摘要目的 探讨罗格列酮对脂多糖(LPS)诱导的体外培养大鼠腹膜间皮细胞CD40和胞间黏附分子1(ICAM-1)表达的影响以及调节机制.方法 分离及培养大鼠原代腹膜间皮细胞.将细胞随机分为正常对照组、LPS(5 mg/L)组、BAY11-7085(NF-κB抑制剂)组(5μmol/L预刺激3 h后加入LPS作用3 h)、不同浓度罗格列酮(过氧化物酶体增殖蛋白激活性受体γ配体)组(10、20 μmol/L分别预处理3 h再加入LPS 5 mg/L)、GW9662(过氧化物酶体增殖蛋白激活性受体γ拮抗剂)预处理组(预处理3 h后加入罗格列酮10 μmol/L,3 h后再加入LPS 5 mg/L)和溶媒对照组.加入LPS后1 h收集细胞检测核因子κB(NF-κB)p65水平;3 h收集细胞分别检测CD40和ICAM-1基因表达;24 h收集细胞分别检测CD40和ICAM-1蛋白表达.RT-PCR法检测基因表达;Western印迹和免疫荧光方法检测蛋白表达及核因子磷酸化.结果 (1)常规培养的腹膜间皮细胞表达基础量CD40和ICAM-1,LPS显著上调其表达(P<0.05);LPS作用1 h时腹膜间皮细胞磷酸化NF-κB p65活化水平显著增高,与对照组差异有统计学意义(1.10±0.17比0.55±0.06,P<0.05).(2)NF-κB抑制剂BAY11-7085预处理后LPS诱导的磷酸化NF-κB p65水平、CD40和ICAM-1表达显著低于LPS组(0.22±0.11比1.10±0.17,P<0.01;0.34±0.02比0.50±0.06,P<0.05;0.35±0.16比0.74±0.03,P<0.05).(3)罗格列酮预处理后,LPS诱导的磷酸化NF-κB p65水平、CD40以及ICAM-1蛋白表达亦显著低于LPS组(0.77±0.08比0.90±0.10,P<0.01;0.79±0.16比0.99±0.06,P<0.05;0.83±0.20比1.22±0.13,P<0.05).GW9662和罗格列酮联合预处理后,LPS诱导的磷酸化NF-κB p65水平与罗格列酮预处理组差异无统计学意义,但CD40和ICAM-1表达显著高于罗格列酮预处理组(0.95±0.19比0.79±0.16;1.04+0.24比0.83±0.20,均P<0.05).结论 NF-κB信号通路参与调节LPS诱导的腹膜间皮细胞表达CD40和ICAM-1.罗格列酮通过NF-κB途径下调CD40和ICAM-1表达,从而发挥抗炎作用.

abstractsObjective To investigate the effect and mechanism by which PPARγ ligand, rosiglitasone, regulates the expression of CD40 and intercellular adhesion molecule 1 (ICAM-1) in the rat peritoneal mesothelial cells (RPMCs) induced by lipopolysaccharide (LPS). Methods RPMCs were harvested from Sprague-Dawley rat peritoneal cavity and maintained under defined in vitro conditions. The cells were randomly divided into groups as follows: medium, LPS (5 mg/L), LPS (5 mg/L)+BAY11-7085(5 μmol/L, NF-κB inhibitor), rosiglitazone (10 μmol/L or 20 μmol/L, peroxisome proliferator-activated receptor γ activator), LPS (5 mg/L)+rosiglitazone (10 μmol/L)+GW9662 (3 μmol/L, peroxisome proliferator-aetivatcd receptor γ antagonist), and LPS (5 mg/L)+vehicle (DMSO 0.2 ml/L). The expressions of CD40 and ICAM-1 RNA in RPMCs were examined by RT-PCR after 3 hour treatment, and the protein expressions of CD40, ICAM-1, p-NF-κB p65 and p-IκBα were examined by Western blot or immunofluorescence after 24 hour treatment. Results Following treatment with LPS, both the expressions of CD40 and ICAM-1 protein in RPMCs were up-regulated significantly (P<0.05), and the phosphoralation of p65 was increased greatly (1.10±0.17 vs 0.55±0.06, P<0.05). BAY11-7085 (5 μmol/L) significantly decreased the protein expression of p-p65 (0.22±0.11 vs 1.10±0.17, P<0.01), CD40 (0.34±0.02 vs 0.50±0.06, P<0.05) and ICAM-1 (0.35±0.16 vs 0.74±0.03, P<0.05). Pretreated with rosiglitazone for 3 h then added with LPS for 1 h, the levels of p-p65, CD40 and ICAM-1 in RPMCs were significantly decreased compared with those of LPS group (0.77±0.08 vs 0.90±0.10, P<O.01; 0.79±0.16 vs 0.99±0.06, P<0.05; 0.83±0.20 vs 1.22±0.13, P<0.05). However, for the group pretreated with rosiglitazone(10 μmol/L) and GW9662(3 μmol/L) for 3 h then added with LPS, the levels of p-p65 in RPMCs did not change significantly compared with those of rosiglitazone-pretreated group. The expressions of CD40 and ICAM-1 in RPMCs were significantly increased compared with those of rosiglitazone-pretreated group (0.95±0.19 vs 0.79±0.16, and 1.04±0.24 vs 0.83±0.20, P<0.05). Conclusion Rosiglitazone can decrease LPS-induced expression of CD40 and ICAM-1 in RPMCs by inhibition of NF-κB activation, which suggests that rosiglitazone may mediate its antiinflammatory effect through a NF-κB dependent mechanism.