OBJECTIVETo study the inhibitory effect of paeoniflorin (PAE) on TNF-α-induced TNF receptor type I (TNFR1)-mediated signaling pathway in mouse renal arterial endothelial cells (AECs) and to explore its underlying molecular mechanisms.

METHODSMouse AECs were cultured in 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 normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 6-h TNF-α 30 ng/mL), the low dose PAE group (cultured by 2-h PAE 0.8 μmo/L plus 6-h TNF-α 30 ng/mL), the middle dose PAE group (cultured by 2-h PAE 8 μmol/L plus 6-h TNF-α 30 ng/mL), the high dose PAE group (cultured by 2-h PAE 80 μmol/L plus 6-h TNF-α 30 ng/mL) with Western blot analysis. Nuclear translocation of transcription factor NF-κB (NE-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-mm TNF-α 30 ng/mL), and the high dose PAE group (cultured by 2-h PAE 80 μmol/L plus 45-min TNF-α 30 ng/mL) 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/mL), the high dose PAE group (cultured by 2-h PAE 80 μmol/L plus 30-min TNF-α 30 ng/mL), 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/mL 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/mL for 30 min) by Western blot.

RESULTSCompared with the normal group, ICAM-1 protein expression levels obviously increased (P < 0.01). Compared with the TNFα group, 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 IκBα degradation 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-α group, while it could be significantly inhibited in the high dose PAE group.

CONCLUSIONSPAE inhibited TNF-α-induced expression of lCAM-1. Its action might be associated with inhibiting TNFR1/NF-κB signaling pathway. p38 participated and mediated these actions.