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目的:应用非选择性β肾上腺素受体激动剂——异丙基肾上腺素(isoproterenol,ISO)连续皮下注射7 d诱导心脏纤维化模型,并观察此模型中炎症调控核转录因子NF-κB的变化。方法:采用10周龄体重为280~320 g的SD大鼠,ISO以0.25 mg/(kg·d)剂量连续背部皮下注射7 d诱导心脏纤维化模型。心脏组织切片进行苦味酸-天狼猩红染色观察纤维化,以胶原容量分数进行胶原面积定量统计。应用real-time PCR法检测心脏组织中Ⅰ型/Ⅲ型胶原及炎症因子IL-6的mRNA表达。组织切片进行HE染色观察心脏病理变化。免疫组织化学方法检测心脏组织中NF-κB在细胞内的定位,Western blot法检测心脏组织中磷酸化的NF-κB水平。结果:ISO诱导的心脏纤维化模型中天狼猩红染色面积明显大于对照(control,CON)组,且ISO组的胶原容积分数显著高于CON组(12.01±1.64 vs.0.95±0.06,P<0.001);ISO组Ⅰ、Ⅲ型胶原的mRNA表达显著高于CON组(Ⅰ型胶原:10.51±0.47 vs.0.98±0.02,P<0.001;Ⅲ型胶原:9.58±1.33 vs.1.02±0.02,P<0.001)。ISO组心脏组织中可见细胞核增多、聚集,提示可能炎症细胞浸润,且炎症因子IL-6的mRNA表达水平明显增加(1.64±0.18 vs.1.04±0.07,P<0.01)。CON组NF-κB主要定位于心肌细胞胞浆中,心肌细胞核中未见NF-κB的定位;ISO组出现明显NF-κB核转位活化,心肌细胞胞浆中的NF-κB较CON组减少,心肌细胞核及间质细胞核均可见明显的NF-κB定位,同时,ISO组心脏组织磷酸化NF-κB的水平明显高于CON组(10.83±2.05 vs.1.05±0.27,P<0.001)。结论:连续7 d皮下注射ISO可成功诱导心脏纤维化模型,且此模型心脏组织中活化的核转录因子NF-κB增加。
OBJECTIVE: To establish a rat model of cardiac fibrosis induced by continuous subcutaneous injection of isoproterenol (ISO), a non-selective β-adrenoreceptor agonist, for 7 days. The inflammatory regulatory NF-κB Variety. Methods: 10-week-old SD rats weighing 280-320 g were used. ISO was injected subcutaneously at the dose of 0.25 mg / (kg · d) for 7 days to induce cardiac fibrosis model. Cardiac tissue sections were subjected to picric acid-Sirius red staining to observe the fibrosis, and the collagen area fraction was used for quantitative statistics. Real-time PCR was used to detect the mRNA expression of type I / type III collagen and inflammatory cytokine IL-6 in heart tissue. Tissue sections were stained with HE to observe the changes of heart pathology. Immunohistochemistry was used to detect the localization of NF-κB in the heart tissue. The level of phosphorylated NF-κB in the heart tissue was detected by Western blot. RESULTS: The Sirius red stained area in ISO-induced cardiac fibrosis model was significantly larger than control (CON) group, and the collagen volume fraction in ISO group was significantly higher than that in CON group (12.01 ± 1.64 vs.0.95 ± 0.06, P <0.001 ). The mRNA expression of type I and type III collagen in ISO group was significantly higher than that in CON group (type I collagen: 10.51 ± 0.47 vs.0.98 ± 0.02, P <0.001; type Ⅲ collagen: 9.58 ± 1.33 vs 1.02 ± 0.02, P < 0.001). In the ISO group, the nuclei increased and aggregated, suggesting the possibility of infiltration of inflammatory cells, and the mRNA expression of IL-6 was significantly increased (1.64 ± 0.18 vs.1.04 ± 0.07, P <0.01). NF-κB in CON group was mainly located in the cytoplasm of cardiomyocytes, and there was no NF-κB localization in the nucleus of cardiomyocytes. Significant NF-κB nuclear translocation was found in the ISO group and decreased in the cytoplasm of cardiomyocytes (P <0.001). The phosphorylation of NF-κB in cardiac tissue of ISO group was significantly higher than that of CON group (10.83 ± 2.05 vs.1.05 ± 0.27, P <0.001). CONCLUSION: The model of cardiac fibrosis induced by subcutaneous injection of ISO for 7 days can be successfully induced, and the activation of NF-κB in cardiac tissue increased in this model.