补阳还五汤苷类组分介导NF-κB通路抗动脉粥样硬化炎症反应的机制研究

Mechanism of Buyang Huanwu Decoction glycosides against atherosclerotic inflammation through NF-κB signaling pathway

探讨补阳还五汤苷类组分调节核因子κB(nuclear factor kappa-B,NF-κB)信号通路对载脂蛋白E^(-/-)(apolipoprotein E^(-/-),ApoE^(-/-))小鼠及RAW264.7细胞炎症反应的影响。体内实验,采用高脂饲料连续喂养ApoE^(-/-)小鼠12周,建立动脉粥样硬化动物模型,75μg·mL-1氧化低密度脂蛋白(oxidized low-density lipoprotein, Ox-LDL)孵育RAW264.7细胞24 h建立动脉粥样硬化细胞模型,利用全自动生化分析仪、苏木素-伊红(hematoxylin-eosin, HE)染色、酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)、Western blot及droplets digital PCR方法,检测血脂水平、主动脉内膜厚度、炎症因子含量及NF-κB通路相关蛋白及mRNA表达水平,评价动脉粥样硬化病变情况、药物抗动脉粥样硬化作用机制。结果表明,ApoE^(-/-)小鼠高脂饲料连续喂养12周后成功建立动脉粥样硬化模型。与对照组比较,模型组小鼠血浆中总胆固醇(total cholesterol, TC)、甘油三酯(triglyceride, TG)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol, LDL-C)含量显著升高,血管内膜增厚,炎症因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6, IL-6)含量增加,NF-κB及IκBα蛋白及mRNA表达显著增加;与模型组相比,补阳还五汤苷类组分高剂量组显著降低小鼠血浆中TC、TG、LDL-C含量,减轻斑块面积和厚度,减少炎症因子TNF-α的含量,抑制NF-κB、IκBα蛋白及mRNA表达,且作用与补阳还五汤一致。体外实验,采用75μg·mL^(-1)Ox-LDL刺激RAW264.7细胞24 h成功建立泡沫细胞模型。与对照组相比,模型组细胞NF-κB的入核量、IκBα蛋白及相关mRNA的表达增加;与模型组比较,补阳还五汤苷类组分中、高剂量组减少NF-κB的入核量、IκBα蛋白及相关mRNA的表达。以上结果表明,苷类组分是补阳还五汤抗动脉粥样硬化的主要有效成分,可抑制NF-κB通路,减轻炎症反应,发挥母方补阳还五汤同效的抗动脉粥样硬化炎症反应的作用。

This study aims to explore the effect of Buyang Huanwu Decoction glycosides on the inflammatory response of apolipoprotein E^(-/-)(ApoE^(-/-)) mice and RAW264.7 cells through nuclear factor kappa-B(NF-κB) signaling pathway. In the in vivo experiment, ApoE^(-/-)mice were fed with high-fat diets for 12 weeks to induce the animal model of atherosclerosis, and 75 μg·mL-1oxidized low-density lipoprotein(Ox-LDL) incubated RAW264.7 cells for 24 h to establish the atherosclerosis cell model. Automatic biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay(ELISA), Western blot, and droplet digital polymerase chain reaction(PCR) were used to determine the blood lipid levels, aortic intimal thickness, inflammatory factor content, NF-κB pathway-related proteins, and mRNA expression levels, and evaluate arterial atherosclerotic lesions and anti-atherosclerotic mechanisms of the drug. The model of atherosclerosis was successfully established in ApoE^(-/-)mice after 12 weeks of feeding with high-fat diets. In the model group, the plasma levels of total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-C) were increased(P<0.01), the intima of the blood vessels was thickened, the levels of inflammatory factors tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) were increased, and the protein and mRNA expressions of NF-κB and inhibitor of NF-κB(IκBα) were significantly increased as compared with the control group. Compared with the model group, the high-dose Buyang Huanwu Decoction glycoside group decreased the plasma levels of TC, TG, and LDL-C, reduced the plaque area and thickness and the content of inflammatory factor TNF-α, and inhibited the protein and mRNA expressions of NF-κB and IκBα, with the effect same as Buyang Huanwu Decoction. In the in vivo experiment, 75 μg·mL^(-1)Ox-LDL stimulated RAW264.7 cells for 24 h to successfully establish a foam cell model. As compared with the control group, the nuclear amount of NF-κB and the protein and mRNA expressions of IκBα in the model group increased. Compared with the model group, the middle-dose and high-dose Buyang Huanwu Decoction glycoside groups decreased the nuclear amount of NF-κB and the protein and mRNA expressions of IκBα. The above results show that the glycosides are the main effective substances of Buyang Huanwu Decoction against atherosclerosis, which inhibit the NF-κB pathway and reduce the inflammatory response, thus playing the role against atherosclerotic inflammation same as Buyang Huanwu Decoction.