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目的研究乌司他丁对脓毒症大鼠血管内皮细胞(VEC)损伤保护作用及机制研究。方法 40只SD雄性大鼠按体重随机分为4组:假手术组,模型组及2个剂量实验组(乌司他丁,10×10~4,20×10~4U·kg~(-1)),其中模型组及实验组应用盲肠结扎穿孔(CLP)构建脓毒症大鼠模型。免疫酶联吸附法(ELISA)检测血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)及IL-6含量;试剂盒法检测血清一氧化氮(NO)及诱导型一氧化氮合酶(iNOS)含量;流式细胞术检测VEC凋亡情况;分光光度法检测血清中含半胱氨酸的天冬氨酸水解酶3(Caspase 3)活性;免疫印迹法分析各组B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)含量变化及核因子-κB(NF-κB)信号通路的激活状况。结果与假手术组比较,模型组中TNF-α、I L-1β、IL-6、NO、iNOS含量与VEC凋亡率及Caspase 3的活性均显著提高,Bax表达量明显上升,而Bcl-2表达量明显下降,NF-κB p65及IκBα磷酸化水平明显提高(均P<0.01);与模型组比较,2个剂量实验组皆能显著抑制上述指标的变化(P<0.05)。其中Caspase 3活性比值为(1.00±0.21,3.70±0.25,1.83±0.14,1.53±0.13)。结论乌司他丁对脓毒症大鼠VEC损伤具有保护作用,这可能与NF-κB信号通路有关。
Objective To study the protective effect and mechanism of ulinastatin on vascular endothelial cells (VEC) injury in septic rats. Methods 40 SD male rats were randomly divided into 4 groups according to body weight: sham-operation group, model group and 2 dose groups (ulinastatin, 10 × 10-4, 20 × 10-4 U · kg -1 )), In which the model group and experimental group using cecal ligation and puncture (CLP) to construct septic rat model. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 were detected by enzyme linked immunosorbent assay (ELISA); serum nitric oxide (NO) The content of nitric oxide synthase (iNOS) was detected by flow cytometry. The apoptosis of VEC was detected by flow cytometry. The activity of caspase 3 in serum was detected by spectrophotometry. The changes of Bcl-2, Bcl-2 and Bax in each group and the activation of nuclear factor-kappa B signal pathway were observed. Results Compared with the sham operation group, the contents of TNF-α, IL-1β, IL-6, NO and iNOS, the apoptosis rate of VEC and the activity of Caspase 3 in model group were significantly increased, while the expression of Bax was significantly increased (P <0.01). Compared with the model group, the two experimental groups were able to significantly inhibit the changes of these indexes (P <0.05). The activity ratio of Caspase 3 was (1.00 ± 0.21, 3.70 ± 0.25, 1.83 ± 0.14, 1.53 ± 0.13). Conclusion Ulinastatin has a protective effect on VEC injury in sepsis rats, which may be related to NF-κB signaling pathway.