乌司他丁负向调控EMT过程对大鼠晚期放射性肺损伤的影响

The effects of ulinastatin on advanced radiation-induced lung injury in rats by negatively regulating the EMT process

目的观察乌司他丁对大鼠晚期放射性肺损伤的影响,并探讨其可能的分子机制。方法根据随机数字表法将48只健康雌性SD大鼠分为对照组、模型组和乌司他丁组,每组16只。模型组和乌司他丁组均接受全胸6MV-X射线单次照射20 Gy,制造放射性肺损伤模型,对照组不做处理。照射后第1天,分别开始对3组大鼠进行药物干预。乌司他丁组乌司他丁尾静脉注射(10万U/kg,1次/d),对照组和模型组同等体积生理盐水尾静脉注射(1次/d)。8周后留取肺组织,Masson染色观察肺组织胶原纤维增生情况,碱水法测定大鼠肺组织羟脯氨酸含量,qRT-PCR和Western blot测定转化生长因子β1(TGF-β1)及上皮间充质转化(EMT)相关表型如E-钙黏蛋白(E-cadherin)、β-链蛋白(β-catenin)、波形蛋白(Vimentin)、α-平滑肌肌动蛋白(α-SMA)和锌指转录因子(Snail)的表达水平。结果 Masson染色结果示乌司他丁组大鼠肺胶原纤维含量较模型组显著减轻,肺组织羟脯氨酸含量明显降低(P<0.05)。与模型组相比,乌司他丁组肺组织TGF-β1表达水平显著降低(P<0.05),上皮细胞表型E-cadherin和β-catenin显著上升(P<0.05),间质细胞表型Vimentin、α-SMA和Snail表达水平降低(P<0.05);乌司他丁组与对照组相关基因表达无明显差异(P>0.05)。结论乌司他丁可通过干扰TGF-β1的表达抑制EMT过程的发生,延缓胸部放射性损伤后大鼠肺组织纤维化改变,为临床防治晚期放射性肺损伤提供一定的理论基础。

Objective To observe the effects of ulinastatin on advanced radiation - induced lung injury in rats and to investigate its possible mechanism. Methods Forty - eight healthy female SD rats were randomly divided into control group, model group and ulinastatin group according to random number table method. Radiation - induced lung in- jury rat models were established with whole - thorax irradiation of 6MV - X - ray by 20 Gy in model group and ulinastatin group. On the first day after irradiation, the rats in the ulinastatin group were treated with ulinastatin （100,000 U/Kg, 1 time/d） ; in control group and model group were given with the same volume normal saline. After 8 weeks, the lung tis- sues were taken and Masson staining was used to observe the proliferation of collagen fibers in lung tissues. The content of hydroxyproline in rat lung tissue was determined by alkaline method. The expression of TGF - β1 and EMT - related phe- notypes such as E -cadherin, β -catenin, Vimentin, α-SMA and Snail were assessed by qRT -PCR and Western blot. Results The results of Masson staining showed that the content of collagen fibers ulinastatin group was significantly lower than that in model group, so was the content of hydroxyproline （P 〈 0. 05）. Compared with model group, the expression of TGF - β1 in ulinaxatin group was significantly lower （ P 〈 0. 05 ）, with significantly up - regulation of E - cadherin and β- catenin （ P 〈 0. 05 ） and down - regulation of Vimentin, ct - SMA and Snail decreased （ P 〈 0. 05 ）. There was no sig- nificant difference in the expression of related genes between ulinastatin group and control group （P 〉 0. 05 ）. Conclusion Ulinastatin can inhibit the occurrence of EMT by interfering with the expression of TGF - β1, delay the changes of lung tissue fibrosis after thoracic radiation injury, and provide a theoretical basis for clinical prevention and treatment of advanced radioactive lung injury.