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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 investigate the inhibitory effect of paeoniflorin (PAE) on TNFR1-mediated signaling pathway in renal artery endothelial cells induced by TNF-α in mice and to explore its molecular mechanism. Methods Mouse arterial endothelial cells were cultured in vitro. Western blot was used to detect the expression level of TNF-α in normal serum (serum-free medium), TNF-α (serum TNF-α30ng / m L for 2 h and TNF- 30ng / m L for 6 h), PAE concentration group (PAE 8 μmol / L for 2 h plus TNF-α 30 ng / m L for 6 h) and PAE high concentration group (PAE 80 μmol / L for 2 h plus TNF-α 30 ng / m L for 6 h). The expression of intercellular cell adhesion molecule-1 (ICAM-1) was detected by immunofluorescence in normal group (serum-free medium), TNF- (30ng / ml for 45 min in culture media), and high concentration of PAE (NF-κB, NF-κB) induced by PAE at 80μmol / L for 2 h and TNF-α at 30ng / Western blot was used to detect the expression of ph-ERK and ph-p38 in normal group (serum-free medium) and PAE high concentration group (PAE 80μmol / L for 2 h) TNF-α group (TNF-α30ng / m L for 30min), TNF-αgroup (TNF-α30ng / m L for 30min), PAE high concentration group PAE80μmol / , P38 inhibitor group (SB group, p38 inhibitor SB238025 25μ (30 mol / L pretreatment for 30 min, PAE 80 μmol / L for 2 h and final TNF-α 30 ng / mL for 30 min) and ERK inhibitor group (PD group, ERK inhibitor PD98059 50 μmol / L for 30 min, PAE 80 μmol / Treated for 2 h, finally TNF-α30 ng / m L for 30 min) IκBα protein expression. Results Compared with normal group, the expression of ICAM-1 in TNF-α group was significantly increased (P <0.01). Compared with TNF-α group, ICAM-1 expression in PAE high concentration group was significantly inhibited (P <0.05). PAE high concentration group ph-p38 and ph-ERK protein expression levels were significantly higher than the normal group (P <0.05). Compared with normal group, the expression of IκBα in TNF-α group decreased (P <0.01). Compared with TNF-α group, PAE high concentration group could significantly inhibit the degradation of IκBα protein induced by TNF-α (P <0.01). SB group could significantly inhibit the inhibitory effect of PAE on IκBα protein degradation (P <0.05). NF-κB / p65 signal in the normal group mainly located in the cytoplasm, TNF-α group stimulated by TNF-α 45 min can induce NF-κB / p65 translocation from the cytoplasm to the nucleus, while PAE high concentration group can significantly inhibit TNF -αinduced NF-κB / p65 nuclear translocation. Conclusion PAE can inhibit the expression of ICAM-1 induced by TNF-α, which is related to the inhibition of TNFR1 / NF-κB signaling pathway. P38 is involved in this effect.