冯杰+++++佟小雅++++++俞佳丽++++++潘祉谕+++++查艳

[摘要]目的 探讨脂多糖（LPS）诱导的急性肾损伤中低氧诱导因子1α（HIF-1α）和血管内皮生长因子（VEGF）的表达及褪黑素（Mel）对其表达的影响。方法 选取3～5周龄的30只Sprague-Dawley（SD）大鼠作为研究对象，随机分为对照组（n=10）、LPS组（n=10，静脉注射500 μg/kg LPS）、Mel+LPS组（n=10，注射LPS之前15 min给予Mel）。48 h后采用TBA法检测肾组织的丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性，分别用RT-qPCR和免疫组化检测HIF-1α、VEGF mRNA和蛋白表达水平。结果 LPS组肾小管上皮细胞肿胀、肾间质水肿，有炎细胞浸润；Mel+LPS组的上述表现明显减轻。LPS组血清BUN和Scr含量[（35.56±1.83）mmol/L和（46.23±2.87）μmol/L]显著高于对照组[（12.69±0.97）mmol/L和（16.73±1.26）μmol/L]以及Mel+LPS组[（23.17±1.33）mmol/L和（35.67±2.25）μmol/L]（P<0.05）。LPS组小鼠肾组织的MDA水平[（697.45±43.13）nmol/（mg·prot）]与对照组[（354.35±28.12）nmol/（mg·prot）]比较急剧增高（P<0.05），Mel+LPS组的MDA[（455.37±30.79）nmol/（mg·prot）]与LPS组比较则明显降低（P<0.05）。LPS组小鼠肾组织的SOD水平[（3.75±0.23）U/（mg·prot）]与对照组[（2.93±0.29）U/（mg·prot）]比较急剧降低（P<0.05），Mel+LPS组的SOD水平[（3.20±0.27）U/（mg·prot）]与LPS组比较明显增高（P<0.05）。LPS组小鼠肾组织的HIF-1α、VEGF mRNA和蛋白水平与对照组相比急剧增高（P<0.05），Mel+LPS组与LPS组比较明显降低（P<0.05）。结论 Mel通过抑制MDA的产生和上调SOD的活性，调控HIF-1α和VEGF的表达，起到了保护LPS诱导的急性肾损伤（AKI）肾脏功能的作用。

[关键词]褪黑素；肾损伤低氧诱导因子1α；血管内皮生长因子

[中图分类号] R-332 [文献标识码] A [文章编号] 1674-4721（2016）10（b）-0012-04

[Abstract]Objective To investigate the expression and influence of melatonin on hypoxia-inducible factor （HIF-1α） and vascular endothelial growth factor （VEGF） in acute kidney injury induced by lipopolysaccharide （LPS）.Methods 30 Sprague-Dawly（3-5 weeks） rats were selected and randomly divided into the control group （n=10），the LPS group （n=10，500 μg/kg LPS was injected intravenously） and the Mel+LPS group （n=10，melatonin was administrated orally，15 min later，LPS was given），followed by 48 hours，the rats were killed.The thiobarbituric acid （TBA） test was given to detect the level of MDA and the activity of superoxide dismutase （SOD）.The mRNAs and proteins of HIF-1α and VEGF were determined by RT-qPCR and immunohistochemistry.Results There was renal tubular epithelial cells swelling，renal interstitial edema and inflammation in the LPS group，those symptoms was attenuant in Mel+LSP group.The level of Scr and BUN in the LPS group [（35.56±1.83）mmol/L and （46.23±2.87）μmol/L] was higher than that in the control group [（12.69±0.97）mmol/L and （16.73±1.26）μmol/L] and the Mel+LPS group [（23.17±1.33）mmol/L and （35.67±2.25）μmol/L] （P<0.05）.The level of MDA in the LPS group [（697.45±43.13）nmol/（mg·prot）] was higher than that in the control group [（354.35±28.12）nmol/（mg·prot）] and the level of MDA in the Mel+LPS group [（455.37±30.79）nmol/（mg·prot）] was lower than that in the LPS group （P<0.05）.The level of SOD in the LPS group [（3.75±0.23）U/（mg·prot）] was lower than that in the control group [（2.93±0.29）U/（mg·prot）] and Mel+LPS group[（3.20±0.27）U/（mg·prot）] （P<0.05）.The level of HIF-1α and VEGF mRNA and protein in the LPS group was higher than that in the control group，and less than that in the Mel+LPS group.Conclusion By inhibiting the production of MDA and up regulating the activity of SOD，Mel can regulate the expression of VEGF and HIF-1α，and plays a role in protecting LPS induced AKI renal function.

[Key words]Melatonin；Renal injury hypoxia inducible factor-1α；Vascular endothelial growth factor

内毒素血症（endotoxemia，ETM）可出现在儿科多系统的多种疾病中，一旦并发肾损伤常难以救治。缺氧诱导因子-1（hypoxia inducible factor-1α，HIF-1α）在多种肾损伤的动物模型中具有肾脏保护作用，其在肾损伤发病过程中的作用在临床研究中也备受关注。血管内皮生长因子（vascular endothelial growth factor，VEGF）是HIF-1α最重要的靶基因之一，在缺氧情况下可被HIF-1α迅速诱导活化。褪黑素（melatonin，Mel）是松果体分泌的吲哚类神经内分泌激素，目前临床上主要用于内分泌和肿瘤等其他疾病的治疗。近年来的研究表明，Mel对ETM肾损伤具有保护作用。本实验通过观察ETM幼年大鼠肾组织中HIF-1α、VEGF的表达，探讨Mel干预对ETM肾损伤的作用及其对HIF-1α介导的VEGF表达的影响。

1材料与方法

1.1材料

血清肌酐（serum creatinine，Scr）、血尿素氮（blood urea nitrogen，BUN）、超氧化物歧化酶（superoxide dismutase，SOD）活性、丙二醛（malondialdehyde，MDA）检测试剂购自Cell Signaling Technology，脂多糖（lipopo-lysaccharide，LPS）购至Sigma， Mel购自Sigma-Aldrich，兔抗HIF-1α抗体、兔抗VEGF抗体和兔抗β-actin抗体购自Abcam，HRP标记的山羊抗兔IgG购自博兴生物，DAB辣根过氧化物酶显色试剂盒购自碧云天，RT-qPCR试剂盒购自Thermo Fisher Scientific。……