脓毒症中血小板活化对肾小管上皮影响的机制研究

目的:探讨脓毒症中血小板活化对肾小管上皮影响的机制。方法: ELISA法检测血小板分泌的炎症因子白介素（IL）-1β和IL-6，应用共培养体系将血小板和肾小管上皮细胞共培养，Western blot检测下室肾小管上皮细胞的p65和p-p65蛋白，qRT-PCR方法检测肾小管上皮细胞中IL-1β和IL-6的mRNA表达水平。构建盲肠结扎穿孔的脓毒症模型，尾静脉注射TLR4特异性抑制剂TAK-242，EdU检测肾小管增殖情况，肾组织石蜡包埋H&E染色后观察脓毒症肾损伤的程度，并检测血清肌酐含量。结果:不同浓度脂多糖（LPS）刺激血小板后，血小板分泌的细胞因子IL-1β、IL-6增加；血小板和肾小管上皮细胞共培养体系中，p-p65蛋白含量增加，TAK-242抑制血小板活化，减少肾小管上皮细胞炎症因子分泌；构建小鼠盲肠结扎穿孔的脓毒症模型后，尾静脉注射TAK-242，抑制血小板表面TLR4的激活，肾小管增殖能力和病理改善，血清肌酐降低。结论:脓毒症患者中血小板聚集并激活，分泌炎症因子增加，激活肾小管上皮细胞NF-κB信号通路，导致肾损伤，TLR4抑制剂可改善肾损伤，有潜在临床应用价值。

Effects of platelet activation on renal tubular epithelial cells in sepsis

AIM: To investigate the effect of platelet activation on renal tubular epithelial cells in sepsis. METHODS: ELISA was used to detect the inflammatory cytokines IL-1β and IL-6 secreted by platelets. Renal tubular epithelial cells and platelets were co-cultured in the co-culture system. Western blot was used to test p65 and p-p65 protein in renal tubular epithelial cells in the lower chamber. IL-1β and IL-6 mRNA expression level was measured by qRT-PCR. Cecal ligation and puncture(CLP) were used to construct the sepsis model, and TLR4 inhibitor TAK-242 was injected in caudal vein. In order to observe injury degree of renal tissue, EdU experiment and serum creatinine detection were carried out. RESULTS: IL-1β and IL-6 cytokines secreted by active platelets were increased in the presence of different concentrations of LPS. In the platelet and renal tubular epithelial cells co-culture system, p-p65 protein was increased in the tubular epithelial cells. TAK-242 could inhibit platelet activation, and reduce the renal tubular epithelial cell inflammatory cytokines secretion.The proliferation and pathological status were improved, and serum creatinine was decreased. CONCLUSION: The platelets in septic patients are aggregated and activated. inflammatory cytokines are increased. These cytokines activate NF-κB signaling pathway of renal tubular epithelial cells, leading to renal injury. TLR4 inhibitors have potential clinical value to improve the renal injury in sepsis.