芍药苷对TNF-α诱导的内皮细胞TNFR1/NF-κB信号通路的抑制作用

Inhibition of Paeoniflorin on TNF-α-induced TNF-α Receptor Type Ⅰ/Nuclear Factor-κB Signal Transduction in Endothelial Cells

目的研究芍药苷(paeoniflorin,PAE)对TNF-α诱导小鼠肾动脉内皮细胞TNFR1介导信号通路的抑制作用,试探讨其作用的分子机制。方法体外培养小鼠动脉内皮细胞。采用Western blot方法检测正常组(无血清培养基培养)、TNF-α组(无血清培养基培养2 h加TNF-α30ng/m L 6 h)、PAE低浓度组(PAE0.8μmol/L培养2 h加TNF-α30ng/m L 6 h)、PAE中浓度组(PAE 8μmol/L培养2 h加TNF-α30ng/m L 6h)及PAE高浓度组(PAE 80μmol/L培养2 h加TNF-α30ng/m L 6 h)细胞间黏附分子-1(intercellular cell adhesion molecule-1,ICAM-1)的蛋白表达;以免疫荧光法检测正常组(无血清培养基培养)、TNF-α组(无血清培养基培养2 h加TNF-α30ng/m L 45 min)、PAE高浓度组(PAE 80μmol/L培养2 h加TNF-α30ng/m L45 min)核因子κB(nuclear factor-κB,NF-κB)的核转位;以Western blot法检测正常组(无血清培养基培养)及PAE高浓度组(PAE 80μmol/L培养2 h)ph-ERK和ph-p38表达;Western blot法检测正常组(无血清培养基培养)、TNF-α组(无血清培养基培养2 h加TNF-α30ng/m L 30 min)、PAE高浓度组PAE 80μmol/L培养2 h加TNF-α30ng/m L 30 min)、p38抑制剂组(SB组,p38抑制剂SB238025 25μmol/L预处理30 min,PAE80μmol/L处理2 h,最后TNF-α30 ng/m L 30 min)及ERK抑制剂组(PD组,ERK抑制剂PD98059 50μmol/L处理30 min,PAE 80μmol/L处理2 h,最后TNF-α30 ng/m L 30 min)IκBα蛋白表达。结果与正常组比较,TNF-α组ICAM-1蛋白表达明显升高(P<0.01);与TNF-α组比较,PAE高浓度组的ICAM-1表达受到显著抑制(P<0.05)。PAE高浓度组ph-p38及ph-ERK蛋白表达水平明显较正常组升高(P<0.05)。与正常组比较,TNF-α组IκBα表达水平下降(P<0.01)。与TNF-α组比较,PAE高浓度组可显著抑制TNF-α诱导的IκBα蛋白降解(P<0.01),SB组可显著阻断PAE对IκBα蛋白降解的抑制作用(P<0.05)。正常组中NF-κB/p65信号主要位于胞浆中,TNF-α组在TNF-α刺激45 min可诱导NF-κB/p65由胞浆向细胞核转位,而PAE高浓度组可显著抑制TNF-α诱导的NF-κB/p65核转位。结论 PAE抑制TNF-α诱导的ICAM-1表达,其作用与抑制TNFR1/NF-κB信号通路有关,p38参与介导此作用。

Objective To study the inhibitory effect of paeoniflorin（ PAE） on TNF-α-induced TNF receptor typeⅠ（ TNFR1）-mediated signaling pathway in mouse renal arterial endothelial cells（ AECs） and to explore its underlying molecular mechanisms. Methods Mouse AECs were culturedin vitro and then they were treated by different concentrations PAE or TNF-α for various time periods. Expression levels of intercellular cell adhesion molecule-1（ ICAM-1） were detected in the normalgroup（ cultured by serum-free culture media）,the TNF-α group（ cultured by 2-h serum-free culture media plus 6-h TNF-α30 ng / m L）,the low dose PAE group（ cultured by 2-h PAE 0. 8 μmol / L plus 6-h TNF-α 30 ng / m L）,the middle dose PAE group（ cultured by 2-h PAE 8 μmol / L plus 6-h TNF-α 30 ng / m L）,the high dose PAE group（ cultured by 2-h PAE 80 μmol /L plus 6-h TNF-α 30 ng / m L） with Western blot analysis. Nuclear translocation of transcription factor NF-κB（ NF-κB） was detected in the normal group（ cultured by serum-free culture media）,the TNF-α group（ cultured by 2-h serum-free culture media plus 45-min TNF-α 30 ng / m L）,and the high dose PAE group（ cultured by 2-h PAE 80 μmol / L plus 45-min TNF-α 30 ng /m L） by immunofluorescent staining. Expression levels of the phosphorylation of extracellular signal-regulated（ protein） kinase（ ph-ERK） and p38（ ph-p38） were detected in the normal group（ cultured by serum-free culture media） and the high dose PAE group（ 2-h PAE 80 μmol / L culture） by Western blot. NF-κB inhibitor-α（ IκBα） protein expressions were detected in the normal group（ cultured by serum-free culture media）,the TNF-α group（ cultured by 2-h serum-free culture media plus 30-min TNF-α 30 ng / m L）,the high dose PAE group（ cultured by 2-h PAE 80 μmol / L plus 30-min TNF-α 30 ng / m L）,the p38 inhibitor group（ SB group,pretreatment with SB238025 25 μmol / L for 30 min,then treated by PAE 80 μmol / L for 2 h,and finally treated by TNF-α 30 ng / m L for 30 min）,the ERK inhibitor group（ PD group,treated by PD98059 50 μmol / L for 30 min,then treated by PAE 80 μmol / L for 2 h,and finally treated by TNF-α 30 ng / m L for 30 min） by Western blot. Results Compared with the normal group,ICAM-1 protein expression levels obviously increased（P〈 0. 01）. Compared with the TNFαgroup,ICAM-1 protein expression levels were obviously inhibited in the high dose PAE group（P 〈0. 05）. Protein expression levels of ph-p38 and ph-ERK were obviously higher in the high dose PAE group（P〈 0. 05）. Compared with the normal group,IκBα protein expression levels obviously decreased in the TNF-α group（P〈 0. 01）. Compared with the TNFα group,TNF-α-induced IκBα degradation could be significantly inhibited in the high dose PAE group（P 〈0. 01）; the inhibition of PAE on IκBαdegradation could be significantly inhibited in the SB group（P 〈0. 05）. NF-κB / p65 signal was mainly located in cytoplasm in the normal group. NF-κB / p65 was translocated from cytoplasm to nucleus after stimulated by 45 min TNF-α in the TNF-αgroup,while it could be significantly inhibited in the high dose PAE group. Conclusions PAE inhibited TNF-α-induced expression of ICAM-1. Its action might be associated with inhibiting TNFR1 / NF-κB signaling pathway. p38 participated and mediated these actions.