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目的观察参附(Shenfu,SF)注射液预处理对大鼠肝移植缺血再灌注后Kupffer细胞中糖皮质激素受体(glucocorticoid receptor,GR)、核转录因子κB(NF-κB)表达的影响,探讨参附预处理保护缺血再灌注损害(ischemia reperfu-sion injury,IRI)的机制。方法将雄性SD大鼠按完全随机分组法分为正常对照组、缺血再灌注组和SF组,其中缺血再灌注组、SF组建立肝移植缺血再灌注模型,于缺血再灌注后6 h分离培养受体移植肝脏的Kupffer细胞。通过RT-PCR、Westernblot和激光共聚焦显微镜技术、免疫荧光法、ELISA等实验技术检测Kupffer细胞GR的mRNA和蛋白表达、NF-κB的蛋白表达、培养上清中TNF-α的含量。结果缺血再灌注组Kupffer细胞GR mRNA和蛋白表达均明显低于正常对照组(P<0.01),缺血再灌注组NF-κB、培养上清TNF-α表达量(830.19±71.34)明显高于正常对照组NF-κB、培养上清TNF-α表达量(65.41±37.63,P<0.01)。与缺血再灌注组相比,SF组GR水平明显上升(P<0.01),NF-κB活性、TNF-α表达量(543.59±41.27)明显下降(P<0.01)。结论缺血再灌注损伤会导致Kupffer细胞GR的表达下调,NF-κB的激活;参附预处理可以改善肝移植缺血再灌注后Kupffer细胞的激活。
Objective To observe the effects of Shenfu (SF) injection on the expression of glucocorticoid receptor (GR) and nuclear factor kappa B (NF-κB) in Kupffer cells after liver ischemia-reperfusion in rat liver transplantation To explore the mechanism of Shenfu preconditioning in protecting ischemia reperfu-sion injury (IRI). Methods Male Sprague-Dawley rats were randomly divided into normal control group, ischemia reperfusion group and SF group. The ischemia-reperfusion group and SF group were given ischemia-reperfusion injury model. After ischemia-reperfusion Six hours later, Kupffer cells in recipient liver were isolated and cultured. The mRNA and protein expression of GR in Kupffer cells, the protein expression of NF-κB and the content of TNF-α in the culture supernatant were detected by RT-PCR, Western blot and laser confocal microscopy, immunofluorescence and ELISA. Results The expression of GR mRNA and protein of Kupffer cells in ischemia-reperfusion group was significantly lower than that in normal control group (P <0.01). The expression of NF-κB in ischemic-reperfusion group and the level of TNF-α in culture supernatant (830.19 ± 71.34) In the normal control group, the expression of TNF-α in the supernatant of NF-κB (65.41 ± 37.63, P <0.01). The level of GR in SF group was significantly higher than that in ischemia / reperfusion group (P <0.01). The activity of NF-κB and the level of TNF-α in serum were significantly decreased (P <0.01). Conclusion Ischemia-reperfusion injury can lead to the down-regulation of GR expression and the activation of NF-κB in Kupffer cells. The pretreatment with Shenfu can improve the activation of Kupffer cells after ischemia-reperfusion injury.