摘要
目的 评价泛素特异性蛋白酶22(USP22)在糖尿病小鼠心肌缺血再灌注损伤中的作用.方法 SPF级雄性C57BL/6小鼠78只,6～8周龄,采用随机数字表法分为6组:假手术组(Sham组,n=12)、1型糖尿病+假手术组(T1D+Sham组,n=12)、心肌缺血再灌注损伤组(I/R组,n=12)、1型糖尿病+心肌缺血再灌注损伤组(DI/R组,n=12)、1型糖尿病+心肌缺血再灌注损伤+空载体质粒组(DI/R+V组,n=15)和1型糖尿病+心肌缺血再灌注损伤+USP22过表达组(DI/R+U组,n=15).采用腹腔注射链脲佐菌素-柠檬酸盐缓冲液的方法制备1型糖尿病模型.采用结扎左冠状动脉的方法制备心肌缺血再灌注损伤模型.DI/R+U组和DI/R+V组于心肌缺血再灌注损伤造模前1 d分别于心脏原位注射USP22过表达质粒或者空载体质粒.心肌缺血再灌注24 h时,采用心脏超声检测心功能(左室射血分数和左室短轴缩短率).随后处死小鼠,取心脏,测定心肌梗死面积,观察心肌组织病理学变化(HE染色),检测细胞凋亡率(TUNEL染色)和ROS活性(DHE染色),采用Western blot法、免疫荧光和免疫组化法检测USP22的表达.蛋白质组学分析USP22调控的下游蛋白质,通过免疫沉淀实验检测蛋白质相互作用.结果 与Sham组相比,I/R组心功能指标下降,心肌细胞凋亡率和ROS活性升高,心肌组织USP22表达下调(P＜0.05);与I/R组相比,DI/R组心肌梗死面积百分比增加,心功能指标下降,心肌细胞凋亡率和ROS活性升高,USP22表达上调(P＜0.05),心肌组织病理学损伤加重;与DI/R+V组相比,DI/R+U组心肌梗死面积百分比减少,心功能指标升高,心肌细胞凋亡率和ROS活性降低,心肌组织USP22表达上调(P＜0.05),心肌组织病理学损伤减轻.蛋白质组学结合免疫沉淀实验结果显示钙调蛋白1(CNN1)与USP22有相互作用.结论 糖尿病小鼠心肌缺血再灌注损伤时,USP22可能为其内源性保护机制;CNN1可能是USP22发挥保护作用的下游机制.
Abstract
Objective To evaluate the role of ubiquitin-specific peptidase 22(USP22)in myocar-dial ischemia-reperfusion(I/R)injury in diabetic mice.Methods Seventy-eight SPF male C57BL/6 mice,aged 6-8 weeks,were divided into 6 groups using a random number table method:sham operation group(Sham group,n=12),type 1 diabetes mellitus+sham operation group(T1D+Sham group,n=12),myo-cardial I/R injury group(I/R group,n=12),type 1 diabetes mellitus+myocardial I/R injury group(DI/R group,n=12),type 1 diabetes mellitus+myocardial I/R injury+empty vector group(DI/R+V group,n=15),and type 1 diabetes mellitus+myocardial I/R injury+USP22 overexpression group(DI/R+U group,n=15).Type 1 diabetes mellitus was induced by intraperitoneal injection of streptozotocin-citrate buffer.Myocardial I/R was induced by ligation of the left coronary artery.At 1 day before developing the myocardial I/R injury model,DI/R+U group and DI/R+V group received an intramyocardial injection of USP22 overex-pression plasmid or empty vector plasmid,respectively.At 24 h of reperfusion,cardiac function was as-sessed using the echocardiography to measure the left ventricular ejection fraction and left ventricular frac-tional shortening.The mice were then sacrificed,and their hearts were harvested for measurement of the my-ocardial infarct size,for microscopic examination of pathological changes(using HE staining)and for deter-mination of the apoptosis rate(TUNEL staining),reactive oxygen species(ROS)activity(DHE stai-ning),and USP22 expression(by Western blot,immunofluorescence,and immunohistochemistry).Pro-teomic analysis was performed to identify downstream proteins regulated by USP22,and protein-protein in-teractions were investigated using co-immunoprecipitation.Results Compared with Sham group,the cardi-ac function indices were significantly decreased,the apoptosis rate of myocardial cells and ROS activity were increased,and USP22 expression in myocardial tissues was down-regulated in I/R group(P＜0.05).Com-pared with I/R group,the percentage of myocardial infarct size was significantly increased,the cardiac func-tion indices were decreased,the apoptosis rate of myocardial cells and ROS activity were increased,and USP22 expression in myocardial tissues was up-regulated(P＜0.05),and the pathological damage to myo-cardial tissues was aggravated in DI/R group.Compared with DI/R+V group,the percentage of myocardial infarct size was significantly decreased,the cardiac function indices were increased,the apoptosis rate of myocardial cells and ROS activity were decreased,and USP22 expression in myocardial tissues was up-regu-lated(P＜0.05),and the pathological damage to myocardial tissues was alleviated in DI/R+U group.The results of proteomics combined with co-immunoprecipitation experiments showed an interaction between cal-ponin 1 and USP22.Conclusions During myocardial I/R injury in diabetic mice,USP22 may act as an endogenous protective mechanism,and calponin 1 might be a downstream mechanism through which USP22 exerts its protective effects.
基金项目
国家自然科学基金青年基金项目(82300414)
无锡市中青年医疗卫生拔尖人才项目(BJ2023045)
无锡市"太湖人才计划"医疗卫生高层次人才项目()
万方数据