0.05) and33.3%(P＜0.01), respectively.Compared with Model group, the atherosclerotic plaque area of aortic root was reducedsignificantly (30.4%,P＜0.05, and54.3%,P＜0.01, in COS-L group and the COS-H group respectively).3. COS treatment reduced the content of macrophages in aortic root plaque: comparedwith Model group, macrophage content in aortic root plaque were decreased by32%(P＜0.01)to55%(P＜0.01) in COS-L group and COS-H-group, respectively.The MMP-9contenton the plaque site showed no significant difference between Model group and COSgroups(P＞0.05).4. COS treatment did not affect plasma IL-6and TNF-α in apoE-/-mice(P＞0.05).5. COS treatment promoted the expression of hepatic LDL-R, and SR-BI significantlyin apoE-/-mice; COS treatment enhanced ABCA1expression significantly in peripheralmacrophages of apoE-/-mice.6. COS treatment increased the cholesterol metabolism-related genes mRNA (ABCA1,SR-BI, LDL-R) expression in HepG2and RAW264.7cells.Conclusion1. COS attenuate atherosclerosis lesions in apoE-/-mice fed HCHF diet;2. The possible mechanism of anti-atherosclerotic effect of COS treatment might berelative to:(1) COS treatment lowered cholesterol and TG contents in Non-HDL in apoE-/-mice;(2) COS treatment decreased macrophage contents in AS plaque;(3) COS treatment enhanced hepatic LDL-R and SR-BI expression in vivo and in vitro;(3) COS treatment enhanced macrophage ABCA1expression in vivo and in vitro.