CD14、NF-κBp65在脓毒症幼鼠急性肾损伤中的表达及乌司他丁对其干预的研究

The experimental study of expression of CD14 and NF-κBp65 in septic immature rats with acute kidney injury and the effect of ulinastatin on its intervention

目的探讨乌司他丁对脓毒症幼鼠急性肾损伤血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平及肾组织白细胞分化抗原14(CD14)、核转录因子-κBp65(NF-κBp65)表达的影响。方法采用盲肠结扎穿孔术(CLP)制备SD幼鼠脓毒症模型。健康清洁级4周龄雄性SD幼鼠128只,实验幼鼠随机分成正常对照组(A组)、假手术组(B组)、脓毒症组(C组)、乌司他丁干预组(D组),每组32只,每组幼鼠再随机分为6h、12h、24h、48h4个亚组(每组各8只)。D组在术后0h、24h经尾静脉注射UT I50mg/kg,其他3组分别注射等量生理盐水。每组幼鼠在4个时间点(6h、12h、24h、48h)被处死后采集血标本和肾组织标本。用酶联免疫吸附法检测血清TNF-α、IL-6水平,用逆转录-聚合酶链反应(RT-PCR)法测定肾组织CD14 mRNA表达和用免疫组织化学法检测肾组织NF-κBp65蛋白表达的水平;HE染色观察肾组织病理改变。结果与A、B组相比较,C、D组幼鼠各时间点血清TNF-α、IL-6水平及肾组织CD14 mRNA、NF-κBp65表达均显著升高,差异有统计学意义(P<0.05),各时间点肾脏组织中可见大量炎性细胞浸润;与C组相比较,D组幼鼠各时间点血清TNF-α、IL-6水平及肾组织CD14mRNA、NF-κBp65表达均明显下降,差异有统计学意义(P<0.05),肾脏组织的炎性病理变化亦明显减轻。结论乌司他丁通过下调肾组织CD14mRNA、NF-κBp65的表达,进而使TNF-α、IL-6等炎症因子的释放受到抑制,对脓毒症致急性肾损伤的早期起到保护作用。

Objective To explore the effect of ulinastatin（UTI） on expression of CD14 and NF-κBp65 in kidey tiusse and serum TNF-α and IL-6 of septic immature rats with acute kidney injury（AKI）. Methods Sepsis model was induced by cecal ligation and puncture（CLP） in Spraugue Dawley（SD） rats. A total of 128 clean 4-week-old healthy male SD rats were randomly divided into the normal control group（group A）,the sham group（group B）,the CLP group（group C） and the UTI treatment group（group D）,32 cases in every group. Each of all groups were respectively subdivided into 4 groups at 6,12,24 and 48 hours,8 rats in every group. The immature rats were injected with UTI（50mg/kg） via the tail vein at 0h,24 h after the group D operated and the immature rats in the other three groups were treated with equal physiological saline. The immature rats were separately killed at 6,12,24 and 48 hours after the operation and blood and renal tissues samples were collected. The contents of tumor necrosis factor-α（TNF-α） and interleukin-6（IL-6） was measured bv enzyme linked immunosorbent assay,and NF-κBp65 were investigated by immunohistochemical staining,meanwhile,the expression of CD14 mRNA in renal tissue was examined with reverse transcription-polymerase chain reaction（RT-PCR） way. Histological alterations of the renal tissue were observed in HE staining. Results Compared with group A and group B,concentration of serum TNF-α and IL-6,and expression of CD14 mRNA and NF-κBp65 of renal tissue in the group C and group D were increased significantly at different time points,and there were significant differences（P〈0.05）;Infiltration of inflammatory cells could be seen in renal tissue at each time point;Compared with group C,content of serum TNF-α and IL-6 as well as expression of CD14 mRNA and NF-κBp65 of renal tissue in the group D were decreased drastically at different time points,and there were significant differences（P〈0.05）;Inflammatory pathological changes of kidney tissue were reduced significantly. Conclusion UTI play early a protective effect on kidney tissues of septic immature rats with AKI,through reducing the expression of CD14 mRNA and NF-κBp65 and inhibiting the contents of TNF-α and IL-6.