中文摘要：

目的：研究Toll样受体4（TLR4）在脂多糖（LPS）诱导肾小管上皮细胞炎症反应中的作用及其可能的分子机制。方法：雄性SD大鼠8只,随机分为假手术组（Sham组）和盲肠结扎穿孔组（CLP组）,各4只。CLP组大鼠采用盲肠结扎穿孔法建立脓毒血症动物模型,Sham组除不行盲肠结扎穿孔外,其余处理同脓毒血症组,造模后24h处死大鼠取肾组织。体外培养大鼠肾小管上皮细胞（NRK-52E）,以1μg/ml LPS刺激不同时间（5,15,30,60min）,采用免疫荧光、Western blot法检测大鼠肾组织及肾小管上皮细胞TLR4的表达及分布,Western blot法检测细胞丝裂原活化蛋白激酶（MAPK）通路（包含p38、JNK两条主要通路）及Iκ-B磷酸化情况。TLR4抑制剂TAK-242（5mol/L）及MAPK通路抑制剂SB202190（10μmol/L）、SP600125（25μmol/L）预处理NRK-52E后,分别检测TLR4蛋白表达、MAPK信号蛋白及I-κB磷酸化水平。结果：1免疫荧光结果显示TLR4主要表达在大鼠肾小管上皮细胞;正常肾小管上皮细胞TLR4主要分布于细胞质。2Western blot结果显示随LPS刺激时间的增加,肾小管上皮细胞TLR4表达显著升高（P〈0.05）。3NRK-52E细胞在受到LPS刺激后其p38、JNK MAPK蛋白及I-κB蛋白磷酸化水平增加（P〈0.05）。4TLR4抑制剂预处理NRK-52E细胞,可显著降低LPS诱导的p38、JNK MAPK信号通路活化及I-κB磷酸化水平（P〈0.05）。5SB202190、SP600125分别预处理NRK-52E细胞,均可显著降低LPS诱导的I-κB磷酸化（P〈0.05）。结论：TLR4通过p38、JNK MAPK信号通路介导LPS诱导的肾小管上皮细胞炎症反应。

英文摘要：

Objective：To investigate the role of Toll-like receptor 4（TLR4）in lipopolysaccharide-induced inflammatory response of NRK-52 E.Methods：Eight male Sprague-Dawley rats were randomly divided into sham operation group（Sham group）and cecal ligation and puncture group（CLP group）（4rats in each group）.The sepsis rat model was established by cecal ligation and puncture in CLP group,and the Sham group didn＇t undergo cecal ligation and puncture processing.The changes kidney structure of the two groups were observed at 24 hours after operation.The NRK-52 Ecells were exposed to 1μg/ml LPS for variable incubation time.The expression of TLR4 was analyzed by immunofluorescence and Western Blot,and Western Blot was also used todetect the p38,JNK MAPK and Iκ-B phosphorylation.TLR4inhibitor（5μmol/L TAK-242）and MAPK pathway inhibitors（10μmol/L SB202190,25μmol/L SP600125）were further introduced to investigate the expression of TLR4 and MAPK signaling proteins and Iκ-B in the process.Results：1TLR4 was localized in renal tubular epithelial cells and mainly distributed in the cytoplasm.2Exposure to LPS stimulated TLR4 expression in a time-dependent manner（P〈0.05）.3The phosphorylation of p38,JNK,Iκ-B was elevated with the stimulating of LPS（P〈0.05）.4Pretreatment with TAK-242 dramatically prevented LPS-induced phosphorylation of p38,JNK and Iκ-B（P〈0.05）.5Pretreatment with SB202190 and SP600125dramatically prevented LPSpromoted phosphorylation of Iκ-B（P〈0.05）.Conclusion：TLR4contributes to LPS-induced inflammatory response of NRK-52 Eby interacting with the p38,JNK MAPK signaling protein.