摘要
目的:观察黄芩-黄连对动脉粥样硬化(AS)小鼠斑块稳定性的影响,并基于Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核转录因子-κB(NF-κB)信号通路探讨其可能的作用机制。方法:10只正常C57BL/6J小鼠作为正常组,同品系的载脂蛋白E基因敲除(ApoE^(-/-))小鼠高脂饲料喂养12周构建动脉粥样硬化模型,随机分为5组,即模型组、阿托伐他汀组、黄芩-黄连低、中、高剂量组,每组10只。正常组、模型组给予等体积生理盐水灌胃,黄芩-黄连低、中、高剂量组分别给予1.95、3.9、7.8 g·kg^(-1)药物灌胃8周。观察小鼠一般状态;苏木素-伊红(HE)染色观测主动脉根部斑块病理情况并计算斑块面积百分比;马松(Masson)染色、油红O染色联合F4/80、α-平滑肌肌动蛋白(α-SMA)免疫组化检测主动脉根部斑块组分并计算斑块易损指数;酶联免疫吸附测定法(ELISA)检测血清肿瘤坏死因子-α(TNF-α)及白细胞介素-6(IL-6)的表达水平;蛋白免疫印迹法(Western blot)检测各组小鼠主动脉组织中基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、TLR4、MyD88、NF-κB p65、p-NF-κB p65蛋白表达水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测MMP-2、MMP-9、TLR4、MyD88、NF-κB p65 mRNA表达水平。结果:与模型组比较,各用药组小鼠一般状态改善,未见明显不良反应;与模型组比较,黄芩-黄连中、高剂量组AS小鼠主动脉根部斑块面积百分比明显降低(P<0.05);黄芩-黄连高剂量组斑块中胶原纤维及平滑肌细胞含量显著增多(P<0.01),脂质及巨噬细胞含量明显减少(P<0.05),黄芩-黄连各剂量组斑块易损指数明显降低,其中黄芩-黄连中、高剂量组显著降低(P<0.01);黄芩-黄连中、高剂量组主动脉组织中MMP-2、MMP-9蛋白及mRNA表达水平明显降低(P<0.05);黄芩-黄连中、高剂量组AS小鼠血清中TNF-α、IL-6水平明显降低(P<0.05);黄芩-黄连中、高剂量组主动脉组织中TLR4、MyD88蛋白及mRNA表达水平明显降低(P<0.05),其NF-κB p65磷酸化水平明显降低(P<0.05)。结论:黄芩-黄连可能通过抑制TLR4/MyD88/NF-κB信号通路发挥抗炎稳斑的作用。
Objective:To observe the effect of Scutellariae Radix-Coptidis Rhizoma on plaque stability in atherosclerotic(AS)mice and to explore its possible mechanism of action based on the Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor kappa B(NF-κB)signaling pathway.Method:Ten normal C57BL/6J mice were used as the normal group,and the same strain of ApoE knockout(ApoE^(-/-))mice were fed with a high-fat diet for 12 weeks to construct an atherosclerosis model.Mice were randomly divided into five groups,namely the model group,the atorvastatin group,and the Scutellariae Radix-Coptidis Rhizoma low-,medium-,and high-dose groups,with ten mice in each group.Then normal and model groups were given equal volume of saline gavage,and the low-,medium-,high-dose Scutellariae Radix-Coptidis Rhizoma groups were given 1.95,3.9,7.8 g·kg^(-1)of the drug by gavage for 8 weeks,respectively.The general state of mice was observed.Hematoxylin-eosin staining was utilized to observe the pathology of aortic root plaques and calculate the percentage of plaque area.Masson staining and oil red O staining combined with immunohistochemistry of F4/80 andα-SMA were used to detect the plaque components of aortic root plaques and calculate the plaque vulnerability index.Enzyme-linked immunosorbent assay was adopted to detect the expression levels of serum tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6).Western blot was applied to detect the protein expression levels of matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9),TLR4,MyD88,NF-κB p65,and phosphorylation(p)-NF-κB p65 in the aortic tissues of mice in each group.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)assay was employed to detect the expression levels of MMP-2,MMP-9,TLR4,and MyD88,NF-κB p65 mRNA.Result:Compared with the model group,the general state of the mice in each medication group was improved,and no obvious side effects were observed.Compared with the model group,the percentage of plaque area in the aortic root of AS mice was significantly reduced in the medium-and high-dose Scutellariae Radix-Coptidis Rhizoma groups(P<0.05).The content of collagen fibers and smooth muscle cells in the plaques of the high-dose Scutellariae Radix-Coptidis Rhizoma group was significantly increased(P<0.01),and the content of lipids and macrophages was significantly reduced(P<0.05),the plaque vulnerability index of each dose group of Scutellariae Radix-Coptidis Rhizoma was significantly reduced,with significant reduction of the medium-and high-dose groups(P<0.01).MMP-2 and MMP-9 protein and mRNA expression levels in aortic tissues were significantly reduced in medium-and high-dose Scutellariae Radix-Coptidis Rhizoma groups(P<0.05).The serum levels of TNF-αand IL-6 were significantly reduced in AS mice in medium-and high-dose Scutellariae Radix-Coptidis Rhizoma groups(P<0.05).In the medium-and high-dose Scutellariae Radix-Coptidis Rhizoma groups,the levels of TLR4,MyD88 protein,and mRNA expression in aortic tissues were significantly reduced(P<0.05),and the level of NF-κB p65 phosphorylation in aortic tissues was significantly reduced(P<0.05).Conclusion:Scutellariae Radix-Coptidis Rhizoma may play an anti-inflammatory and stabilizing role by inhibiting the TLR4/MyD88/NF-κB signaling pathway.
作者
纪凌云
吴俏兰
陈泽涛
葛春蕾
陈维达
宋婷
JI Lingyun;WU Qiaolan;CHEN Zetao;GE Chunlei;CHEN Weida;SONG Ting(Shandong University of Traditional Chinese Medicine(TCM),Jinan 250014,China;Affiliated Hospital of Shandong University of TCM,Jinan 250011,China;Linyi Hospital of TCM,Linyi 276600,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第13期28-36,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
山东省自然科学基金项目(ZR2020MH354)
山东大学科技计划项目(202019038)
齐鲁医派中医学术流派传承项目(202019151)。
