摘要
目的:观察血管软化丸通过对TLR9调控,影响其TLR9下游信号NF-κB和IRF7水平,以及对巨噬细胞(M2/M1)极化平衡状态的影响,研究血管软化丸抗动脉粥样硬化的作用机制。方法:体外实验将巨噬细胞随机分为5组,即对照组(空白血清),血管软化丸中剂量组,血管软化丸高剂量组,IRS组(TLR9拮抗剂:IRS869),ODN组(激动剂:ODN1826)。体内实验将Apo E-/-小鼠分为5组,即对照组(生理盐水,灌胃),IRS组(IRS869,腹腔注射),ODN组(ODN1826,腹腔注射),血管软化丸中剂量组(浓度为1.728 g/m L),血管软化丸高剂量组(浓度为3.456 g/m L)。腹腔注射每周2次,灌胃每日1次,连续给药8周后取材进行检测。采用Western Blot法检测各组细胞TLR9的蛋白含量,主动脉TLR9及其下游分子My D88,p-NF-κB和IRF7的蛋白含量;采用流式细胞术和免疫化学荧光检测各组细胞M1型和M2型的比例;采用RT-PCR和ELISA法检测各组细胞TNF-α的表达;采用RT-PCR法和免疫荧光检测RAW264.7细胞和动脉斑块的M1型和M2型巨噬细胞的相关标志性因子和炎症因子;病理学检测验证血管软化丸抗动脉粥样硬化的疗效。结果:与对照组RAW264.7细胞相比,各中药组和IRS组TLR9蛋白表达量明显下降(P<0.05),ODN组TLR9蛋白表达量明显升高(P<0.05)。与对照组相比,各中药组和IRS组动脉斑块的TLR9、My D88、p-NF-κB和IRF7蛋白表达水平均明显较低(P<0.05)。与对照组相比,ODN组M1型巨噬细胞比例相对增加,各中药组和IRS组M2型比例相对增加,差异均具有统计学意义(P<0.05)。与对照组相比,各中药组和IRS组动脉斑块中i NOS的荧光密度低表达,而CD206的荧光密度为高表达。与对照组相比,各中药组和IRS组斑块形成和面积相对较少(P<0.05)。在RAW264.7细胞实验中,中药组和IRS可逆转ODN1826引起的TNF-α的升高,使其降低(P<0.05)。与对照组比较,各中药组和IRS组斑块形成和面积相对较少(P<0.05)。结论:血管软化丸和TLR9的拮抗剂I
Objective： To study the action mechanism of Vascular Softening Pill on anti-atherosclerosis by observing its regulation for TLR9,which influences the levels of NF-κB and IRF7,as well as its effect on macrophage polarization. Methods： In vitro experiments,macrophages were randomly divided into the control groups（ blank serum）,the Vascular Softening Pill groups（ medium-dose and high-dose）,IRS group（ TLR9 antagonist： IRS869）,ODN group（ Agonist： ODN1826）. In the vivo experiment,Apo E-/-mice were randomly divided into the control group（ normal saline,intragastric administration）,IRS group（ IRS869,intraperitoneal injection）,ODN group（ ODN1826,intraperitoneal injection）,medium-dose Vascular Softening Pill group（ 1. 728 g/m L）,and high-dose Vascular Softening Pill group（ 3. 456 g/m L）; intraperitoneal injection was twice a week,intragastric administration was once per day,the intervention was eight weeks. The protein contents of TLR9,My D88,p-NF-κB and IRF7 were detected by Western Blot method. The ratio of M1 to M2 was tested by flow cytometry and immunochemical fluorescence. TNF-α expression was examined by RT-PCR technique and ELISA method. RT-PCR and immunofluorescence were used to detect the landmark factors and inflammatory factors of the expression of M1 and M2 macrophages in RAW264. 7 cells and arterial plaques. Pathology test was applied to verify the efficacy of Vascular Softening Pill on atherosclerosis.Results： Compared to RAW264. 7 cells in the control group,the protein expressions of TLR9 in TCM groups and IRS group were significantly decreased（ P〈0. 05）,it was significantly increased in ODN group（ P〈0. 05）. Compared to the control group,the protein expressions of TLR9,My D88,p-NF-κB and IRF7 of the plaques were significantly lower in TCM groups and IRS group（ P〈0. 05）. The proportion of M1 macrophages was increased in ODN group,and the proportion of M2 was increased in TCM groups and IRS group（ P〈0. 05）. In TCM groups and IRS group,t
作者
秦合伟
李彦杰
任锟
张志鑫
赵晶
邢若星
原筝
QIN Hewei;LI Yanjie;REN Kun;ZHANG Zhixin;ZHAO Jing;XING Ruoxing;YUAN Zheng(Henan Province Hospital of TCM,The Second Affiliated Hospital of Henan University of Chinaese Medicine,Zhengzhou 450002,China)
出处
《中医药信息》
2018年第4期46-51,共6页
Information on Traditional Chinese Medicine
基金
国家自然科学基金项目(No.81704030)
河南省高校重点科研项目(No.18A360008)
河南省中医药科学研究专项课题(No.2017ZY2067)
河南省中医院专项课题(No.2017YJKT02)
