解耦联蛋白2在漆黄素保护肾缺血/再灌注损伤中的作用及机制

The role of uncoupling protein 2in the protection of fisetin on renal ischemia-reperfusion injury

目的研究解耦联蛋白2(UCP-2)在漆黄素保护肾缺血/再灌注损伤中的作用及机制。方法6~8周的雄性C57BL/6J小鼠随机分为假手术组(n=6)、缺血/再灌注组(n=6)、缺血/再灌注+漆黄素组(n=6)。将人肾小管上皮(HK-2)细胞分别进行缺氧/复氧及缺氧/复氧+漆黄素处理(n=3)。检测血肌酐、尿素氮水平。苏木素-伊红及过碘酸雪夫染色观察肾脏病理损伤并统计损伤指数;二氢乙啶超氧化物阴离子探针检测组织氧化应激水平。检测HK-2细胞丙二醛水平以及还原型谷胱甘肽与氧化型谷胱甘肽比值(GSH/GSSG)以评估细胞内氧化应激水平;原位末端转移酶标记技术(TUNEL)评估细胞凋亡情况;亲脂性羰花青荧光探针(JC-1)检测线粒体膜电位;实时荧光定量聚合酶链式反应(q-PCR)检测解耦联蛋白2(UCP-2)mRNA表达水平;免疫印迹检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(BAX)、前体和激活型半胱氨酸蛋白酶-3(Caspase-3)以及UCP-2的蛋白表达水平。结果与缺血/再灌注组相比,缺血/再灌注+漆黄素组的血肌酐[(48.7±4.4)比(162.1±4.3)μmol/L,P<0.05]、尿素氮[(13.7±0.6)比(25.6±0.6)mmol/L,P<0.05]以及损伤指数(2.0±0.3比3.3±0.2,P<0.05)明显降低,凋亡细胞明显减少(P<0.05),Bcl-2表达升高、BAX与激活型Caspase-3表达降低(均P<0.05)。二氢乙啶染色提示,与缺血/再灌注组相比,缺血/再灌注+漆黄素组氧化应激水平明显降低(P<0.05)。在HK-2细胞中,与缺氧/复氧处理相比,缺氧/复氧+漆黄素处理的细胞中丙二醛水平明显下降(P<0.05)、GSH/GSSG上升(P<0.05)、线粒体膜电位上升(P<0.05)。在HK-2细胞中,与缺氧/复氧处理相比,给予缺氧/复氧+漆黄素处理的细胞UCP-2蛋白(0.7±0.1比0.5±0.1,P<0.05)与mRNA(0.7±0.2比0.2±0.1,P<0.05)的相对表达量均上调;动物模型中,与缺血/再灌注组相比,缺血/再灌注+漆黄素组UCP-2蛋白(0.8±0.1比0.4±0.2,P<0.05)与mRNA表达量(0.8±0.2比0.2±0.1,P<0.05)上升。结论漆黄素保护肾缺血/再灌注损伤导致的线粒体功能障碍与UCP-2的表达上调有关。

Objective To investigate the role of uncoupling protein 2(UCP-2)in the renal protection of fisetin on ischemia-reperfusion injury(I/R)in vivo and vitro.Methods C57 BL/6 mice of 6-8 weeks were randomly divided into Sham group(n=6),I/R group(n=6)and I/R+fisetin group(n=6).HK-2 cells were treated with hypoxia/reperfusion injury(H/R)and H/R+fisetin(n=3),respectively.Serum creatinine and blood urea nitrogen were measured.The pathological change of injured kidney was observed with both hematoxylin and eosin staining and periodic acid Schiff staining,and kidney injure score was calculated.The level of oxidative stress in tissue was detected by dihydroethidium(DHE)of frozen sections and the level of oxidative stress in cells was detected by malondialdehyde(MDA)and reduced glutathione/oxidated glutathione ratio(GSH/GSSG).TUNEL staining was used to evaluate the degree of tissue apoptosis.Mitochondrial membrane potential(MMP)was determined by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl carboeyanine iodide(JC-1),real time fluores-cence quantitative polymerase chain reaction(q-PCR)was used to measure the mRNA expression of UCP-2.The protein expression of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(BAX),pro Caspase-3,active Caspase-3 and UCP-2 were measured by Western-blot.Results The levels of serum creatinine[(48.7±4.4)vs(162.1±4.3)μmol/L,P<0.05]and blood urea nitrogen[(13.7±0.6)vs(25.6±0.6)mmol/L,P<0.05]and kidney injury score(2.0±0.3 vs 3.3±0.2,P<0.05)in I/R+fisetin group were lower(P<0.05)than those in I/R group.Compared with I/R group,the proportion of apoptotic cells reduced(P<0.05),the expression of Bcl-2 increased,while the expression of BAX and active Caspase-3 decreased(P<0.05)in I/R+fisetin group.DHE staining showed that the level of oxidative stress in I/R+fisetin group was lower than that in I/R group(P<0.05).In HK-2 cells,compared with H/R treatment,MDA level decreased(P<0.05),the GSH/GSSG ratio and MMP increased in H/R+fisetin treated cells(P<0.05).Compared with H/R treated HK-2 cells,the protein(0.7±0.1 vs 0.5±0.1,P<0.05)and mRNA(0.7±0.2 vs 0.2±0.1,P<0.05)expression of UCP-2 were higher in H/R+fisetin treated cells.And compared with I/R group,the protein(0.8±0.1 vs 0.4±0.2,P<0.05)and mRNA(0.8±0.2 vs 0.2±0.1,P<0.05)expression of UCP-2 in I/R+fisetin group increased.Conclusion The protective effect of fisetin on the renal mitochondria dysfunction caused by ischemia-reperfusion injury may be related to the upregulation of UCP-2 expression.