罗格列酮对脂多糖诱导肾小管上皮细胞趋化因子分泌的影响及可能机制  

Effect of rosiglitazone on the secretion of chemokines in renal tubular epithelial cells stimulated by lipopolysaccharide and the possible mechanism

作　　者：卢颖[1] 周桥[1] 仲芳[1] 郝旭[1] 李聪[1] 王伟铭[1] 陈楠[1] 

机构地区：[1]上海交通大学医学院瑞金医院肾脏科,200025

出　　处：《中华肾脏病杂志》2010年第12期909-914,共6页Chinese Journal of Nephrology

基　　金：国家自然科学基金（30270613,30771000）;上海市科委项目（08dz1900502,07JC14037）

摘　　要：目的 探讨罗格列酮（RGL）对脂多糖（LPS）诱导人近端肾小管上皮细胞（HK-2）趋化因子分泌的影响及可能机制.方法 用RGL（10 μmol/L）预处理HK-2细胞2 h后加入LPS（1 mg/L）,与单纯LPS组、单纯RGL组及未加任何刺激（CON）组进行比较.用实时定量PCR方法 检测细胞中白细胞介素8（IL-8）和单核细胞趋化蛋白1（MCP-1）mRNA表达水平;用ELISA法检测细胞培养上清中IL-8和MCP-1蛋白水平.通过RNAi技术沉默肾小管上皮细胞过氧化物酶体增殖蛋白激活性受体γ（PPARγ）,观察RGL的作用是否依赖于PPARγ.用 Western印迹法检测细胞核中NF-κB蛋白水平;用EMSA方法 检测NF-κB DNA结合活性.结果 在HK-2细胞中,与CON组相比,单纯LPS刺激使IL-8、MCP-1 mRNA分别升高（4.30±0.45）倍和（4.80±1.29）倍（均P＜0.05）,使培养上清中IL-8、MCP-1分别升高（1.39±0.18）和（2.11±0.47）倍（均P＜0.05）;与LPS组相比,RGL预处理组IL-8和MCP-1在mRNA水平分别下降66.37%和71.88%（均P＜0.05）,在蛋白水平分别下降41.68%和47.87%（均P＜0.05）.在PPARγ沉默的HK-2细胞中,RGL预处理2 h后再加LPS刺激,IL-8和MCP-1mRNA仅分别下降18.16%、16.83%,培养上清中IL-8和MCP-1仅分别下降11.39%、11.86%,与单纯LPS组相比差异无统计学意义.RGL预处理2 h不能抑制LPS诱导的NF-κB核易位,但可显著降低NF-κB DNA结合活性.结论 RGL以PPARγ依赖的方式抑制LPS诱导HK-2细胞分泌IL-8和MCP-1,其机制可能与降低NF-κB DNA结合活性有关.Objective To investigate the inhibitory effect and mechanism of rosiglitazone on chemokines secretion in renal tubular epithelial cells （HK-2） stimulated by lipopolysaccharide （LPS）. Methods Cells were divided into four groups： control （CON）, LPS （1 mg/L）,rosiglitazone （10 μmol/L）, rosiglitazone （10 μmol/L） ＋LPS （1 mg/L）. MCP-1 and IL-8 expression was measured using real time PCR and ELISA. PPARγ was knockdown by RNAi to investigate whether the inhibitory effect of rosiglitazone was PPARγ-dependent or -independent. The NF-κB in nucleus was detected by Western blotting. The DNA binding activity of NF-κB was determined by electrophoretic mobility shift assay. Results Compared with CON group, the expressions of IL-8 and MCP-1 were increased by （4.30±0.45） and （4.80±1.29） times in mRNA level, （1.39±0.18）and （2.11 ±0.47） times in protein level, respectively, in LPS-stimulated HK-2 cells （P〈0.05）.Application of rosiglitazone followed by LPS significantly reduced IL-8 and MCP-1 secretion compared with LPS group （decreasing by 66.37% and 71.88% in mRNA levels, while 41.68% and 47.87% in protein levels） （P〈0.05）. In pcDNATM 6.2-GW/EmGFP-miPPARγ transfected cells, IL-8and MCP-1 only were decreased by 18.16% and 16.83% in mRNA level, while 11.39% and 11.86%% in protein level in rosiglitazone pretreated group, showing no significant difference compared with LPS group. Rosiglitazone did not block NF-κB nuclear translocation while significantly inhibiting the DNA binding activity of NF-κB. Conclusions Rosiglitazone inhibits the expressions of MCP-1 and IL-8 via a PPARγ-dependent mechanism in HK-2 cells, resulting from inhibition the DNA binding activity of NF-κB.

关 键 词：脂多糖类 白细胞介素8 趋化因子CCL2 PPA脚 罗格列酮 肾小管上皮细胞 NF—κB 

分 类 号：R692[医药卫生—泌尿科学]