昼夜节律调控Nrf2蛋白表达在大鼠心肌缺血再灌注后急性肾损伤中的作用及机制

Circadian rhythm regulates Nrf2 expression in acute kidney injury following myocardial ischemia⁃reperfusion in rats

目的探索脑和肌肉芳香烃受体核转位样蛋白-1(Bmal1)基因调控促红细胞生成素2相关因子2(Nrf2)蛋白表达在大鼠心肌缺血再灌注(MIR)后急性肾损伤中的作用。方法采用随机数字表法将SD大鼠分为假手术组和MIR组,每组8只。所有大鼠使用戊巴比妥麻醉后插管,进行机械通气;MIR组通过左前降支冠状动脉闭塞缺血(30 min),然后移除微血管夹进行2 h再灌注,建立MIR模型;两组大鼠再分别在光照时间点(ZT0)和黑暗时间点(ZT12)收紧结扎线。采用苏木精-伊红(HE)染色评估心肌和肾脏组织学损伤。检测肌酸激酶同工酶(CK⁃MB)、乳酸脱氢酶(LDH)以评估心肌受损程度。检测血清肌酐(Scr)、尿素氮(BUN)、中性粒细胞明胶酶相关脂质运载蛋白(NGAL)和肾组织超氧化物歧化酶(SOD)、丙二醛(MDA)以评估肾脏受损程度。通过Western blot法检测Bmal1、Nrf2蛋白表达水平。结果与假手术组比较,MIR组在ZT0和ZT12的肾组织学改变显著并且肾脏组织病理学评分显著增高,差异均有统计学意义(t=25.62、31.97,P均<0.05);且MIR组ZT12肾脏组织病理学评分显著高于ZT0,差异有统计学意义(t=7.79,P<0.05)。与假手术组ZT0比较,MIR组ZT0血清CK⁃MB、LDH、Scr、BUN、NGAL、MDA水平显著升高,SOD活性显著降低,差异均有统计学意义(t=40.20、31.81、15.89、11.70、12.50、6.58、7.12,P均<0.05);与假手术组ZT12比较,MIR组ZT12血清CK⁃MB、LDH、Scr、BUN、NGAL、MDA水平显著升高,SOD活性显著降低,差异均有统计学意义(t=49.33、53.61、22.90、17.20、16.82、11.23、9.96,P均<0.05);且MIR组ZT12 CK⁃MB、LDH、Scr、BUN、NGAL、MDA水平显著高于ZT0,SOD活性显著低于ZT0,差异均有统计学意义(t=8.07、23.41、7.76、5.38、5.68、5.16、4.05,P均<0.05)。与假手术组ZT0比较,MIR组ZT0 Bmal1、Nrf2蛋白表达显著升高,差异均有统计学意义(t=17.80、18.14,P均<0.05);与假手术组ZT12比较,MIR组ZT12 Bmal1、Nrf2蛋白表达显著升高,差异均有统计学意义(t=13.18、13.82,P均<0.05);且MIR组ZT12 Bmal1、Nrf2蛋白表达显著低于ZT0,差异均有统计学意义(t=9.30、10.62,P均<0.05)。结论大鼠夜间MIR加重肾脏损伤,与夜间Bmal1调控Nrf2蛋白降低相关。

Objective To explore the role of brain and muscle ARNT⁃like protein 1(Bmal1)regulating nuclear factor erythropoietin⁃2⁃related factor 2(Nrf2)protein in acute kidney injury following myocardial ischemia⁃reperfusion(MIR)in rats.Methods SD rats were divided into sham group and MIR group by random number table method,8 in each group.All rats were anesthetized with pentobarbital and intubated for mechanical ventilation.The MIR model was established by occluding the left anterior descending coronary artery for ischemia(30 min),followed by removal of the microvascular clamp for 2 h of reperfusion.The ligatures were tightened at the light time point(ZT0)and the dark time point(ZT12)in both groups.Hematoxylin⁃eosin(HE)staining was used to evaluate myocardial and renal histological damage.Creatine kinase isoenzyme(CK⁃MB)and lactate dehydrogenase(LDH)were detected to evaluate the degree of myocardial damage.Serum creatinine(Scr),urea nitrogen(BUN),neutrophil gelatinase⁃associated lipocalin(NGAL)and renal tissue superoxide dismutase(SOD)and malondialdehyde(MDA)were detected to evaluate the degree of renal damage.Western blot was used to detect the changes in Bmal1 and Nrf2 protein expression levels.Results Compared with the sham group,the MIR group had significant renal histologic changes and higher renal histologic grading scores at ZT0 and ZT12,with statistically significant differences(t=25.62,31.97;both P<0.05);the renal histologic grading scores at ZT12 of the MIR group were significantly higher than those at ZT0(t=7.79,P<0.05).Compared with at ZT0 in the sham group,serum CK⁃MB,LDH,Scr,BUN,NGAL,MDA levels were significantly higher and SOD activity was significantly lower at ZT0 of the MIR group(t=40.20,31.81,15.89,11.70,12.50,6.58,7.12;all P<0.05);compared with at ZT12 in the sham group,serum CK⁃MB,LDH,Scr,BUN,NGAL,MDA levels were significantly higher and SOD activity was significantly lower at ZT12 of the MIR group(t=49.33,53.61,22.90,17.20,16.82,11.23,9.96;all P<0.05);the levels of CK⁃MB,LDH,Scr,BUN,NGAL and MDA at ZT12 of the MIR group were significantly higher than that at ZT0,and the activity of SOD was significantly lower than that at ZT0(t=8.07,23.41,7.76,5.38,5.68,5.16,4.05;all P<0.05).Compared with at ZT0 in the sham group,the protein expression of Bmal1 and Nrf2 was significantly higher at ZT0 of the MIR group(t=17.80,18.14;both P<0.05);compared with at ZT12 in the sham group,Bmal1 and Nrf2 protein expression was significantly elevated at ZT12 of the MIR group(t=13.18,13.82;both P<0.05);Bmal1 and Nrf2 protein expression at ZT12 was significantly lower than that at ZT0 in MIR group(t=9.30,10.62;both P<0.05).Conclusion In brief,MIR aggravated renal injury in rats at night,which was related to the decrease of Nrf2 protein regulated by Bmal1 at night.