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（5mg／L）组、BAY11-7085（NF-κB抑制剂）组（5μmol／L预刺激3h后加入LPS作用3h）、不同浓度罗格列酮（过氧化物酶体增殖蛋白激活性受体1配体）组（10、20μmol／L分别预处理3h再加入LPS5mg／L）、GW9662（过氧化物酶体增殖蛋白激活性受体v拮抗剂）预处理组（预处理3h后加入罗格列酮10μmol／L，3h后再加入LPS5mg／L）和溶媒对照组。加入LPS后1h收集细胞检测核因子κB（NF—κB）p65水平；3h收集细胞分别检测CD40和ICAM-1基囚表达；24h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1h时腹膜间皮细胞磷酸化NF-κBp65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-κBp65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-κBp65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-κBp65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。
Objective To investigate the effect and mechanism by which PPARγ ligand, rosiglitazone, 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 （10μmol/L or 20μmol/L, peroxisome proliferator-activated receptor γ activator）, LPS （5 mg/L）±rosiglitazone （10 μmol/L）± GW9662 （3 μmol/L, peroxisome proliferator-activated receptor γ antagonist）, and LPS （5 rag/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）. BAYll-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〈0.O1; 0.79±0.16 vs 0.99±0.06, P〈0.05; 0.83±0.20 vs 1.22±0.13, P〈O.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-KB activation, which suggests that rosiglitazone may mediate its antiinflammatory effect through a NF-κB dependent mechanism.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2009年第6期430-436,共7页
Chinese Journal of Nephrology
关键词
NF-κB
脂多糖类
抗原
CD40
胞问黏附分子1
罗格列酮
腹膜间皮细胞
NF-kappa B
Lipopolysaccharides
Antigens, CD40
Intercellular adhesion molecule 1
Rosiglitazone
Peritoneal mesothelial cells
