肾胺酶通过激活转录因子Nrf2对急性缺血性肾损伤起到保护作用
发布时间：2018-08-23 15:06
【摘要】：目的观察肾胺酶在急性缺血性肾损伤中的保护作用;并探讨其减轻急性缺血性肾损伤的可能机制。方法动物实验:选取成年雄性Sprague-Dawley(SD)大鼠24只,随机予假手术(游离出双侧肾蒂,不夹闭肾动脉)或缺血再灌注处理(夹闭双侧肾蒂1h后再灌注24h),在术前30分钟随机予肾胺酶(Renalase,Ren)1mg/kg或生理盐水1ml腹腔注射。共分为3组(n=8):假手术组(Shame组):仅游离出双侧肾蒂,不夹闭肾动脉,术前30分钟予生理盐水1ml腹腔注射;缺血组(IR组):缺血处理前30分钟予生理盐水1ml腹腔注射;干预组(IR+Ren组):缺血处理前30分钟予Ren 1mg/kg腹腔注射。缺血再灌注24h后处死大鼠,收集血清检测血肌酐、尿素氮水平;取肾组织观察病理学改变并进行损伤评分,检测肾组织内氧化应激相关指标丙二醛(Malondialdehyde,MDA)及超氧化物歧化酶(Superoxide dismutase,SOD)水平,应用免疫印迹法测定肾组织内核因子E2相关因子2(Nuclear factor-erythroid 2 related factor 2,Nrf2)及相关蛋白表达水平,主要包括血红素氧合酶-1(Hemeoxygenase,HO-1)以及醌氧化还原酶(NADPH quinine oxidoreductase,NQO-1)。细胞实验:用双氧水(0.5mmol/l)刺激人肾小管上皮细胞(human renal proximal epithelial tubular cells,h RPTEC)8小时诱导急性氧化应激损伤,分别加入1ug/ml或5ug/ml Ren共孵育,检测细胞内SOD及MDA水平,并检测Nrf2及相关蛋白水平。结果与假手术组相比,缺血再灌注24h后大鼠的血肌酐、尿素氮明显上升,肾小管损伤严重,肾组织内MDA水平升高,SOD水平下降(P0.05),Nrf2、HO-1、NQO-1的蛋白表达水平上升(P0.05);而腹腔注射Ren后,大鼠血肌酐、尿素氮较缺血组明显下降,肾脏病理损伤显著改善,MDA水平下降,SOD水平上升(P0.05);肾脏Nrf2、HO-1及NQO-1的蛋白表达水平较缺血组升高(P0.05)。在双氧水诱导的人肾小管上皮细胞氧化应激损伤模型中,加入Ren后,MDA水平下降,SOD水平上升(P0.05),人肾小管上皮细胞Nrf2、HO-1及NQO-1的蛋白表达水平升高(P0.05)。结论肾胺酶对急性缺血性肾损伤具有保护作用,其机制可能予激活转录因子Nrf2,抑制氧化应激反应相关。
[Abstract]:Objective to investigate the protective effect of reninase on acute ischemic renal injury and its possible mechanism. Methods Animal experiment: 24 adult male Sprague-Dawley (SD) rats were randomly given sham-operated (free bilateral renal pedicle). Renal artery was not clipped or ischemia reperfusion (1 h after bilateral renal pedicle was clipped for 24 h). Renase Ren 1mg/kg or normal saline 1ml were injected intraperitoneally 30 minutes before operation. The rats were divided into 3 groups: sham operation group (Shame group): only bilateral renal pedicle was free, renal artery was not clamped, saline 1ml was injected intraperitoneally 30 minutes before operation, and ischemia group (IR group) received intraperitoneal injection of normal saline 1ml 30 minutes before ischemic treatment. Intervention group (IR Ren group): 30 minutes before ischemic treatment Ren 1mg/kg intraperitoneal injection. 24 hours after ischemia reperfusion, the rats were killed, the serum levels of creatinine and urea nitrogen were collected, the pathological changes of renal tissue were observed and the injury score was evaluated. The levels of malondialdehyde (MDA) and superoxide dismutase (Superoxide) in renal tissue were measured. The expression of Nuclear factor-erythroid 2 related factor 2nrf2 and related protein in renal tissue was measured by Western blot. It mainly includes heme oxygenase 1 (heme oxygenase 1) and quinone oxidoreductase (NADPH quinine oxidoreductase1 NQO-1). Cell experiment: human renal tubular epithelial cells (human renal proximal epithelial tubular cells / RPTEC) were stimulated with hydrogen peroxide (0.5mmol/l) for 8 hours to induce acute oxidative stress injury, then were incubated with 1ug/ml or 5ug/ml Ren respectively. The levels of SOD and MDA, Nrf2 and related proteins were detected. Results compared with sham operation group, serum creatinine, urea nitrogen and renal tubule injury were significantly increased after 24 hours of ischemia reperfusion in rats. The level of MDA in renal tissue increased and decreased (P0.05), and the protein expression level of Nrf2HO-1NQO-1 increased after intraperitoneal injection of Ren (P0.05). The levels of serum creatinine and urea nitrogen in rats were significantly lower than those in ischemic group, and the levels of MDA and NQO-1 in renal pathological injury were significantly improved (P0.05), while the expression of Nrf2HO-1 and NQO-1 in kidney was higher than that in ischemic group (P0.05). In the oxidative stress injury model of human renal tubular epithelial cells induced by hydrogen peroxide, the level of Ren decreased and the level of SOD increased (P0.05), and the protein expression of Nrf2HO-1 and NQO-1 in human renal tubular epithelial cells increased (P0.05). Conclusion Reninase has protective effect on acute ischemic renal injury, and its mechanism may be related to the activation of transcription factor Nrf2 and the inhibition of oxidative stress response.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.5
，
本文编号：2199436
[Abstract]:Objective to investigate the protective effect of reninase on acute ischemic renal injury and its possible mechanism. Methods Animal experiment: 24 adult male Sprague-Dawley (SD) rats were randomly given sham-operated (free bilateral renal pedicle). Renal artery was not clipped or ischemia reperfusion (1 h after bilateral renal pedicle was clipped for 24 h). Renase Ren 1mg/kg or normal saline 1ml were injected intraperitoneally 30 minutes before operation. The rats were divided into 3 groups: sham operation group (Shame group): only bilateral renal pedicle was free, renal artery was not clamped, saline 1ml was injected intraperitoneally 30 minutes before operation, and ischemia group (IR group) received intraperitoneal injection of normal saline 1ml 30 minutes before ischemic treatment. Intervention group (IR Ren group): 30 minutes before ischemic treatment Ren 1mg/kg intraperitoneal injection. 24 hours after ischemia reperfusion, the rats were killed, the serum levels of creatinine and urea nitrogen were collected, the pathological changes of renal tissue were observed and the injury score was evaluated. The levels of malondialdehyde (MDA) and superoxide dismutase (Superoxide) in renal tissue were measured. The expression of Nuclear factor-erythroid 2 related factor 2nrf2 and related protein in renal tissue was measured by Western blot. It mainly includes heme oxygenase 1 (heme oxygenase 1) and quinone oxidoreductase (NADPH quinine oxidoreductase1 NQO-1). Cell experiment: human renal tubular epithelial cells (human renal proximal epithelial tubular cells / RPTEC) were stimulated with hydrogen peroxide (0.5mmol/l) for 8 hours to induce acute oxidative stress injury, then were incubated with 1ug/ml or 5ug/ml Ren respectively. The levels of SOD and MDA, Nrf2 and related proteins were detected. Results compared with sham operation group, serum creatinine, urea nitrogen and renal tubule injury were significantly increased after 24 hours of ischemia reperfusion in rats. The level of MDA in renal tissue increased and decreased (P0.05), and the protein expression level of Nrf2HO-1NQO-1 increased after intraperitoneal injection of Ren (P0.05). The levels of serum creatinine and urea nitrogen in rats were significantly lower than those in ischemic group, and the levels of MDA and NQO-1 in renal pathological injury were significantly improved (P0.05), while the expression of Nrf2HO-1 and NQO-1 in kidney was higher than that in ischemic group (P0.05). In the oxidative stress injury model of human renal tubular epithelial cells induced by hydrogen peroxide, the level of Ren decreased and the level of SOD increased (P0.05), and the protein expression of Nrf2HO-1 and NQO-1 in human renal tubular epithelial cells increased (P0.05). Conclusion Reninase has protective effect on acute ischemic renal injury, and its mechanism may be related to the activation of transcription factor Nrf2 and the inhibition of oxidative stress response.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.5
，
本文编号：2199436
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