补肾活血方对去卵巢小鼠冠脉微循环的保护作用及机制研究
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摘要
目的探讨心肌巨噬细胞(macrophages,M)表型M1/M2极化在去卵巢小鼠冠脉微循环障碍(CMD)中的作用及补肾活血方的影响。方法将90只雌性C57BL/6小鼠按随机数字表法分为9组:6周正常组(6WC组)、6周假手术组(6WS组)、6周模型组(6WM组,去卵巢6W)、8周正常组(8WC组)、8周假手术组(8WS组)、8周模型组(8WM组,去卵巢8W)、巨噬细胞清除组(简称CM组,腹腔注射氯膦酸二钠脂质体0. 01 m L/kg,每周1次)、补肾活血组[简称BG组,补肾活血方灌胃,5 m L/(kg·d),每天1次]及miR-155抑制组(简称MI组,尾静脉注射miR-155抑制剂20 mg/kg,每周1次),每组10只,持续干预8周。采用小动物超声成像系统检测左室短轴缩短率(LVFS)和左室射血分数(LVEF),HE染色观察心肌组织结构,麦胚凝集素染色检测心肌细胞横截面积,免疫荧光染色检测冠脉微血管密度和CD68+表达;实时荧光定量PCR检测心肌miR-155、IL-1β、TNF-α、IL-10及血管内皮生长因子(VEGF) mRNA表达。结果与6WC组比较,6WS和6WM组心肌LVEF和LVFS值、炎症细胞浸润、心肌细胞横截面积、冠脉微血管密度差异均无统计学意义(P> 0. 05);与8WC和8WS组比较,8WM组心肌LVEF和LVFS值明显下降,心肌细胞肿胀和炎症细胞浸润增加,心肌细胞横截面积增大,冠脉微血管密度减少,CD68+表达增加,miR-155、IL-1β和TNF-αmRNA表达增加,IL-10和VEGF mRNA表达减少(P <0. 05,P <0. 01);与8WM组比较,BG组心肌LVEF、LVFS值和冠脉微血管密度增加,炎症细胞浸润、细胞横截面积减小,CM和BG组心肌组织炎症细胞浸润、CD68+表达均减少,CM、BG和MI组心肌miR-155、IL-1β和TNF-αmRNA表达均减少; BG和MI组心肌IL-10和VEGF mRNA表达均增加,差异均有统计学意义(P <0. 05,P <0. 01);与MI组比较,BG组心肌miR-155、IL-1β、TNF-α、IL-10、VEGF mRNA表达差异无统计学意义(P> 0. 05)。结论补肾活血方可能通过下调心肌miR-155调控M1/M2极化抑制冠脉微血管炎症反应。
Objective To observe the effect of myocardial macrophage phenotype M1/M2 polarization in ovariectomized mice coronary microvascular dysfunction( CMD) model and the protective role of Bushen Huoxue Recipe. Methods Totally 90 female C57 BL/6 mice were assigned to 9 groups by random digit table: 6-week control group( 6 WC),6-week sham group( 6 WS),6-week model group( 6 WM,ovariectomized 6 weeks),8-week control group( 8 WC),8-week sham group( 8 WS),8-week model group( 8 WM,ovariectomized 8 weeks),clear macrophage group( CM,intraperitoneally injected with Clophosome,0. 01 ml/kg,once a week),Bushen Huoxue group[BG,given Bushen Huoxue Recipe by gavage,5 m L/( kg·d),once a day]and miR-155 inhibitor group( MI,injected miR-155 antagomir,20 mg/kg,once a week). 10 in each group,sustained intervention for 8 weeks. Small animal ultrasound imaging system was used to collect echocardiography to analyze left ventricular fraction shortening( LVFS) and left ventricular ejection fraction( LVEF). HE staining was used to observe the myocardial structure. Wheat germ agglutinin staining was used to observe myocyte cross-sectional area.Immunofluorescence was used to observe coronary microvascular density,the expression of CD68+. Real-time quantitative PCR was used to detect the expression of microRNA-155( miR-155),interleukin( IL)-1β,tumor necrosis factor( TNF)-α,IL-10 and vascular endothelial growth factor( VEGF) mRNA. Results Compared with 6 WC group,there was no statistically significant difference in LVFS and LVEF levels,the myocardial cell swelled,inflammatory cells infiltration,the cross-sectional area of cardiac cells,and coronary microvascular density in 6 WS and 6 WM groups(P >0. 05). Compared with 8 WC and 8 WS group,LVFS and LVEF levels decreased,and the myocardial cell swelled,inflammatory cells infiltration increased,the crosssectional area of cardiac cells increased,coronary microvascular density decreased,the expression of CD68+,miR-155,IL-1β,TNF-α mRNA increased,the expression of IL-10 and VEGF mRNA decreased in 8 WM group,all the above differences were statistically significant(P < 0. 05,P < 0. 01). Compared with 8 WM group,LVFS and LVEF levels,coronary microvascular density increased,inflammatory cells infiltration and the cross-sectional area of cardiac cells decreased in BG group; inflammatory cells infiltration and the expression of CD68+decreased in CM and BG group; the expression of miR-155,IL-1β,TNF-αmRNA decreased in CM,BG and MI group; the expression of IL-10 and VEGF mRNA increased in BG and MI group; all the above differences were statistically significant(P <0. 05,P <0. 01). Compared with MI group,there was no statistically significant difference in the expression of miR-155,IL-1β,TNF-α,IL-10 and VEGF mRNA in BG group(P >0. 05). Conclusion Bushen Huoxue Recipe may inhibit the inflammatory response of coronary microcirculation by down-regulating the expression of miR-155 regulating M1/M2 polarization.
Objective To observe the effect of myocardial macrophage phenotype M1/M2 polarization in ovariectomized mice coronary microvascular dysfunction( CMD) model and the protective role of Bushen Huoxue Recipe. Methods Totally 90 female C57 BL/6 mice were assigned to 9 groups by random digit table: 6-week control group( 6 WC),6-week sham group( 6 WS),6-week model group( 6 WM,ovariectomized 6 weeks),8-week control group( 8 WC),8-week sham group( 8 WS),8-week model group( 8 WM,ovariectomized 8 weeks),clear macrophage group( CM,intraperitoneally injected with Clophosome,0. 01 ml/kg,once a week),Bushen Huoxue group[BG,given Bushen Huoxue Recipe by gavage,5 m L/( kg·d),once a day]and miR-155 inhibitor group( MI,injected miR-155 antagomir,20 mg/kg,once a week). 10 in each group,sustained intervention for 8 weeks. Small animal ultrasound imaging system was used to collect echocardiography to analyze left ventricular fraction shortening( LVFS) and left ventricular ejection fraction( LVEF). HE staining was used to observe the myocardial structure. Wheat germ agglutinin staining was used to observe myocyte cross-sectional area.Immunofluorescence was used to observe coronary microvascular density,the expression of CD68+. Real-time quantitative PCR was used to detect the expression of microRNA-155( miR-155),interleukin( IL)-1β,tumor necrosis factor( TNF)-α,IL-10 and vascular endothelial growth factor( VEGF) mRNA. Results Compared with 6 WC group,there was no statistically significant difference in LVFS and LVEF levels,the myocardial cell swelled,inflammatory cells infiltration,the cross-sectional area of cardiac cells,and coronary microvascular density in 6 WS and 6 WM groups(P >0. 05). Compared with 8 WC and 8 WS group,LVFS and LVEF levels decreased,and the myocardial cell swelled,inflammatory cells infiltration increased,the crosssectional area of cardiac cells increased,coronary microvascular density decreased,the expression of CD68+,miR-155,IL-1β,TNF-α mRNA increased,the expression of IL-10 and VEGF mRNA decreased in 8 WM group,all the above differences were statistically significant(P < 0. 05,P < 0. 01). Compared with 8 WM group,LVFS and LVEF levels,coronary microvascular density increased,inflammatory cells infiltration and the cross-sectional area of cardiac cells decreased in BG group; inflammatory cells infiltration and the expression of CD68+decreased in CM and BG group; the expression of miR-155,IL-1β,TNF-αmRNA decreased in CM,BG and MI group; the expression of IL-10 and VEGF mRNA increased in BG and MI group; all the above differences were statistically significant(P <0. 05,P <0. 01). Compared with MI group,there was no statistically significant difference in the expression of miR-155,IL-1β,TNF-α,IL-10 and VEGF mRNA in BG group(P >0. 05). Conclusion Bushen Huoxue Recipe may inhibit the inflammatory response of coronary microcirculation by down-regulating the expression of miR-155 regulating M1/M2 polarization.
引文
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[21]Weisser SB,Mclarren KW,Kuroda E,et al.Generation and characterization of murine alternatively activated macrophages[J].Methods Mol Biol,2013,946:225-239.
[22]Gordon S,Martinez FO.Alternative activation of macrophages:mechanism and functions[J].Immunity,2010,32(5):593-604.
[23]Mulder R,Banete A,Basta S.Spleen-derived macrophages are readily polarized into classically activated(M1)or alternatively activated(M2)states[J].Immunobiology,2014,219(10):737-745.
[24]张卫萍,刘超永,周娟.淫羊藿苷对脂蛋白多糖诱导的巨噬细胞RAW264.7细胞M1/M2炎症表型转化的影响[J].中华中医药杂志,2016,31(10):4239-4242.
[25]Elton TS,Selemon H,Elton SM,et al.Regulation of the MIR155 host gene in physiological and pathological processes[J].Gene,2013,532(1):1-12.
[26]Biethahn K,Orinska Z,Vigorito E,et al.miRNA-155controls mast cell activation by regulating the PI3Kgamma pathway and anaphylaxis in a mouse model[J].Allergy,2014,69(6):752-762.
[2]Kakuta K,Dohi K,Sato Y,et al.Chronic inflammatory disease is an independent risk factor for coronary flow velocity reserve impairment unrelated to the processes of coronary artery calcium deposition[J].J Am Soc Echocardiogr,2016,29(2):173-180.
[3]Kono Y,Fukuda S,Hanatani A,et al.Remote ischemic conditioning improves coronary microcirculation in healthy subjects and patients with heart failure[J].Drug Des Devel Ther,2014,8:1175-1181.
[4]Mills CD,Ley K.M1 and M2 macrophages:the chicken and the egg of immunity[J].J Innate Immun,2014,6(6):716-726.
[5]Laffont B,Rayner KJ.MicroRNAs in the pathobiology and therapy of atherosclerosis[J].Can J Cardiol,2017,33(3):313-324.
[6]汶医宁,马静,史凡凡,等.补肾活血颗粒治疗绝经后微血管性心绞痛的疗效及其对血清NO、ET-1的影响[J].陕西中医,2016,37(8):987-988.
[7]张玉芬,王玉珍,赵洁,等.补肾活血汤对去卵巢大鼠心脏微血管内皮细胞形态及血清E2、ET、NO、PGI2、TXA2含量的影响[J].现代生物医学进展,2014,14(31):6044-6049.
[8]韩为华,乔黎焱,刘镘利,等.二仙汤加味对去卵巢大鼠心肌NF-κBp50、IκBα的影响[J].中国中医急症,2011,20(9):1440-1441.
[9]Lv LL,Tang PM,Li CJ,et al.The pattern recognition receptor,Mincle,is essential for maintaining the M1 macrophage phenotype in acute renal inflammation[J].Kidney Int,2017,91(3):587-602.
[10]Game X,Roumiguie M,Bouali O,et al.Vaginal lubrication after cervicovaginal stimulation is facilitated by phosphodiesterase type 5 inhibition in ovariectomized mice[J].J Sex Med,2013,10(6):1452-1460.
[11]Cao H,Huang Y,Wang L,et al.Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8production in M2 macrophages[J].Oncotarget,2016,7(40):65441-65453.
[12]Zhou S,Wang Y,Meng Y,et al.In Vivo therapeutic success of microRNA-155 antagomir in a mouse model of lupus alveolar hemorrhage[J].Arthritis Rheumatol,2016,68(4):953-964.
[13]Grabner A,Amaral AP,Schramm K,et al.Activation of cardiac fibroblast growth factor receptor 4 causes left ventricular hypertrophy[J].Cell Metab,2015,22(6):1020-1032.
[14]Weidner N.Intratumor microvessel density as a prognostic factor in cancer[J].Am J Pathol,1995,147(1):9-19.
[15]卢健棋,温志浩.中医药干预冠脉微循环障碍的研究近况[J].中国中医基础医学杂志,2013,19(5):597-600.
[16]张玉芬,陈艳秋,陈钧,等.二仙汤加味治疗绝经后微血管性心绞痛疗效观察[J].辽宁中医药大学学报,2012,14(10):96-98.
[17]王玉珍,刘海涛,宋欢欢,等.补肾活血汤对去卵巢大鼠心肌微血管和CYP2J3的影响[J].心脏杂志,2015,25(5):540-546.
[18]方喜业主编.医学实验动物学[M].北京:人民卫生出版社,1995:177-178.
[19]Pinto AR,Godwin JW,Rosenthal NA.Macrophages in cardiac homeostasis,injury responses and progenitor cell mobilisation[J].Stem Cell Res,2014,13(3 Pt B):705-714.
[20]Edvinsson A,Brann E,Hellgren C,et al.Lower inflammatory markers in women with antenatal depression brings the M1/M2 balance into focus from a new direction[J].Psychoneuroendocrinology,2017,80:15-25.
[21]Weisser SB,Mclarren KW,Kuroda E,et al.Generation and characterization of murine alternatively activated macrophages[J].Methods Mol Biol,2013,946:225-239.
[22]Gordon S,Martinez FO.Alternative activation of macrophages:mechanism and functions[J].Immunity,2010,32(5):593-604.
[23]Mulder R,Banete A,Basta S.Spleen-derived macrophages are readily polarized into classically activated(M1)or alternatively activated(M2)states[J].Immunobiology,2014,219(10):737-745.
[24]张卫萍,刘超永,周娟.淫羊藿苷对脂蛋白多糖诱导的巨噬细胞RAW264.7细胞M1/M2炎症表型转化的影响[J].中华中医药杂志,2016,31(10):4239-4242.
[25]Elton TS,Selemon H,Elton SM,et al.Regulation of the MIR155 host gene in physiological and pathological processes[J].Gene,2013,532(1):1-12.
[26]Biethahn K,Orinska Z,Vigorito E,et al.miRNA-155controls mast cell activation by regulating the PI3Kgamma pathway and anaphylaxis in a mouse model[J].Allergy,2014,69(6):752-762.