从丹皮酚对TLR-NF-κB信号通路的影响探讨高血压病血瘀证的形成机制
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摘要
【目的】通过观察活血化瘀中药牡丹皮的提取物丹皮酚(paeonol,Pae)对高血压病血瘀证患者血清损伤的血管内皮细胞TLR-NF-κB信号通路的影响,探讨高血压病血瘀证的形成机制。【方法】以体积分数10%的高血压病血瘀证、非血瘀证患者及健康人(正常组)血清干预人脐静脉内皮细胞(HUVEC-C)24 h,采用实时荧光定量(PCR)法检测Toll样受体4(TLR4)mRNA表达。以不同浓度的丹皮酚(大、中、小剂量分别为400μg/mL、200μg/mL、100μg/mL)干预脂多糖(LPS)诱导的HUVEC-C TLR4表达(24 h)及核转录因子(NF-κB)活化(2 h)。采用PCR法检测TLR4 mRNA及NF-κB mRNA的表达,免疫印迹(Western blot)技术检测TLR4蛋白及NF-κB蛋白(以I-κBα蛋白的降解反映)的表达。【结果】血清作用24 h后,TLR4 mRNA的表达按正常组、非血瘀证组、血瘀证组的顺序逐渐增高,组间差异有统计学意义(P<0.01)。丹皮酚可呈剂量依赖性减少LPS诱导的TLR4 mRNA高表达(P<0.001),抑制LPS诱导的TLR4蛋白高表达和I-κBα蛋白的降解。【结论】TLR-NF-κB信号途径介导的炎症反应和免疫紊乱是高血压病血瘀证形成的机制之一。
Objective To investigate the pathogenic mechanism of blood stasis syndrome(BSS) associated with essential hypertension by observing the effect of paeonol on Toll-like receptor-necrosis factor kappa B(TLRNF-κB) signal pathway. Methods The human umbilical vein endothelial cells(HUVEC-C) were cocultured with 10% serum for 24 h isolated separately from BSS hypertension patients, non-BSS hypertension patients and healthy volunteers. We observed the effect of paeonol at the concentrations of 400, 200 and 100 μg / mL on the levels of 24-hour TLR4 expression and 2-hour NF-κB activation in HUVEC-C activated by lipopolysaccharide( LPS). The expression levels of TLR4 mRNA and NF-κB mRNA in HUVEC-C were detected by real time polymerase chain reaction(PCR), and the expression levels of TLR4 protein and NF-κB protein were detected by Western blotting method. The NF-κB expression was reflected through the degradation of I-κBα. Results After cocultured with the serum for 24 h, the TLR4 mRNA expression level in HUVEC-C was increased in healthy volunteers group, non-BSS hypertension group and BSS hypertension group in increasing sequence, and the inter-group differences were significant(P<0.01). Paeonol showed a dose-dependent effect on decreasing TLR4 mRNA high expression induced by LPS( P <0.01), and inhibiting LPS-induced TLR4 protein high expression and I-κBα protein degradation. Conclusion It is indicated that inflammatory reaction and immune disorder mediated by TLR-NF-κB signal pathway probably are the pathogenic mechanism of BSS associated with hypertension.
Objective To investigate the pathogenic mechanism of blood stasis syndrome(BSS) associated with essential hypertension by observing the effect of paeonol on Toll-like receptor-necrosis factor kappa B(TLRNF-κB) signal pathway. Methods The human umbilical vein endothelial cells(HUVEC-C) were cocultured with 10% serum for 24 h isolated separately from BSS hypertension patients, non-BSS hypertension patients and healthy volunteers. We observed the effect of paeonol at the concentrations of 400, 200 and 100 μg / mL on the levels of 24-hour TLR4 expression and 2-hour NF-κB activation in HUVEC-C activated by lipopolysaccharide( LPS). The expression levels of TLR4 mRNA and NF-κB mRNA in HUVEC-C were detected by real time polymerase chain reaction(PCR), and the expression levels of TLR4 protein and NF-κB protein were detected by Western blotting method. The NF-κB expression was reflected through the degradation of I-κBα. Results After cocultured with the serum for 24 h, the TLR4 mRNA expression level in HUVEC-C was increased in healthy volunteers group, non-BSS hypertension group and BSS hypertension group in increasing sequence, and the inter-group differences were significant(P<0.01). Paeonol showed a dose-dependent effect on decreasing TLR4 mRNA high expression induced by LPS( P <0.01), and inhibiting LPS-induced TLR4 protein high expression and I-κBα protein degradation. Conclusion It is indicated that inflammatory reaction and immune disorder mediated by TLR-NF-κB signal pathway probably are the pathogenic mechanism of BSS associated with hypertension.
引文
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[12]胡小勤,陈利国,屈援.生化汤对血瘀证大鼠血管内皮细胞黏附分子表达的影响[J].中成药,2006,28(9):1330.
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[14]陈利国,屈援,葛红颖,等.补阳还五汤对血瘀证大鼠血管内皮细胞黏附分子表达的影响[J].中草药,2005,36(5):706.
[2]成威,舒春明,周胜华.冠心病和高血压病的免疫机制进展[J].现代生物医学进展,2009,9(2):362.
[3]宋卫华,党爱民,刘国仗.原发性高血压相关基因研究进展[J].中国分子心脏病学杂志,2005,5(2):500.
[4]衷敬柏.内皮素与血瘀证相关性研究[J].中国中西医结合杂志,2002,22(8):624.
[5]李民,朱平,张旭.麦冬药血清对血管内皮细胞损伤的机制研究[J].传染病药学,2003,13(2):4.
[6]张兰凤,王阶.血瘀证的细胞学和分子学研究进展[J].中国中医基础医学杂志,2003,9(1):71.
[7]马晓娟,殷惠军,陈可冀.血瘀证与炎症相关性的研究进展[J].中国中西医结合杂志,2007,27(7):669.
[8]陈可冀,李连达,翁维良,等.血瘀证与活血化瘀研究[J].中西医结合心脑血管病杂志,2005,3(1):1.
[9]袁肇凯,黄献平,谭光波,等.冠心病血瘀证血管内皮细胞功能的检测分析[J].中国中西医结合杂志,2006,26(5):407.
[10]Bulut Y,Faure E,Thomas L,et al.Chlamydial heats hock protein 60 activates macrophages and endothelial cells through toll-like receptor 4 and MD2 in a MyD88-dependent pathway[J].J Immunol,2002,168:143.
[11]陈利国,屈援,胡小勤,等.犀角地黄汤对肾上腺素与低温处理大鼠血管内皮细胞黏附分子表达的影响[J].中国病理生理杂志,2006,22(3):547.
[12]胡小勤,陈利国,屈援.生化汤对血瘀证大鼠血管内皮细胞黏附分子表达的影响[J].中成药,2006,28(9):1330.
[13]陈利国,陈畅宏,屈援,等.血府逐瘀汤对血瘀证大鼠血管内皮细胞黏附分子表达的影响[J].山东中医药大学学报,2006(5):395.
[14]陈利国,屈援,葛红颖,等.补阳还五汤对血瘀证大鼠血管内皮细胞黏附分子表达的影响[J].中草药,2005,36(5):706.