腎胺酶通過激活轉(zhuǎn)錄因子Nrf2對(duì)急性缺血性腎損傷起到保護(hù)作用
發(fā)布時(shí)間：2018-08-23 15:06
【摘要】：目的觀察腎胺酶在急性缺血性腎損傷中的保護(hù)作用;并探討其減輕急性缺血性腎損傷的可能機(jī)制。方法動(dòng)物實(shí)驗(yàn):選取成年雄性Sprague-Dawley(SD)大鼠24只,隨機(jī)予假手術(shù)(游離出雙側(cè)腎蒂,不夾閉腎動(dòng)脈)或缺血再灌注處理(夾閉雙側(cè)腎蒂1h后再灌注24h),在術(shù)前30分鐘隨機(jī)予腎胺酶(Renalase,Ren)1mg/kg或生理鹽水1ml腹腔注射。共分為3組(n=8):假手術(shù)組(Shame組):僅游離出雙側(cè)腎蒂,不夾閉腎動(dòng)脈,術(shù)前30分鐘予生理鹽水1ml腹腔注射;缺血組(IR組):缺血處理前30分鐘予生理鹽水1ml腹腔注射;干預(yù)組(IR+Ren組):缺血處理前30分鐘予Ren 1mg/kg腹腔注射。缺血再灌注24h后處死大鼠,收集血清檢測血肌酐、尿素氮水平;取腎組織觀察病理學(xué)改變并進(jìn)行損傷評(píng)分,檢測腎組織內(nèi)氧化應(yīng)激相關(guān)指標(biāo)丙二醛(Malondialdehyde,MDA)及超氧化物歧化酶(Superoxide dismutase,SOD)水平,應(yīng)用免疫印跡法測定腎組織內(nèi)核因子E2相關(guān)因子2(Nuclear factor-erythroid 2 related factor 2,Nrf2)及相關(guān)蛋白表達(dá)水平,主要包括血紅素氧合酶-1(Hemeoxygenase,HO-1)以及醌氧化還原酶(NADPH quinine oxidoreductase,NQO-1)。細(xì)胞實(shí)驗(yàn):用雙氧水(0.5mmol/l)刺激人腎小管上皮細(xì)胞(human renal proximal epithelial tubular cells,h RPTEC)8小時(shí)誘導(dǎo)急性氧化應(yīng)激損傷,分別加入1ug/ml或5ug/ml Ren共孵育,檢測細(xì)胞內(nèi)SOD及MDA水平,并檢測Nrf2及相關(guān)蛋白水平。結(jié)果與假手術(shù)組相比,缺血再灌注24h后大鼠的血肌酐、尿素氮明顯上升,腎小管損傷嚴(yán)重,腎組織內(nèi)MDA水平升高,SOD水平下降(P0.05),Nrf2、HO-1、NQO-1的蛋白表達(dá)水平上升(P0.05);而腹腔注射Ren后,大鼠血肌酐、尿素氮較缺血組明顯下降,腎臟病理損傷顯著改善,MDA水平下降,SOD水平上升(P0.05);腎臟Nrf2、HO-1及NQO-1的蛋白表達(dá)水平較缺血組升高(P0.05)。在雙氧水誘導(dǎo)的人腎小管上皮細(xì)胞氧化應(yīng)激損傷模型中,加入Ren后,MDA水平下降,SOD水平上升(P0.05),人腎小管上皮細(xì)胞Nrf2、HO-1及NQO-1的蛋白表達(dá)水平升高(P0.05)。結(jié)論腎胺酶對(duì)急性缺血性腎損傷具有保護(hù)作用,其機(jī)制可能予激活轉(zhuǎn)錄因子Nrf2,抑制氧化應(yīng)激反應(yīng)相關(guān)。
[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.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692.5
，
本文編號(hào)：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.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692.5
，
本文編號(hào)：2199436
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