泻心汤含药血清对RAW264.7源性泡沫细胞TLR9/MyD88/NF-κB p65信号通路的影响
|
摘要
目的:通过体外细胞实验研究泻心汤对巨噬细胞源性泡沫细胞Toll样受体9(TLR9)信号通路的影响,探讨泻心汤抗动脉粥样硬化的作用机制。方法:50只SPF级雄性SD大鼠随机分为泻心汤低、中、高剂量组(1. 4,4. 2,12. 6 g·kg~(-1)·d~(-1))及正常组,除正常组20只外,其余每组10只,正常组给予等体积的纯水灌胃,各组连续灌胃7 d,末次给药1 h后,分离血清,制备泻心汤低、中、高剂量组含药血清及正常组血清。用氧化低密度脂蛋白(ox-LDL)干预RAW264. 7巨噬细胞分化为泡沫细胞。采用油红O染色法鉴定细胞泡沫化;噻唑蓝(MTT)比色法观察含药血清对巨噬细胞源性泡沫细胞增殖的影响后,选择各剂量浓度为20%的含药血清作用于泡沫细胞模型,采用酶联免疫吸附测定(ELISA)检测白细胞介素-1β(IL-1β),γ-干扰素(INF-γ)的含量;实时荧光定量PCR(Real-time PCR)及蛋白免疫印迹法(Western blot)分别测定TLR9,髓样分化因子88(MyD88),核转录因子-κB(NF-κB) mRNA及蛋白表达。结果:油红O染色显示出ox-LDL干预后细胞内红色颗粒明显,泡沫细胞模型制备成功; MTT比色法结果显示,与正常组血清比较,泻心汤高剂量组含药血清在10%～30%浓度范围内,其细胞增殖差异无显著性。后续选择各剂量浓度为20%的含药血清干预经ox-LDL诱导的泡沫细胞,与正常组比较,模型组用ox-LDL干预后诱导了TLR9,MyD88,NF-κB p65,IL-1β,INF-γ的高表达(P <0. 05,P <0. 01);与模型组比较,不同剂量组泻心汤含药血清干预后降低了TLR9,MyD88,NF-κB p65,IL-1β,INF-γ的表达(P <0. 05,P <0. 01),且部分干预作用呈现出剂量依赖性。结论:泻心汤含药血清可抑制泡沫细胞TLR9/MyD88/NF-κB p65通路以及促炎因子IL-1β,INF-γ的转录和过表达,这可能是泻心汤抗动脉粥样硬化重要作用机制之一。
Objective: The effect of Xiexintang on Toll like receptor 9( TLR9) signaling pathway in macrophage derived foam cells was studied by in vitro cell experiments. Method: The fifty SPF male SD rats were randomly divided into low,medium and high dose groups( 1. 4,4. 2,12. 6 g·kg~(-1)·d~(-1)) and normal groups.Except 20 rats in the normal group,10 rats in each group were given equal volume of pure water gavage in the blank group. After the last Administration for 7 days,serum was separated,and the serum containing drugs in the low,medium and high dose groups of Xiexintang was prepared. Oxidized low density lipoprotein( ox-LDL) was used to intervene the differentiation of RAW264. 7 macrophages into foam cells. The cell foam was identified by oil red O staining. After observing the effect of drug containing serum on the proliferation of macrophage derived foam cells by methye thiazolye telrazlium( MTT) method,the serum containing 20% concentration of each drug was selected to act on the foam cell model. The expression of interleukin( IL)-1β and interferon( INF)-γ was determined by enzyme-linked immunosorbent assay( ELISA) and real-time fluorescence quantitative PCR( Realtime PCR). TLR9,myeloid differentiation factor 88( MyD88) and nuclear factor( NF)-κB were detected by Western blot. Result: Oil red O staining showed that the red particles were obvious after ox-LDL intervention. The foam cell model was successfully prepared. MTT results showed that there was no significant difference in cell proliferation between the high dose group of Xiexintang in the 10% ～ 30% concentration range and the normal group serum. Follow up selection of the serum containing 20% concentration of each dose intervened the foam cells induced by ox-LDL. Compared with the normal group,the model group after ox-LDL intervention induced the high expression of TLR9,My D88,NF-κB p65,IL-1β,INF-γ( P < 0. 05,P < 0. 01). Compared with model group,the serum of Xiexintang containing different dosage groups reduced the expression of TLR9,MyD88,NF-κB p65,IL-1β,INF-γ( P < 0. 05, P < 0. 01), and part of the intervention was dose-dependent. Conclusion:Xiexintang containing serum can inhibit ox-LDL-induced RAW264. 7 macrophage foaming,and its mechanism may involve regulation of TLR9/MyD88/NF-κB p65 signaling pathway and inhibition of IL-1β and INF-γoverexpression. This may be one of its mechanisms of against atherosclerosis.
Objective: The effect of Xiexintang on Toll like receptor 9( TLR9) signaling pathway in macrophage derived foam cells was studied by in vitro cell experiments. Method: The fifty SPF male SD rats were randomly divided into low,medium and high dose groups( 1. 4,4. 2,12. 6 g·kg~(-1)·d~(-1)) and normal groups.Except 20 rats in the normal group,10 rats in each group were given equal volume of pure water gavage in the blank group. After the last Administration for 7 days,serum was separated,and the serum containing drugs in the low,medium and high dose groups of Xiexintang was prepared. Oxidized low density lipoprotein( ox-LDL) was used to intervene the differentiation of RAW264. 7 macrophages into foam cells. The cell foam was identified by oil red O staining. After observing the effect of drug containing serum on the proliferation of macrophage derived foam cells by methye thiazolye telrazlium( MTT) method,the serum containing 20% concentration of each drug was selected to act on the foam cell model. The expression of interleukin( IL)-1β and interferon( INF)-γ was determined by enzyme-linked immunosorbent assay( ELISA) and real-time fluorescence quantitative PCR( Realtime PCR). TLR9,myeloid differentiation factor 88( MyD88) and nuclear factor( NF)-κB were detected by Western blot. Result: Oil red O staining showed that the red particles were obvious after ox-LDL intervention. The foam cell model was successfully prepared. MTT results showed that there was no significant difference in cell proliferation between the high dose group of Xiexintang in the 10% ～ 30% concentration range and the normal group serum. Follow up selection of the serum containing 20% concentration of each dose intervened the foam cells induced by ox-LDL. Compared with the normal group,the model group after ox-LDL intervention induced the high expression of TLR9,My D88,NF-κB p65,IL-1β,INF-γ( P < 0. 05,P < 0. 01). Compared with model group,the serum of Xiexintang containing different dosage groups reduced the expression of TLR9,MyD88,NF-κB p65,IL-1β,INF-γ( P < 0. 05, P < 0. 01), and part of the intervention was dose-dependent. Conclusion:Xiexintang containing serum can inhibit ox-LDL-induced RAW264. 7 macrophage foaming,and its mechanism may involve regulation of TLR9/MyD88/NF-κB p65 signaling pathway and inhibition of IL-1β and INF-γoverexpression. This may be one of its mechanisms of against atherosclerosis.
引文
[1] Conti P,Shaik-Dasthagirisaeb Y. Atherosclerosis:a chronic inflammatory disease mediated by mast cells[J]. Cent Eur J Immunol,2016,40(3):380-386.
[2] Moriya J. Critical roles of inflammation in atherosclerosis[J]. J Cardiol,2019,73(1):22-27.
[3] Hovland A,Jonasson L,Garred P, et al. The complement system and toll-like receptors as integrated players in the pathophysiology of atherosclerosis[J].Atherosclerosis,2015,241(2):480-494.
[4] Koulis C,CHEN Y C,Hausding C,et al. Protective role for toll-like receptor-9 in the development of atherosclerosis in apolipoprotein e-deficient mice[J].Arterioscler Thromb Vasc Biol,2014,34(3),516-525.
[5] Krogmann A O,Lüsebrink E,Steinmetz M,et al.Proinflammatory stimulation of toll-like receptor 9 with high dose Cp G ODN 1826 impairs endothelial regeneration and promotes atherosclerosis in mice[J].PLo S One,2016,11(1):e0146326.
[6] Hirata Y,Kurobe H,Higashida M,et al. HMGB1 plays a critical role in vascular inflammation and lesion formation via toll-like receptor 9[J]. Atherosclerosis,2013,231(2):227-233.
[7]殷小杰,马晓静,王岚,等.三黄泻心汤活血化瘀优势方抗动脉粥样硬化的作用机制[J].中国实验方剂学杂志,2018,24(22):83-88.
[8]吴智春,吴凯,王浩,等.金匮泻心汤抗动脉粥样硬化的实验研究[J].中国老年学杂志,2003,23(7):461-462.
[9]苗婷婷.三黄泻心汤研究进展[J].湖南中医杂志,2016,32(3):190-192.
[10]张智伟,蔡琨,于红红,等.紫花地丁含药血清对巨噬细胞炎症因子分泌的影响[J].免疫学杂志,2014,30(1):53-56.
[11]张君涛,王平,刘爱峰,等.中药含药血清制备方法的研究概述[J].中华中医药杂志,2015,30(11):4006-4009.
[12] HU J,XI D,ZHAO J,et al. Crosstalk between highdensity lipoprotein and inflammation and it's significance to atherosclerosis[J]. Am J Med Sci,2016,352(4):408-415.
[13] Hegyi L,Skepper J N,Cary N R,et al. Foam cell apoptosis and the development of the lipid core of human atherosclerosis[J]. J Pathol,2015,180(4):423-429.
[14] Falckhansen M,Kassiteridi C,Monaco C. Toll-like receptors in atherosclerosis[J]. Biochem Soc T,2007,35(6):1453-1455.
[15]于红红,吴玛莉,冷泠,等.自噬与动脉粥样硬化的关系及中药的干预作用[J].中国动脉硬化杂志,2014,22(7):736-740.
[2] Moriya J. Critical roles of inflammation in atherosclerosis[J]. J Cardiol,2019,73(1):22-27.
[3] Hovland A,Jonasson L,Garred P, et al. The complement system and toll-like receptors as integrated players in the pathophysiology of atherosclerosis[J].Atherosclerosis,2015,241(2):480-494.
[4] Koulis C,CHEN Y C,Hausding C,et al. Protective role for toll-like receptor-9 in the development of atherosclerosis in apolipoprotein e-deficient mice[J].Arterioscler Thromb Vasc Biol,2014,34(3),516-525.
[5] Krogmann A O,Lüsebrink E,Steinmetz M,et al.Proinflammatory stimulation of toll-like receptor 9 with high dose Cp G ODN 1826 impairs endothelial regeneration and promotes atherosclerosis in mice[J].PLo S One,2016,11(1):e0146326.
[6] Hirata Y,Kurobe H,Higashida M,et al. HMGB1 plays a critical role in vascular inflammation and lesion formation via toll-like receptor 9[J]. Atherosclerosis,2013,231(2):227-233.
[7]殷小杰,马晓静,王岚,等.三黄泻心汤活血化瘀优势方抗动脉粥样硬化的作用机制[J].中国实验方剂学杂志,2018,24(22):83-88.
[8]吴智春,吴凯,王浩,等.金匮泻心汤抗动脉粥样硬化的实验研究[J].中国老年学杂志,2003,23(7):461-462.
[9]苗婷婷.三黄泻心汤研究进展[J].湖南中医杂志,2016,32(3):190-192.
[10]张智伟,蔡琨,于红红,等.紫花地丁含药血清对巨噬细胞炎症因子分泌的影响[J].免疫学杂志,2014,30(1):53-56.
[11]张君涛,王平,刘爱峰,等.中药含药血清制备方法的研究概述[J].中华中医药杂志,2015,30(11):4006-4009.
[12] HU J,XI D,ZHAO J,et al. Crosstalk between highdensity lipoprotein and inflammation and it's significance to atherosclerosis[J]. Am J Med Sci,2016,352(4):408-415.
[13] Hegyi L,Skepper J N,Cary N R,et al. Foam cell apoptosis and the development of the lipid core of human atherosclerosis[J]. J Pathol,2015,180(4):423-429.
[14] Falckhansen M,Kassiteridi C,Monaco C. Toll-like receptors in atherosclerosis[J]. Biochem Soc T,2007,35(6):1453-1455.
[15]于红红,吴玛莉,冷泠,等.自噬与动脉粥样硬化的关系及中药的干预作用[J].中国动脉硬化杂志,2014,22(7):736-740.