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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。
AIM: To investigate the role of Toll-like receptor 4 (TLR4) in the inflammatory response of tubular epithelial cells induced by lipopolysaccharide (LPS) and its possible molecular mechanism. Methods: Eight male Sprague-Dawley rats were randomly divided into sham operation group (Sham group) and cecal ligation and puncture group (CLP group), 4 rats in each group. CLP group rats were established by cecal ligation and perforation animal model of sepsis, Sham group without cecal ligation and puncture, the other with the same sepsis group, rats were sacrificed 24 hours after modeling of kidney tissue. The rat renal tubular epithelial cells (NRK-52E) were cultured in vitro and stimulated with 1μg / ml LPS for different time (5, 15, 30, 60min). Immunofluorescence and Western blot were used to detect the expression of TLR4 Western blot was used to detect the mitogen-activated protein kinase (MAPK) pathway (including p38 and JNK major pathways) and IκB phosphorylation. NRK-52E cells were pretreated with TAK-242 (5mol / L) and SB202190 (10μmol / L) and SP600125 (25μmol / L) for 24 hours. The expressions of TLR4 protein, MAPK signal protein and I- Level Results: 1 Immunofluorescence results showed that TLR4 mainly expressed in rat renal tubular epithelial cells; normal renal tubular epithelial cells TLR4 mainly distributed in the cytoplasm. 2Western blot results showed that the expression of TLR4 in renal tubular epithelial cells was significantly increased with the increase of LPS stimulation time (P <0.05). The phosphorylation of p38, JNK MAPK and I-κB protein in 3NRK-52E cells increased (P <0.05) after stimulated by LPS. 4TLR4 inhibitor pretreatment NRK-52E cells can significantly reduce LPS-induced activation of p38, JNK MAPK signaling pathway and I-κB phosphorylation (P <0.05). 5SB202190, SP600125 pretreatment NRK-52E cells were significantly reduced LPS induced I-κB phosphorylation (P <0.05). CONCLUSION: TLR4 mediates LPS-induced tubular epithelial cell inflammation through p38 and JNK MAPK signaling pathways.