摘要
目的:探讨低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)激动剂罗沙司他改善小鼠肺缺血再灌注损伤(ischemia-reperfusion injury,IRI)的作用和机制。方法:本研究分为动物实验和细胞实验两部分。研究对象为6~8周雄性C57/BL6小鼠共36只。动物实验中,选取20只小鼠通过左肺动脉结扎构建缺血再灌注(ischemia reperfusion,I/R)模型,分为缺血再灌注0、1、2和4 h组(IR-0、1、2、4 h组,每组n=4),并设立对照组(sham组,n=4)。检测病肺组织中HIF-1α表达时空变化。随后选取16只小鼠随机分为对照组(sham组,n=4)、I/R模型组(I/R+DMSO组,n=4)、低剂量或高剂量罗沙司他(25 mg/kg或50 mg/kg)治疗组(I/R+ROX LD组,I/R+ROX HD组,每组n=4)。Sham组实施开胸假手术,I/R+DMSO组、I/R+ROX LD组和I/R+ROX HD组实施左肺动脉I/R术;于术前5 d每天分别注射25 mg/kg罗沙司他溶液(I/R+ROX LD)、50 mg/kg罗沙司他溶液(I/R+ROX HD)以及二甲基亚砜(dimethyl sulfoxide,DMSO)溶液(I/R+DMSO组)。检测病肺组织损伤、炎症反应及肺血管内皮细胞凋亡情况。细胞实验中,人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)进行低氧-复氧(hypoxia-reoxygenation,H/R)处理,探究不同时间HUVEC细胞中HIF-1α表达情况;使用罗沙司他(25μmol)预处理HUVEC后进行H/R,检测细胞中HIF-1α表达和凋亡情况。siRNA沉默HIF-1αmRNA,使用罗沙司他(25μmol)预处理并进行H/R,检测细胞中HIF-1α表达和凋亡情况。结果:动物实验结果显示,小鼠再灌注后肺HIF-1αmRNA表达持续升高,而蛋白表达随再灌注时间先升高后下降(P<0.05);免疫荧光染色显示I/R小鼠HIF-1α定位于肺内皮细胞;I/R+ROX(LD、HD)组小鼠肺组织HIF-1α较I/R+DMSO组表达上调;细胞实验显示HIF-1αmRNA在H/R后表达持续上升,蛋白表达先上升后下降(P<0.05);罗沙司他预处理组和H/R组相比,Bcl-2表达增加而Bax和Cleaved-Caspase-3的表达降低(P<0.05);siRNA沉默HIF-1αmRNA后,Bcl-2表达较罗沙司他治疗组表达下降而Bax和Cleaved-Caspase-3表达增加。结论:HIF-1α激动剂可抑制血管内皮细胞凋亡,减轻血管内皮损伤,减少小鼠缺血再灌注后肺部炎性细胞浸润,降低炎症水平。
Objective To investigate the effects and underlying mechanisms of the hypoxia⁃inducible factor⁃1α(HIF⁃1α)agonist Roxadustat in alleviating pulmonary ischemia⁃reperfusion injury(IRI)in mice.Method This study consisted of both in vivo and in vitro experiments.Thirty⁃six male C57/BL6 mice(6~8 weeks old)were used.In the animal experiments,20 mice underwent left pulmonary artery ligation to establish the IRI model and were divided into reperfusion groups of 0,1,2,or 4 hours(IR⁃0/1/2/4 h,n=4 each),with a sham group(n=4)as control.Temporal and spatial changes in pulmonary HIF⁃1αexpression were analyzed.Another 16 mice were randomized into four groups:sham(n=4),I/R+vehicle(DMSO,n=4),and I/R+Roxadustat treatment at 25 mg/kg or 50 mg/kg(I/R+ROX⁃LD,I/R+ROX⁃HD,n=4 each).Roxadustat or DMSO was administered intraperitoneally once daily for 5 days before surgery.Lung injury,inflammation,and endothelial apoptosis were subsequently assessed.In the cell experiments,human umbilical vein endothelial cell(HUVEC)was subjected to hypoxia⁃reoxygenation(H/R)to determine the time course of HIF⁃1αexpression.Cells pretreated with Roxadustat(25µmol)were then exposed to H/R,and HIF⁃1αexpression and apoptosis were analyzed.To verify the role of HIF⁃1α,siRNA knockdown of HIF⁃1αmRNA was performed before Roxadustat pretreatment and H/R exposure.Result In vivo,pulmonary HIF⁃1αmRNA expression increased progressively after reperfusion,while protein expression peaked early and subsequently declined(P<0.05).Immunofluorescence staining revealed HIF⁃1αpredominantly localized to pulmonary endothelial cells following I/R.Compared with the I/R+DMSO group,Roxadustat(both doses)upregulated HIF⁃1αexpression in lung tissue.In vitro,HIF⁃1αmRNA expression increased continuously after H/R,while protein levels first rose and then decreased(P<0.05).Roxadustat pretreatment upregulated Bcl⁃2 and downregulated Bax and cleaved caspase⁃3 compared with the H/R group(P<0.05).HIF⁃1αknockdown reversed these effects,resulting in decreased Bcl⁃2 and increased Bax and cleaved caspase⁃3 expression relative to the Roxadustat⁃treated group.Conclusion The HIF⁃1αagonist Roxadustat inhibits vascular endothelial apoptosis,alleviates endothelial injury,reduces inflammatory cell infiltration in lung tissue,and lowers inflammatory responses in mice with pulmonary ischemia–reperfusion injury.
作者
刘言
邹正浩澜
何凯扬
陈天瑞
丁祥超
林慧庆
Liu Yan;Zou Zhenghaolan;He Kaiyang;Chen Tianrui;Ding Xiangchao;Lin Huiqing(Department of Thoracic Surgery,Renmin Hospital of Wuhan University,Wuhan 430060,China)
出处
《中华器官移植杂志》
2025年第11期779-788,共10页
Chinese Journal of Organ Transplantation
基金
国家自然科学基金面上项目(82170106)
湖北省国际科技合作项目(2023EHA068)
武汉市知识创新专项(2023020201010167)。
关键词
肺移植
缺血再灌注损伤
低氧诱导因子
细胞凋亡
血管内皮细胞损伤
Lung transplantation
Ischemia⁃reperfusion injury
Hypoxia inducible factor
Apoptosis
vascular endothelial cell injury
