摘要
目的探讨制首乌有效成分大黄素在抗动脉粥样硬化(AS)方面的作用机制,以ApoE-/-小鼠为研究对象,观察大黄素对AS模型中mTOR信号通路的调控作用。方法72只成年雄性ApoE-/-小鼠随机分成6组:模型组、高、中、低剂量组、阳性对照组和大黄素中剂量+雷帕霉素(联合用药)组,每组12只。所有小鼠用高脂饲料饲养9 w,并每隔1 d腹部皮下注射脂多糖(LPS)25μg/只。成模后停止LPS注射,并随机处死1只小鼠,进行主动脉油红O染色,确定成模。从第10周起,基于高脂饮食,高、中、低剂量组分别按照40、20、10 mg/kg灌服大黄素,阳性对照组按200 mg/kg灌服血脂康,联合用药组给予20 mg/kg大黄素及腹部皮下注射雷帕霉素1 mg/kg。所有药物体积为0.5 ml,持续给药6 w后取材。检测血清中总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)的浓度。取小鼠主动脉近心端2 cm进行切片苏木素-伊红(HE)染色,并在显微镜下进行病理观察。用实时荧光定量聚合酶链反应(qRT-PCR)检测心肌组织中磷酸肌醇3激酶(PI3K)、蛋白激酶B(AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)mRNA的表达。Western印迹检测心肌组织中mTOR、p-mTOR、PI3K、AKT蛋白表达。结果给药各组TC、TG、LDL-C水平均明显降低(均P<0.05)。除低剂量组外其余给药组HDL-C水平均明显高于模型组(均P<0.05)。模型组内膜明显增厚,可见大的脂质核心,斑块纤维帽变薄并伴有破裂;高剂量组内膜稍有增厚;中剂量组内膜增厚,稍有破裂,但细胞排列整体有序;低剂量组内膜变薄,有脂质堆积,见玻璃样变形成;阳性对照组内膜增厚,斑块纤维帽较薄;联合用药组内膜稍增厚,未见脂质。与模型组比较,高、中剂量组、阳性对照组和联合用药组mTOR mRNA水平明显降低(P<0.05);高、中、低剂量组和联合用药组PI3K mRNA水平明显降低(P<0.05)。高、中剂量组、联合用药组和阳性对照组AKT mRNA水平明显降低(均P<0.05)。与模型组比较,高、中剂量组、联合用药组PI3K蛋白表达水平明显降低(P<0.05);高、中剂量组AKT蛋白表达水平明显下降(P<0.05)。高、中、低剂量组、阳性对照组和联合用药组mTOR蛋白表达水平明显降低(P<0.05)。高、中、低剂量组和联合用药组p-mTOR蛋白表达水平明显降低(P<0.05)。结论一定剂量制首乌大黄素对ApoE-/-小鼠AS模型病变有显著影响,其机制可能与抑制mTOR信号通路有关。
Objective To investigate the mechanism of emodin,an effective component of Shouwu,in anti-atherosclerosis(AS).Methods ApoE-/-mice were used as the research object to observe the regulation of emodin on mTOR signaling pathway in AS model.Seventy-two adult male ApoE-/-mice were randomly divided into 6 groups:model group,high,middle and low dose of emodin,positive groups and combination group(middle dose of emodin+rapamycin),12 in each group.The normal control group was 12 C57BL/6J mice.Except the normal control group,the other 6 groups were fed with high-fat diet for 9 weeks,and subcutaneously injected with 25μg of lipopolysaccharide every two days.LPS injection was stopped after the model was formed,and one mouse was randomly sacrificed in each group to perform aortic oil red O staining to determine the model.From the 10th week,based on the high-fat diet,40 mg/kg,20 mg/kg,10 mg/kg emodin was administered to high,middle and low dose of emodin group respectively.200 mg/kg Xuezhikang was given in positive group,20 mg/kg emodin was administered and rapamycin 1 mg/kg was injected subcutaneously into the abdomen in combination group.The normal control group was given normal diet and administrated saline.All drug volumes were 0.5 ml and sample was taken after 6 weeks of continuous administration.TC,TG,LDL-C and HDL-C were detected.2 cm of the aorta of the mouse heart′s proximal end for section was taken for HE staining and pathological observation under the microscope.PI3K,AKT and mTOR mRNA in myocardial tissue were detected by real-time fluorescent quantitative PCR.mTOR,p-mTOR,PI3K and AKT protein in myocardial tissue were detected by Western blot.Results Compared with the model group,the levels of TC,TG,and LDL-C all decreased with different degrees(P<0.01),HDL-C significantly increased except for the low dose of emodin group(P<0.01).Observed by HE staining microscopy,high and middle dose of emodin groups and the combination group could effectively improve the occurrence and development of AS and delay the formation of plaques.The effect of low dose of emodin was not obvious.Compared with model group,mTOR,PI3K,and AKT mRNA in cardiac tissue in the normal control group,high,middle and low dose of emodin groups,positive group,and combination group were decreased(P<0.05),expression of mTOR protein was significantly lower in the high,middle dose of emodin and combination groups(P<0.01),expression of p-mTOR protein in the middle dose of emodin group was significantly lower than that of model group(P<0.01),expression of PI3K protein was significantly lower in the high and middle dose of emodin groups(P<0.01),expression of AKT protein in high dose of emodin group was significantly lower(P<0.01).Conclusions Shouwu emodin has a significant effect on the lesions of ApoE-/-mice AS model at a certain dose,and its mechanism might be related to the inhibition of mTOR signaling pathway.
作者
张磊
田维毅
王和生
赖陈岑
李妹娟
夏丽
冷传龙
ZHANG Lei;TIAN Wei-Yi;WANG He-Sheng(Guizhou University of Traditional Chinese Medicine,Guiyang 550002,Guizhou,China)
出处
《中国老年学杂志》
CAS
北大核心
2021年第13期2769-2773,共5页
Chinese Journal of Gerontology
基金
国家自然科学基金项目(81760790,81460670)。
