| Atherosclerosis is a common disorder characterized by the accumulation of macrophages and products of lipid and protein oxidation in blood vessels. Oxidative stress, chronic inflammation and endothelial dysfunction play key roles in the development and progression of atherosclerosis.Procyanidins, a group of flavonoids that are commonly found in red wine, grapes, cocoa, and apples, have a broad range of biological activities such as antioxidant, antigenotoxic, antiatherogenic and antihyperglycemic function.Procyanidins extracted from the litchi pericarp (LPPC) used in the present study is thought to be a new source of procyanidins and have been proven to possess strong antioxidant activities in vitro.In the present study, we sought to determined the effects of oral administration of LPPC on the developemt of atherosclerosis in apolipoprotein E knockout (ApoE KO) mice fed a high fat diet for 24 weeks and then explored the underlying mechanisms. Part One Effects of LPPC on Atherosclerotic Lesion Formation in ApoE KO MiceObjective:To determine the ameliorating effects of LPPC on atherosclerosis in apolipoprotein E knockout (ApoE KO) mice.Methods:A total of 50 eight-week-old 18-22 g male ApoE KO mice were randomly divided into two groups with 25 mice in each group:the ApoE KO group and ApoE KO+ LPPC group.25 wild-type (WT, C57BL/6J) mice were considered to be the control group.All ApoE KO mice were fed on a high fat diet (HFD, based on Harlan Teklad diet TD 88137 comprised of 21% fat by weight [42% of calories] and 0.15% cholesterol by weight) for 24 weeks. WT mice were fed a chow diet. Mice in LPPC group were orally administrated with LPPC (100 mg/kg body mass daily) dissolved in distilled water freshly prepared every day throughout the whole experiment. Food consumption and body weight changes were recorded weekly. At the end of the experiment, twelve mice in each group were perfused with PBS via the left ventricle after pentobarbital sacrifice. The aortas (from the bifurcation off the aortic arch to the branching point of the right subclavian and common carotid artery) of four mice in each group were opened longitudinally to expose the endothelial surface for enface Oil Red O staining. The aortic arch of the other four mice in each group were removed and snap-frozen in Tissue-Tek OCT compund for 10μm thickness serial sections, and then stained with Oil red O for atherosclerotic lesion quantification. Aortic arch of another four mice in each group were stored in 10% buffered formalin overnight and embedded in paraffin for the immunohistochemical detection.Results:The ApoE KO mice resulted in a significant increase in mean lesion size compared with the control WT mice. Histomorphometric analysis revealed that LPPC significantly decreased atherosclerotic lesion size in the ApoE KO+LPPC group than in the ApoE KO group (ApoE KO:56.9%±8.1%, ApoE KO+LPPC:32.6%±9.2%, P< 0.01). The atherosclerotic lesion formation in the transverse section of aortic arch stained with Oil red O showed that LPPC intervention also significantly decreased atherosclerotic lesion formation.Conclusion:The present study demonstrated that the supplementation of LPPC to ApoE KO mice ameliorated atherosclerotic plaque development. Part Two Mechanism studySection 1 Effects of LPPC on Lipid disorder in ApoE KO MiceObjective:To investigate whether LPPC could provide protection against atherosclerosis through regulating lipid metabolism in ApoE KO MiceMethod:At the end of experiment, blood samples and liver tissues were collected. Plasma total cholesterol (TC), triglyceride (TG) levels, low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), hepatic TC and TG contents were determined. The mRNA expressions of key genes involved in lipid metabolism were quantified by real-time RT-PCRResults:Compared with WT mice in the control group, ApoE KO mice showed a marked increase in TC (P< 0.01) and LDL-C levels (P< 0.05), while after LPPC administration, the TC and LDL-C contents in plasma were significantly decreased (P< 0.05). Although plasma TG level was diminished a little, the difference had no statistical meaning (P>0.05). LPPC also decrease the plasma TG level significantly; For HDL-C levels, there was no significant difference between ApoE KO and ApoE KO+LPPC group. LPPC also decreased hepatic TC and TG contents significantly. Moreover, LPPC could significantly increase mRNA expressions of farnesoid X receptor (FXR) and small heterodimer partner (SHP) which emerge as key regulators of lipid homeostasis at the transcriptional level, decrease mRNA expressions of 3-hydroxy-3-Methylglutaryl (HMG)-CoA reductase which mediates cholestrol biosynthesis, and increase mRNA expressions of ATP-binding cassette transporter-1 (ABCA1) which modulates cholesterol efflux in the liver of ApoE KO mice.Conclusion:LPPC can effectively regulate key gene expressions involved in lipid metabolism system and thus alleviate the lipid disorder especially hypercholesteromia in ApoE KO mice. These findings indicated that LPPC ameliorated atherosclerosis via regulating gene expression involved in hepatic lipid homeostasis in ApoE KO mice fed a HFD. Section 2 Effect of LPPC on NO, iNOS and eNOS in ApoE KO MiceObjective:To investigate whether LPPC could provide protection against atherosclerosis through regulating the nitric oxide system in ApoE KO MiceMethods:Plasma NO levels and iNOS activity were determined using appropriate kits according to the manufacturer's instructions. Aortic mRNA expression of iNOS and eNOS were quantified by real-time RT-PCR. Aortic protein expression of iNOS and eNOS were quantified by western blot test. The concentrations of ET-1 and vascular cell adhesion molecule-1 (VCAM) in plasma were measured using ELISA assays.Results:The plasma NO contents in ApoE KO mice were markedly higher than that in control WT mice. LPPC intervention significantly lowered the NO levels (P< 0.01 versus ApoE KO group). Interestingly, plasma concentrations of ET-1 were significantly lower in the HFD ApoE mice compared to the chow WT mice while LPPC intervention reversed this decrease significantly. LPPC also significantly reduce plasma iNOS activities when compared with ApoE KO group (P< 0.01). PCR and Western blotting showed that control WT mice had minimal iNOS mRNA expression and iNOS protein contents in aorta, while ApoE KO mice showed significantly higher iNOS mRNA expression and iNOS protein contents than WT mice. However, LPPC supplementation exhibited significantly lower iNOS mRNA expression and protein contents in aortas than ApoE KO mice. Aortic sections from ApoE KO mice showed greater staining for iNOS than those of WT mice, whereas in ApoE KO mice treated with LPPC, iNOS protein was significantly decreased. Conversely, the protein expression of eNOS was lower in aortic sections of ApoE KO than in control WT mice, however in ApoE KO mice treated with LPPC the expression was increased. Moreover, the mRNA and protein expression of eNOS in aortas of ApoE KO mice were increased by LPPC which further explain the endothelial protective actions of LPPC.Conclusion:These results elucidated that that the excess NO production found in ApoE KO mice may be due to the elevated iNOS activity as well as gene and protein expression, and the antiatherogenetic effect of LPPC may be at least partly through the inhibition of iNOS. Section 3 Effect of LPPC on Oxidative Stress and Antioxidant Systems in ApoE KO MiceObjective:To determine the effect of LPPC on oxidative stress and antioxidant systems in ApoE KO mice.Methods:The GSH levels, MDA contents and the activities of SOD,GPx and CAT in plasma were measured according to the appropriate methods. The mRNA expression of NADPH oxidases ubtypes (p22phox, p47phox, p67phox, NOX-1, NOX-2/gp91phox and NOX-4) in aortas of four mice in each group were detected by RT PCR.Results:LPPC intervention significantly increases the plasma GSH level when compared with ApoE KO group (P<0.01). MDA contents in plasma were significantly elevated in ApoE KO mice than control WT mice. LPPC intervention brought down the MDA levels significantly. There was also quite a deal of difference in the alterations of activities of SOD between control and ApoE KO mice group. Comparing to control, ApoE KO mice had significantly lower SOD enzyme activities in plasma, however, LPPC intervention resulted in no significant change in the activities of SOD. LPPC intervention significantly increases the plasma CAT activities when compared with ApoE KO group (P < 0.05), and also increases the CAT/SOD ratio significantly (P<0.01).The mRNA expression (expressed as percent of control) for NAD(P)H oxidase (p47phox, p67phox, NOX-2/gp91phox and NOX-4) from aortas were higher in ApoE KO mice than those in control mice, however LPPC significantly decreased the expression of NAD(P)H oxidase in ApoE KO miceConclusion:In this study, LPPC showed profoundly antioxidative effects in ApoE KO mice, and the antiatherogenetic effect of LPPC may be partly due to the inhibition of NADPH oxidase-derived oxidative stress.
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