抑制TRAF6的E3泛素连接酶活性减轻野百合碱诱导的肺动脉高压大鼠肺血管重构  

Inhibition of TRAF6 E3 ubiquitin ligase activity attenuates pulmonary vascular remodeling in monocrotaline-induced pulmonary hypertensive rats

作　　者：许正秋 邱莉华 陈睿[1] 马倩 李冰冰[1] XU Zhengqiu;QIU Lihua;CHEN Rui;MA Qian;LI Bingbing(Department of Anesthesiology,Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine,Nanjing 210008,China)

机构地区：[1]南京中医药大学附属鼓楼临床医学院麻醉科,江苏南京210008

出　　处：《中国病理生理杂志》2023年第10期1773-1779,共7页Chinese Journal of Pathophysiology

基　　金：南京市卫健委科技重点发展项目(No.ZKX20017);南京市青年卫生人才第一层次培养项目(No.QRX17013)。

摘　　要：目的:探讨抑制肿瘤坏死因子受体相关因子6(TRAF6)的E3泛素连接酶活性对野百合碱(MCT)诱导的肺动脉高压(PH)大鼠肺血管重构以及相关信号通路的影响。方法:采用随机数字表法将42只SPF级健康成年雄性Sprague-Dawley大鼠分为4组:对照组(n=6)、MCT模型组(n=15)、C25-140(TRAF6特异性E3泛素连接酶活性抑制剂)组(n=6)和MCT+C25-140组(n=15)。MCT及MCT+C25-140组大鼠于实验开始时颈后皮下单次注射MCT60 mg/kg,建立PH模型,其他2组大鼠皮下注射5%DMSO作为对照。C25-140组及MCT+C25-140组大鼠于第14天、21天和28天腹腔注射9.8 mg/kg C25-140。第28天行超声心动图检测评估大鼠肺动脉压力和心室收缩力变化,之后对大鼠进行安乐死并取肺组织。通过HE染色和α-平滑肌肌动蛋白(α-SMA)免疫荧光染色评估大鼠肺血管形态;采用免疫共沉淀检测大鼠肺动脉平滑肌细胞(PASMCs)中信号转导及转录激活因子3(STAT3)的K63泛素化水平;Western blot检测大鼠肺组织中TRAF6、STAT3、p-STAT3及cyclin D1的表达水平;免疫荧光染色观察大鼠肺血管TRAF6和STAT3的共定位情况。结果:(1)与对照组比较,MCT组大鼠肺动脉血流加速时间(PAAT)和三尖瓣环平面收缩期位移(TAPSE)明显减少(P<0.05),血管中膜厚度明显增加(P<0.05),α-SMA免疫荧光染色显示肺小血管明显肌化,提示造模成功。(2)与对照组相比,PH大鼠PASMCs中STAT3的K63泛素化明显升高。(3)MCT干预后28天,大鼠肺组织中TARF6、p-STAT3/STAT3比值及cyclin D1水平相对于对照组显著升高(P<0.05);与MCT组相比,MCT+C25-140组大鼠PAAT、TAPSE明显上升(P<0.05),血管中膜厚度明显减小(P<0.05)。给予C25-140处理后,肺组织中TARF6、p-STAT3/STAT3比值及cyclin D1水平相对于MCT组显著降低(P<0.05)。(4)免疫荧光染色的结果显示MCT组大鼠肺小动脉中膜TRAF6与STAT3存在显著共定位,C25-140处理能显著抑制两者的相互作用。结论:抑制TRAF6的E3泛素连接酶活性可�AIM:To investigate the impact of inhibiting E3 ubiquitin ligase activity targeting tumor necrosis factor receptor-related factor 6(TRAF6)on pulmonary vascular remodeling and related signaling pathways in monocrota‐line(MCT)-induced pulmonary hypertensive rats.METHODS:A total of 42 healthy adult male Sprague-Dawley rats,graded SPF,were randomly allocated into four groups:the control(CON)group(n=6),the MCT model group(n=15),the C25-140 group(n=6),and the MCT+C25-140 group(n=15).Rats in the MCT and MCT+C25-140 groups received a single subcutaneous injection of 60 mg/kg MCT on day 0 to establish the pulmonary hypertension model,while rats in the other two groups were subcutaneously injected with 5%DMSO as a control.Rats in the C25-140 and MCT+C25-140 groups received intraperitoneal injections of 9.8 mg/kg C25-140(a TRAF6-specific E3 ubiquitin ligase activity inhibitor)on the 14th,21st and 28th day after MCT administration.Echocardiography was used to assess changes in pulmonary ar‐tery pressure and ventricular contractility on day 28.Afterward,the rats were euthanized,and lung tissue was extracted.Hematoxylin-eosin(HE)staining andα-smooth muscle actin(α-SMA)immunofluorescence staining were employed to evaluate the morphology of pulmonary vessels,while the K63 ubiquitin level of STAT3 in rat pulmonary arterial smooth muscle cells(PASMCs)was detected by immunoprecipitation.Western blot was used to assess the expression levels of TRAF6,signal transducer and activator of transcription 3(STAT3),p-STAT3 and cyclin D1 in lung tissue.Immunofluo‐rescence staining was performed to observe the co-localization of TRAF6 and STAT3 in pulmonary vessels.RESULTS:In comparison to the CON group,the MCT group rats exhibited a significant decrease in pulmonary artery flow acceleration time(PAAT)and tricuspid annular plane systolic excursion(TAPSE)(P<0.05),along with a notable increase in vascu‐lar media thickness(P<0.05).α-SMA immunofluorescence staining revealed evident muscularization of pulmonary arteri‐oles,indicating su

关 键 词：肺动脉高压 肿瘤坏死因子受体相关因子6 信号转导及转录激活因子3 细胞周期蛋白D1 细胞凋亡 血管重构 

分 类 号：R363.2[医药卫生—病理学] R285.5[医药卫生—基础医学] R544.16