图1 各组小鼠主动脉斑块染色图
Published:30 September 2024,
Received:19 March 2024,
Revised:18 August 2024
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To investigate the effects of Yiqi tongmai formula on atherosclerosis (AS) in ApoE-/- mice and its mechanism.
Forty ApoE-/- mice were randomly divided into model group, positive control group [atorvastatin calcium, 2.6 mg/(kg·d)], and low-dose, medium-dose and high-dose groups of Yiqi tongmai formula [0.46, 0.91, 1.82 g/(kg·d), by raw material], with 8 mice in each group. Eight C57BL/6J mice were selected as the normal group. Except for the normal group, the other groups were given a high-lipid diet and relevant drug or normal saline intragastrically, once a day, for 12 consecutive weeks. After the last medication, the serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) as well as the contents of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were measured in mice. The proportion of aortic plaque area in each group of mice was detected and calculated, and the pathological morphological changes of the aortic sinus were observed; the protein phosphorylation levels of aortic phosphoinositide 3-kinase (PI3K), protein kinase B (aka Akt) and mammalian target of rapamycin (mTOR) were examined.
Compared with the model group, the serum levels of TC, TG and LDL-C and the contents of TNF-α, IL-1β and MCP-1 (including low-dose group) were decreased significantly in medium-dose and high-dose groups of Yiqi tongmai formula, while the content of HDL-C in high-dose group was increased significantly (P<0.05 or P<0.01); aortic plaques of the mice were reduced in Yiqi tongmai formula groups to different extents, and pathological changes such as lipid deposition and inflammatory cell infiltration were relieved to different extents; the proportion of aortic plaque area, the protein phosphorylation levels of PI3K, Akt and mTOR in aortic tissue were significantly reduced in medium-dose and high-dose groups of Yiqi tongmai formula (P<0.05 or P<0.01).
Yiqi tongmai formula can improve lipid metabolism, reduce inflammatory response, and delay plaque development in AS mice. Its effect may be related to the inhibition of PI3K/Akt/mTOR signaling pathway activation.
atherosclerosis;
inflammation response;
PI3K/Akt/mTOR signaling pathway
动脉粥样硬化(atherosclerosis,AS)是一种慢性炎症性、代谢性疾病,以脂质沉积、平滑肌细胞增殖、血管内膜增厚和斑块形成为主要特征[
益气通脉方由红参、三七、酒丹参、土鳖虫、水蛭、酒大黄6味中药组成,具有健脑益气、化瘀通脉、改善循环、消除斑块及血栓之功。方中,君药红参可补益元气、祛瘀通络;臣药三七、酒丹参意在活血祛瘀、行气止痛;臣药水蛭、土鳖虫通经活络,专于行药力;佐药酒大黄泻下祛浊、引药下行,具去瘀血、生新血之功[
磷脂酰肌醇3激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,又称“Akt”)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路是经典的细胞内信号转导通路,可参与细胞增殖、血管生成和炎症反应等多个生物学过程[
本研究所用主要仪器包括170-4070型凝胶电泳仪、Mini TransBlot型转膜仪、Chemi Doc XRS+型凝胶扫描成像系统(美国Bio-Rad公司),SMZ745T型体视显微镜(日本Nikon公司),Multiskan FC型酶标仪[赛默飞世尔(上海)仪器有限公司]等。
红参、三七、酒丹参、酒大黄饮片(批号分别为2108171、2111079、21121303、2107122)均购自安徽普仁中药饮片有限公司;水蛭、土鳖虫饮片(批号分别为22010301、21110201)均购自亳州市先锋中药饮片有限公司。所有饮片经河南省中医院/河南中医药大学第二附属医院药学部黄晓敏副主任药师鉴定均为真品。
阿托伐他汀钙片(批号8148435,规格20 mg)购自晖致制药(大连)有限公司;饱和油红O染液、苏木精-伊红(HE)染色试剂盒(批号分别为CR2305087、2309004)均购自北京索莱宝科技有限公司;低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C)、总胆固醇(total cholesterol,TC)检测试剂盒(批号均为20230916)和甘油三酯(triglyceride,TG)检测试剂盒(批号20230918)均购自南京建成生物工程研究所;肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)、单核细胞趋化蛋白1(monocyte chemoattractant protein-1,MCP-1)酶联免疫吸附测定(ELISA)试剂盒(批号分别为ZD0226041375、DP07P0F89596、FU02F6487219)均购自武汉伊莱瑞特生物科技股份有限公司;兔源PI3K抗体和鼠源Akt、mTOR、磷酸化mTOR(phosphorylated mTOR,p-mTOR)、β-肌动蛋白(β-actin)抗体(货号分别为20584-1-AP、60203-2-Ig、66888-1-Ig、67778-1-Ig、66009-1-Ig)及辣根过氧化物酶标记的山羊抗兔、山羊抗鼠IgG二抗(货号分别为SA00001-1、SA00001-2)均购自武汉三鹰生物技术有限公司;兔源磷酸化PI3K(phosphorylated PI3K, p-PI3K)、磷酸化Akt(phosphorylated Akt,p-Akt)抗体(货号分别为YP0765、YP0006)均购自苏州睿瀛生物技术有限公司。
SPF级雄性ApoE-/-小鼠40只和雄性C57BL/6J小鼠8只,体重18~22 g,均购自斯贝福(北京)生物技术有限公司,动物生产许可证号为SCXK(京)2019-0010。所有小鼠均分笼饲养于河南省中医院/河南中医药大学第二附属医院中心实验室SPF级动物房(温度23~25 ℃,相对湿度50%~70%,每12 h昼夜循环),自由摄食、饮水。本实验方案经该院伦理委员会批准(伦理批准文号PZ-HNSZYY-2022-044)。普通饲料与高脂饲料(含83.75%基础饲料+15%猪油+1.25%胆固醇)均购自斯贝福(北京)生物技术有限公司。
益气通脉方由红参、三七、酒丹参、酒大黄、水蛭、土鳖虫饮片按质量比1∶1∶3∶2∶1∶0.6配伍。酒丹参、酒大黄用80%乙醇水浴回流提取3次,每次1 h,过滤浓缩得干浸膏;红参、三七、土鳖虫和水蛭打成细粉,混合均匀。灌胃前,将上述干浸膏及细粉混于一定量生理盐水中,制得混悬液,备用。阿托伐他汀钙片用生理盐水配制成0.26 mg/mL的混悬液,备用。
参考文献方法[
末次给药后,每组取6只小鼠禁食、不禁水12 h,腹腔注射1%戊巴比妥钠50 mg/kg麻醉,摘眼球取血。血样于室温下静置2 h后离心,取上层血清,于-80 ℃下保存,备用。取血后,迅速解剖小鼠,随机取各组3只小鼠的心脏及主动脉,浸泡于4%多聚甲醛中固定24 h;取各组剩余3只小鼠的主动脉,迅速放入液氮中,于-80 ℃下保存,备用。
取“2.3”项下各组小鼠冻存的血清样品,室温下解冻后,按照相应试剂盒说明书方法操作,以酶标仪检测其血清中TC、TG、LDL-C、HDL-C水平。
取“2.3”项下各组小鼠冻存的血清样品,室温下解冻后,按照相应ELISA试剂盒说明书方法操作,以酶标仪检测其血清中TNF-α、IL-1β、MCP-1含量。
取“2.3”项下各组小鼠经固定的主动脉,用磷酸盐缓冲液清洗3次,剥离多余的结缔组织后沿纵轴剖开,放入45 ℃预热的油红O染液(饱和油红O染液与水的体积比为3∶2)中孵育1 h,再用75%乙醇分化5 min×3次,用水润洗后置于黑色橡胶垫上,以体视显微镜观察、拍照。利用Image J软件分析斑块,并按下式计算斑块面积占比:斑块面积占比=斑块面积/主动脉总面积×100%。
取“2.3”项下各组小鼠经固定的心脏,经常规石蜡包埋后制作主动脉窦切片(厚约3 μm),经HE染色后脱水、封片,使用显微镜观察其主动脉窦病理形态学变化情况。
取“2.3”项下各组小鼠冻存的主动脉,提取蛋白后以BCA法测定蛋白浓度,并作变性处理。取变性蛋白适量,进行电泳分离并转膜,用5%脱脂奶粉封闭2 h;加入PI3K、p-PI3K、Akt、p-Akt、mTOR、p-mTOR、β-actin一抗(稀释比例分别为1.5∶1 000、1∶1 000、1∶1 000、1∶1 000、0.4∶1 000、2∶1 000、0.2∶1 000),于4 ℃下孵育过夜;加入相应二抗(稀释比例均为1∶5 000),于37 ℃下孵育1 h;采用ECL Plus超敏发光液曝光后,于凝胶扫描成像系统下成像。使用Image Lab 5.2软件分析蛋白的条带灰度值,以目的蛋白与内参蛋白(β-actin)的灰度值比值表示目的蛋白的表达量,再以p-PI3K与PI3K、p-Akt与Akt、p-mTOR与mTOR的表达量比值表示PI3K、Akt、mTOR蛋白的磷酸化水平。
采用GraphPad Prism 8软件对数据进行统计分析。数据以x±s表示,多组间比较采用单因素方差分析,进一步两两比较采用Tukey’s多重比较检验。检验水准α=0.05。
与正常组比较,模型组小鼠血清中TC、TG、LDL-C水平均显著升高(P<0.01),HDL-C水平显著降低(P<0.01);与模型组比较,益气通脉方中、高剂量组及阳性对照组小鼠血清中TC、TG、LDL-C水平均显著降低(P<0.05或P<0.01),益气通脉方高剂量组小鼠血清中HDL-C水平显著升高(P<0.05)。结果见
组别 | TC | TG | LDL-C | HDL-C |
---|---|---|---|---|
正常组 | 4.84±1.64 | 1.32±0.43 | 8.46±1.31 | 4.84±0.49 |
模型组 | 34.85±2.61a | 2.65±0.29a | 28.89±2.27a | 2.03±0.45a |
益气通脉方低剂量组 | 32.68±1.65 | 2.18±0.39 | 25.42±2.04 | 2.54±0.47 |
益气通脉方中剂量组 | 27.47±3.67b | 1.78±0.42c | 20.92±4.66c | 2.87±0.51 |
益气通脉方高剂量组 | 26.84±4.97c | 1.80±0.35c | 22.12±4.44c | 2.92±0.49b |
阳性对照组 | 25.86±4.63c | 1.66±0.37c | 22.39±1.77b | 2.76±0.47 |
a:与正常组比较,P<0.01;b:与模型组比较,P<0.05;c:与模型组比较,P<0.01。
与正常组比较,模型组小鼠血清中TNF-α、IL-1β、MCP-1含量均显著升高(P<0.01);与模型组比较,益气通脉方中、高剂量组及阳性对照组小鼠血清中TNF-α、IL-1β、MCP-1含量以及益气通脉方低剂量组小鼠血清中MCP-1含量均显著降低(P<0.05或P<0.01)。结果见
组别 | TNF-α | IL-1β | MCP-1 |
---|---|---|---|
正常组 | 74.60±12.12 | 92.10±11.28 | 149.20±57.43 |
模型组 | 274.10±44.05a | 193.20±21.06a | 405.00±56.52a |
益气通脉方低剂量组 | 232.00±27.47 | 172.70±9.87 | 331.40±28.97b |
益气通脉方中剂量组 | 205.10±23.90c | 160.30±13.45b | 313.50±24.52c |
益气通脉方高剂量组 | 193.00±37.99c | 148.40±23.00c | 297.30±23.50c |
阳性对照组 | 207.30±28.69c | 144.30±26.17c | 294.50±45.08c |
a:与正常组比较,P<0.01;b:与模型组比较,P<0.05;c:与模型组比较,P<0.01。
与正常组比较,模型组小鼠主动脉斑块明显,其斑块面积占比显著增大(P<0.01);与模型组比较,各药物组小鼠主动脉斑块均有不同程度减少,其中益气通脉方中、高剂量组及阳性对照组小鼠主动脉斑块面积占比均显著减小(P<0.01)。结果见
图1 各组小鼠主动脉斑块染色图
Ⅰ:正常组;Ⅱ:模型组;Ⅲ:益气通脉方低剂量组;Ⅳ:益气通脉方中剂量组;Ⅴ:益气通脉方高剂量组;Ⅵ:阳性对照组。
图2 各组小鼠主动脉斑块面积占比比较(x±s,n=3)
Ⅰ:正常组;Ⅱ:模型组;Ⅲ:益气通脉方低剂量组;Ⅳ:益气通脉方中剂量组;Ⅴ:益气通脉方高剂量组;Ⅵ:阳性对照组;a:与正常组比较,P<0.01;b:与模型组比较,P<0.01。
正常组小鼠主动脉外膜、中膜、内膜层次清晰,血管内膜平滑完整,未见斑块沉积;模型组小鼠主动脉内膜明显增厚,斑块明显且斑块内可见大量胆固醇脂质沉积、炎症细胞浸润及由泡沫细胞组成的脂质条纹;与模型组比较,益气通脉方低、中、高剂量组和阳性对照组小鼠主动脉内斑块呈不同程度减少,脂质沉积、炎症细胞及泡沫细胞亦有所减少。结果见
图3 各组小鼠主动脉窦病理形态学改变的显微图(HE染色)
箭头:斑块。
与正常组比较,模型组小鼠主动脉中PI3K、Akt、mTOR蛋白的磷酸化水平均显著升高(P<0.01);与模型组比较,益气通脉方中、高剂量组及阳性对照组小鼠主动脉中PI3K、Akt、mTOR蛋白的磷酸化水平均显著降低(P<0.05或P<0.01)。结果见
图4 各组小鼠主动脉中PI3K/Akt/mTOR信号通路相关蛋白表达的电泳图
Ⅰ:正常组;Ⅱ:模型组;Ⅲ:益气通脉方低剂量组;Ⅳ:益气通脉方中剂量组;Ⅴ:益气通脉方高剂量组;Ⅵ:阳性对照组。
组别 | p-PI3K/PI3K | p-Akt/Akt | p-mTOR/mTOR |
---|---|---|---|
正常组 | 0.49±0.04 | 0.43±0.10 | 0.46±0.09 |
模型组 | 1.09±0.22a | 1.00±0.04a | 1.01±0.15a |
益气通脉方低剂量组 | 0.83±0.07 | 0.89±0.06 | 0.80±0.18 |
益气通脉方中剂量组 | 0.70±0.05b | 0.67±0.07b | 0.63±0.14c |
益气通脉方高剂量组 | 0.59±0.10b | 0.62±0.12b | 0.59±0.13c |
阳性对照组 | 0.59±0.02b | 0.65±0.14b | 0.50±0.10b |
a:与正常组比较,P<0.01;b:与模型组比较,P<0.01;c:与模型组比较,P<0.05。
AS是一种常见的血管内膜疾病,可累及主动脉、冠状动脉等所有血管,以内膜斑块增厚为主要特征,发病机制复杂[
脂质代谢失衡和炎症反应是AS发展的关键因素,改善脂质沉积和炎症反应对AS的治疗具有重要意义[
PI3K/Akt/mTOR是一条细胞内信号转导通路,PI3K的激活可促使下游Akt发生磷酸化,后者可调控一系列因子的表达,进而触发mTOR的磷酸化[
综上所述,益气通脉方可改善AS小鼠的脂质代谢,减轻炎症反应,延缓斑块发展,上述作用可能与抑制PI3K/Akt/mTOR信号通路激活有关。但本研究尚未采用PI3K/Akt/mTOR信号通路激活剂进行验证,后续本课题组将进一步完善实验设计,深入探讨益气通脉方干预AS的作用机制,为该方的临床应用提供参考依据。
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