白鲜皮水提物对ApoE-/-小鼠动脉粥样硬化早期病变形成的抑制作用

Cortex Dictamni Inhibits Formation of Early Atherosclerotic Lesions in ApoE-Deficient Mice

目的研究白鲜皮水提物对载脂蛋白E基因缺损小鼠主动脉弓粥样硬化早期病变形成的影响。方法将40只ApoE-/-小鼠随机分成空白对照组和白鲜皮高、中、低三剂量组(白鲜皮水提物3.2、1.6、0.8 g/kg)给药,计算各组主动脉弓粥样硬化病变的面积。体内检测血清中的脂质含量以及抗氧化指标丙二醛(MDA)含量、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽-S转移酶(GST)的活性变化。测定白鲜皮水提物对Cu2+介导的人血清低密度脂蛋白(LDL-C)氧化易感性的影响。结果与对照组相比,白鲜皮各给药组小鼠动脉粥样硬化早期病变面积均小于对照组,血清中MDA的含量降低,SOD、CAT活性增高,GST变化不明显,血清脂质含量组间没有显著差异。白鲜皮水提物明显延长Cu2+介导的人血清LDL氧化滞留时间和达峰时间。结论白鲜皮水提物对ApoE-/-小鼠动脉粥样硬化早期病变形成具有显著的抑制作用,其作用机制可能是通过对抗脂蛋白的过氧化作用而实现。

Objective Cortex Dictamni （CD） is a commonly used Chinese herb medicine in the treatment of skin diseases. In a previous study we had shown that CD has an anti-inflammatory effect and regulates immune function. The aim of this study was to determine whether CD has any effect on the early formation of aortic atherosclerotic lesions in ApoE-/- mice and explore the possible mechanisms involved. Methods 40 female apoE-/- mice （ weighing 15-16 g,6 weeks of age ） were kept in a temperature-controlled facility with a 12-h light /12-h dark photoperiod,and free access to food and water. The mice were randomly divided into four groups： the control group （n = 10）,high dose CD group （ n = 10）,medium dose CD group （n = 10） and low dose CD group （n = 10）. All animals were fed a high fat diet and the three CD groups were orally administered with CD at different doses （3. 2 mg /g,1. 6 mg /g,0. 8 mg /g） for 6 weeks while the control group got the same volume of distilled water. The fresh blood of all overnight fasted animals were obtained from the eye vein at 0 week and 6 weeks and serum was obtained by low-speed centrifugation. The serum levels of lipids including total cholesterol （ TC ）, triglyceride （ TG ）, high-density lipoprotein cholesterol （ HDL-C ）, low-density lipoprotein cholesterol （LDL-C） were determined. Serum malondialdehyde （MDA） content and the activities of superoxide dismutase （SOD）,catalase （CAT） and glutathione S-transferase （GST） were also tested. At the end of the experiment,the heart and proximal aorta were removed from the mice. The hearts were then cut directly under and parallel to the aortic cusps and the upper portions were embedded in OCT compound. The samples were cut into a total of 80 sections （6 μm） and then stained with oil red-O every fifth section （16 samples from each mouse）. The size of the atherosclerotic lesion in each aortic section was evaluated on the basis of oil red-O-staining using Image-Pro Plus 6. 0 software. In vitro,the human low- density lipoproteins （density 1. 063 g /mL） was isolated from the human serum by density gradient ultracentrifugation. The influence of CD at different concentrations （0 mg,1. 6 mg,2. 4 mg,2. 8 mg and 3. 2 mg） on the susceptibility of human LDL oxidation induced by Cu2 ＋ were examined. Lag time and Tmax were used as indices of LDL oxidation susceptibility. Result In vivo experiment： （1） There was no significant difference in the intake of food and water between the three CD groups and the control group. （2） At 0 week and 6 weeks of the experiment,the average weights of the four groups were not significantly different （P 0. 05）. （3） Although the serum levels of TC,TG,HDL-C,LDL-C in the three CD groups were decreased in comparison with that in the control group,there was statistically no significant difference （ P 0. 05）. （4） The high fat diet for 6 weeks caused early formation of aortic atherosclerotic lesions in ApoE-/-mice and the control mice showed advanced atherosclerotic lesions in the aortic root. Comparing with the control group, the atherosclerotic lesions of all the CD groups were significantly reduced. Quantitative analysis showed that atherosclerotic lesions of the control group,the high,medium and low dose CD groups showed significant differences （P 0. 01） between the three CD groups and the control group. The inhibitory effect had a positive correlation with the dose of CD. In vitro experiment： （1） At the beginning and end of the experiment,the serum lipid peroxidation contents and antioxidant enzyme activities in all mice were determined. The data indicated that the MDA content in the control group was highly increased. SOD,CAT and GST activities were all reduced because of the high fat diet. In the high,medium and low dose CD groups, the MDA contents were significantly decreased comparing with that in the control group （ P 0. 05） and the SOD,CAT activities in the high and medium dose groups were significantly increased compared with that in the control group （ P 0. 05）. But the GST activity had no significant differences among the four groups （P 0. 05）. （2） The lag time and Tmax of Cu2 ＋ induced LDL oxidation in the CD-treated mice were prolonged. The LDL oxidation susceptibility was reduced after the CD administeration and this inhibition of oxidation was positively correlated with the concentration of CD. Conclusion CD can significantly inhibit the formation of early aortic atherosclerotic lesions in ApoE-/-mice. It has no influence on the weight and the serum lipid levels in the apoE-/-mice. The possible mechanism of action may be via the inhibition of lipid peroxidation by increasing the activities of antioxidant enzyme activities.