NF-κB及下游通路与大鼠急性期放射性心肌纤维化相关性研究

Association of NF-κB and its downstream pathway with acute radiation-induced myocardial fibrosis in rats

目的 建立急性放射性心脏损伤大鼠模型,观察急性心肌组织炎症反应和纤维化等病理学表现,探讨NF-κB及其下游通路是否与心肌纤维化相关.方法 成年雄性SD大鼠14只,完全随机法均分为对照组和照射组.照射组采用6MVX线单次20 Gy经心前区照射构建放射性心脏损伤模型,照射后第14天应用HE染色观察心肌细胞及细胞间质形态学改变;Masson染色观察胶原纤维分布情况,并以CVF作半定量分析指标进行成组f检验.蛋白印记法及qPCR法分别检测大鼠心肌组织NF-κB基团成员p50、p65及其下游通路HIF-1d、CTGF、COL-1蛋白及mRNA表达量变化.结果 大鼠心脏局部照射后第14天,照射组较对照组心肌细胞水肿明显、排列紊乱,部分心肌细胞断裂、心肌细胞核轻度固缩、核染色加深,少量异形细胞核,心肌间质可见大量炎症细胞浸润、成纤维细胞增多.Masson染色显示胶原纤维广泛分布于心肌细胞间质,与对照组相比照射组大鼠心肌胶原明显增多,正常心肌细胞排列紊乱、有被胶原纤维替代的趋势.半定量分析结果显示照射组CVF明显高于对照组（22.05％：3.76％,P=0.003）.蛋白印记法及qPCR法结果显示心肌组织p50、p65、HIF-1 α、CTGF、COL-1蛋白表达及mRNA水平照射组较对照组均明显升高（P均＜0.05）.结论 急性放射性心脏损伤病理学表现为心肌细胞水肿、细胞间质大量炎症细胞浸润、成纤维细胞增多、胶原纤维增多.其损伤机制可能与心肌组织NF-κB激活有关,同时放射线可引起其下游通路HIF-1α、CTGF在蛋白和基因水平表达上调,可能在放射性心肌炎症向纤维化发展过程中起到重要作用.

Objective To examine the pathological changes in the myocardial tissues such as inflammatory response and fibrosis in a rat model of acute radiation-induced heart damage （RIHD）,and to explore whether NF-κB and its downstream pathway are associated with acute radiation-induced myocardial fibrosis.Methods Fourteen nale adult Sprague-Dawley rats were randomly divided into control group and radiation group.Local heart irradiation was delivered to the precordial region of rats to establish an RIHD model in a single fraction with a dose of 20 Gy generated by a 6 MV linear accelerator.At 14 days after irradiation,the histopathological changes in myocardial and interstitial tissues were examined by HE staining;the distribution of collagen fibers was observed by Masson staining,and collagen volume fraction （CVF） was used as a semi-quantitative evaluation for myocardial collagen deposition,which was defined as the percentage of collagen area occupied in total area,and was compared using the independent-samples t test.The protein and mRNA expression levels of the NF-κB members p50 and p65 and the downstream pathway members hypoxia-inducible factor 1α（HIF-1o）,connective tissue growth factor （CTGF）,and type I （COL-1） were quantitatively analyzed by Western blot and qPCR,respectively.Results At 14 days after local heart irradiation,the radiation group showed significant myocardial edema and derangement,rupturc of some myocardial ceils,mild nuclear pyknosis,darkened nuclear staining,a small number of irregular nuclei,and myocardial interstitial inflammatory cell infiltration accompanied by increased fibroblast,as compared with the control group.The Masson staining showed that the collagen fibers in radiation group were widely distributed at the interstitial tissue and increased significantly compared with those in the control group;normal myocardial cells were in disordered array and had a tendency to be replaced by collagen fibers.The semi-quantitative analysis showed that radiation induced a significant increase in CVF （22.05％ vs.3.76％,P =0.003）.Western blot and qPCR revealed that the protein and mRNA expression of p50,p65,HIF-1 α,CTGF,and COL-1 was significantly higher in the radiation group than in the control group （all P ＜ 0.05）.Conclusions The pathological features of acute RIHD include significant myocardial edema and myocardial interstitial inflammatory cell infiltration accompanied by increased fibroblasts and collagen fibers.Radiation exposure can activate NF-κB and cause the upregulation of HIF-1α and CTGF at both protein and mRNA levels,which may play an important role in the progression of radiation-induced myocardial inflammation to fibrosis.