OSGEP在小鼠肝缺血再灌注损伤中的作用:与氧化应激反应的关系  

Role of O-sialoglycoprotein endopeptidase in hepatic ischemia-reperfusion injury in mice: relationship with oxidative stress

作　　者：张藤娟 周婉晴 陈诚[1] 张倩[1] 赵燕飞 何德浩 叶治[1] 夏萍萍[1] Zhang Tengjuan;Zhou Wanqing;Chen Cheng;Zhang Qian;Zhao Yanfei;He Dehao;Ye Zhi;Xia Pingping(Department of Anesthesiology,Affiliated Xiangya,Hospital of Central South University,Changsha 410078,China)

机构地区：[1]中南大学湘雅医院麻醉科,长沙410078

出　　处：《中华麻醉学杂志》2024年第1期85-90,共6页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(82001393,82171467);湖南省自然科学基金(2021JJ3119)。

摘　　要：目的评价O型唾液酸糖蛋白内肽酶(OSGEP)在小鼠肝缺血再灌注损伤中的作用及其与氧化应激反应的关系。方法实验ⅠSPF级健康雄性C57BL/6小鼠24只,野生型12只和OSGEP敲减型12只,6~8周龄,体质量18~22 g,采用随机数字表法分为4组(n=6):野生型假手术组(Sham组)、野生型HIRI组(HIRI组)、敲减OSGEP+假手术组(Sham+KD组)和敲减OSGEP+HIRI组(HIRI+KD组)。阻断肝动脉与肝门静脉60 min恢复灌注的方法制备缺血再灌注损伤模型,Sham组和Sham+KD组仅暴露血管,不进行夹闭;HIRI组和HIRI+KD组夹闭60 min恢复灌注。于再灌注6 h后处死小鼠提取肝组织标本,HE染色后光学显微镜下观察病理学结果进行Suzuki评分,检测血清ALT和AST浓度,DCFH-DA荧光探针法检测ROS水平,比色法测定肝组织MDA和谷胱甘肽(GSH)含量,采用Western blot法检测OSGEP表达水平。实验Ⅱ将生长良好的AML12细胞采用随机数字表法分为4组(n=30):对照组(C组)、氧糖剥夺/复糖复氧组(OGD/R组)、氧糖剥夺/复糖复氧组+敲减OSGEP组(OGD/R+KD组)和氧糖剥夺/复糖复氧+敲减OSGEP阴性对照组(OGD/R+NC组)。C组在正常条件下培养,OGD/R组氧糖剥夺6 h后复糖复氧24 h,OGD/R+KD组先构建稳定转染OSGEP的AML12细胞,余同OGD/R组。采用CCK-8法检测细胞存活率,检测LDH释放量,DCFH-DA法检测ROS水平,比色法测定MDA和GSH含量。结果实验Ⅰ与Sham组比较,HIRI组OSGEP表达下调,血清AST和ALT浓度、Suzuki评分、肝组织ROS水平和MDA含量升高,GSH含量降低(P<0.05),Sham+KD组各指标差异无统计学意义(P>0.05);与HIRI组比较,HIRI+KD组血清AST和ALT浓度、Suzuki评分、肝组织ROS水平和MDA含量升高,GSH含量降低(P<0.05)。实验Ⅱ与C组比较,OGD/R组OSGEP表达下调,细胞存活率和GSH含量降低,LDH释放量、ROS水平和MDA含量升高(P<0.05);与OGD/R组比较,OGD/R+KD组细胞存活率和GSH含量降低,LDH释放量、ROS水平和MDA含量升高(P<0.05),OGD/R+NC组各指标差异无统计学�Objective To evaluate the role of O-sialoglycoprotein endopeptidase(OSGEP)in hepatic ischemia-reperfusion injury(HIRI)and the relationship with oxidative stress in mice.Methods ExperimentⅠTwenty-four SPF healthy male C57BL/6 mice,12 wild-type and 12 OSGEP knockdown,aged 6-8 weeks,weighing 18-22 g,were divided into 4 groups(n=6 each)by the random number table method:wild-type shamoperation group(Sham group),wild-type HIRI group(HIRI group),OSGEP knockdown+sham operation group(Sham+KD group)and OSGEP knockdown+HIRI group(HIRI+KD group).Ischemia-reperfusion model was prepared by blocking the hepatic artery and portal vein for 60 min followed by reperfusion in anesthetized animals,the blood vessels were only exposed without occlusion in Sham group and Sham+KD group,and the blood vessels were clamped for 60 min followed by reperfusion in HIRI group and HIRI+KD group.The mice were sacrificed after 6-h reperfusion to extract liver tissue samples for microscopic examination of histopathological changes(with an optical microscope after HE staining)which were evaluated using Suzuki score and for determination of the serum concentrations of alanine aminotransferase(ALT)and aspartate aminotransferase(AST),level of reactive oxygen species(ROS)(using the DCFH-DA fluorescent probe method),contents of malondialdehyde(MDA)and glutathione(GSH)in liver tissues(using a colorimetric method)and expression of OSGEP(using Western blot).ExperimentⅡThe well-growing AML12 cells were divided into 4 groups(n=30 each)using a random number table method:control group(C group),oxygen-glucose deprivation/restoration(OGD/R)group,OGD/R+OSGEP knockdown group(OGD/R+KD group),and OGD/R+OSGEP knockdown negative control group(OGD/R+NC group).Group C was cultured under normal conditions.Group OGD/R was subjected to O2-glucose deprivation for 6 h followed by restoration of O2-glucose supply for 24 h in OGD/R group.In OGD/R+KD group,stable transfection of AML12 cells with OSGEP knockdown was performed prior to the experiment,and the other procedures we

关 键 词：内肽酶类 肝 再灌注损伤 氧化性应激 

分 类 号：R614[医药卫生—麻醉学]