Erbin在肾缺血再灌注损伤中的表达和作用

Expression and function of Erbin protein in renal ischemia-reperfusion injury

目的探讨ErbB2相互作用蛋白（Erbin）在体内外肾缺血再灌注损伤（IRI）模型中的表达变化及体外细胞转染Erbin对IRI的影响。方法（1）体内实验：建立肾脏IRI小鼠模型，分为假手术组和再灌注3、6、12、24、48h模型组。收集并检测各组血清中BUN、Scr水平，采用PAS染色观察肾组织病理变化，TUNEL染色检测细胞凋亡，免疫组化检测Erbin的分布，Western印迹检测Erbin及NF—KBp65的表达变化。（2）体外实验：建立肾小管上皮细胞IRI模型，分别在更换正常含血清培养基后3、6、12、24h收集细胞，Western印迹检测Erbin的表达变化，流式细胞术及ELISA分别检测细胞凋亡和IL-6、TNF．仪炎性因子分泌。质粒Prk5．myc—Erbin瞬时转染建立Erbin过表达模型，分为对照组、IRI组、Erbin组和Erbin＋IRI组，分别检测各组细胞Erbin表达、NF-KB激活、细胞凋亡及炎性因子分泌。结果（1）与假手术组比较，模型组小鼠血Scr、BUN随再灌注时间延长而逐渐升高，24h达到峰值（P〈0．05）；同时再灌注6、12、24、48h模型组肾小管上皮细胞脱落坏死，管型形成，肾损伤评分和上皮细胞凋亡指数均高于假手术组（均P〈0．05），以24h最为显著；24h模型组肾小管内Erbin及细胞核内NF-KBp65的表达高于假手术组（均P〈0．05）。（2）与对照组比较，IRI组细胞核内NF—KBp65、细胞凋亡率及炎性因子（IL-6、TNF-a）分泌均增加（均P〈0．05）；再灌注12、24h模型组Erbin表达均高于对照组（均P〈0．05），24h组最为显著。与IRI组比较，Erbin＋IRI组细胞核内NF—κBp65、细胞凋亡率及IL-6、TNF-α分泌均降低（均P〈0．05）。结论Erbin在肾脏IRI中的表达增加。过表达Erbin可抑制IRI组中NF-KB的激活和细胞凋亡及炎性因子的分泌，进而减少肾损伤程度。

Objective To investigate the expression of ErbB2 interacting protein （Erbin） in renal ischemia-reperfusion injury （IRI） in vivo and in vivo, and the effect of Erbin over-expression on IRI. Methods （1） In vivo, the model of renal IRI was constructed in mice, and set up sham group and reperfusion 3, 6, 12, 24 and 48 h IRI group. BUN and Ser were detected and PAS staining was used to observe the pathology change of renal tissues. Cell apoptosis was detected by TUNEL staining. Erbin and NF - κB expression in renal tissue was detected by Western blotting, and immunohistochemistry was used to detect the distribution of Erbin. （2） In vivo, IRI model in HK2 cells was constructed and cells were harvested after culturing in normal medium for 3, 6, 12 and 24 h. Erbin expression was detected by Western blotting. Flow cytometry and ELISA were used to evaluate the level of cell apoptosis and inflammatory cytokine secretion respectively. HK2 cells were transiently transfected with Prk5-myc-Erbin plasmid via lipofectamine 2000, and were divided into control group, IRI group, Erbin group and Erbin ＋ IRI group. The protein expression of Erbin and NF-κB, cell apoptosis and inflammatory cytokine secretion was detected. Results （1） Compared with sham group, serum BUN and Scr were dramatically increased in IRI model, especially in 24 h after reperfusion （P 〈 0.05）. Moreover, PAS staining showed that a lot of renal tubular epithelial ceils were necrosis and fell off, and many protein cast were formed, renal injury score and apoptotic index were higher in 6 h, 12 h, 24 h, 48 h IRI model than those in sham group （all P 〈 0.05）. The expression of Erbin, which was expressed in renal tubules, and nuclear NF-κB in 24 h IRI model were significantly increased, as compared with sham group （all P 〈 0.05）. （2） Compared to those in control group, nuclear NF-KB expression, apoptosis and inflammatory cytokine secretion were significantly increased in IRI group. Meanwhile, Erbin expression was also induced and peaked at 24 h （P 〈 0.05）. Compared to those in IRI group, cell apoptosis, the expression of nuclear NF-κB, inflammatory cytokine IL-6 and TNF-α were decreased in Erbin＋IRI group （all P 〈 0.05）. Conclusions Erbin expression is up- regulated in renal IRI, and over-expression of Erbin can partly inhibit NF-κB activation, cell apoptosis and inflammatory cytokine secretion in IRI group, which indicates Erbin may playing a protective role in renal IRI.