全反式维甲酸抑制核因子E2相关因子2和血红素加氧酶1加重大鼠肾脏缺血再灌注损伤表达

ATRA inhibits Nrf2 and HO-1 expression to aggravate the renal ischemia-reperfusion injury in rats

目的 观察用全反式维甲酸(all-trans retinoic acid,ATRA)抑制核因子E2相关因子2(Nrf2)和血红素加氧酶1(HO-1)是否会加重大鼠肾脏缺血再灌注损伤(IRI)表达。方法 2020年10月至2022年2月选取24只健康成年雄性SD大鼠切除右肾,并按随机数字表法分为3组(n=8),即假手术组(Sham)、肾脏缺血再灌注(I/R)组、全反式维甲酸(I/R+ATRA)组。其中Sham组和I/R组给予腹腔注射玉米油(1 m L·kg^(-1)·d^(-1))1周,ATRA组则给予腹腔注射溶于玉米油的ATRA(10 mg·kg^(-1)·d^(-1))1周后,3组大鼠用10%的水合氯醛(0.3 m L/100 g)进行麻醉后固定四肢,将其在37℃条件下沿腹中线打开腹腔并分离出左肾动脉。其中Sham组切除右肾,不予以夹闭左肾动脉;I/R组和ATRA组采用右肾切除联合左肾动脉夹闭45 min后再灌注24 h的方法建立大鼠肾脏I/R模型。实验结束后收集3组大鼠血清及肾组织标本,用比色法检测血清肌酐(Scr)、血尿素氮(BUN)浓度;HE染色法观察肾组织病理改变;TUNEL法进行肾组织细胞凋亡的检测;二氢乙啶(DHE)荧光染色评估肾组织活性氧的表达情况;通过比色法检测肾组织丙二醛(MDA)浓度、超氧化物歧化酶(SOD)活性;用蛋白质印迹法分别对Nrf2、HO-1的表达进行检测。结果 与Sham组相比,I/R组大鼠肾组织Nrf2、HO-1蛋白表达量均增加(P<0.01),活性氧表达量增加,SOD活性下降,MDA含量、血清Scr、BUN浓度升高(P<0.01),肾小管损伤评分及凋亡细胞表达较高(P<0.05),其中Nrf2蛋白和HO-1蛋白分别为0.52±0.09和0.37±0.79,高于Sham组的0.06±0.01和0.02±0.01。与I/R组相比,I/R+ATRA组大鼠肾组织Nrf2、HO-1蛋白显著减少(P<0.01),活性氧表达量明显增加,SOD活性严重下降,MDA含量、血清Scr、BUN浓度明显升高(P<0.01),肾小管损伤评分显著增加,肾脏凋亡细胞表达亦增高(P<0.05),其中I/R+ATRA组Nrf2蛋白和HO-1蛋白分别为0.29±0.04和0.15±0.03,显著低于I/R组。结论 Nrf2/HO-1通路参与肾脏缺血再灌注损伤过程,ATRA可能通过抑制Nrf2/HO-1通路,加重氧化应激,进一步加重肾脏缺血再灌注损伤。

Objective To investigate whether inhibition of nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)expression by all-trans retinoic acid(ATRA)aggravates the renal ischemia-reperfusion injury(I/R)in rats.Methods From Oc-tober 2020 to February 2022,24 healthy adult male SD rats were selected to remove their right kidneys,and they were randomly as-signed into three groups:sham operation group(Sham),I/R group,and ATRA group,with 8 rats in each group.The rats in Sham and I/R groups were given intraperitoneal injection of corn oil(1 mL·kg^(-1)·d^(-1))for 1 week.The rats in the ATRA group were injected intraperito-neally with ATRA dissolved in corn oil(10 mg·kg^(-1)·d^(-1))for 1 week.3 groups of rats were anesthetized with 10%chloral hydrate(0.3 mL/100 g)and fixed limbs.The abdominal cavity was opened along the abdominal midline and the left renal artery was separated at 37℃.In the Sham group,the right kidney was excised without blocking the left renal artery.Renal I/R model was established by right nephrectomy combined with left renal artery occlusion for 45 min followed by 24 h reperfusion in I/R and ATRA groups.After the ex-periment,three groups of rats serum and kidney tissue samples were collected.The concentrations of serum creatinine(Scr)and blood urea nitrogen(BUN)were detected by colorimetric method.HE staining was used to observe the pathological changes of kidney tissue.The apoptosis of renal tissue cells was detected by TUNEL staining.The expression level of reactive oxygen species(ROS)in renal tis-sue was evaluated by dihydroethidium(DHE)fluorescence staining.A colorimetric method was used to determine the contents of super-oxide dismutase(SOD)and malondialdehyde(MDA)in the kidney tissue.The protein expression levels of Nrf2 and HO-1 were detected by Western blotting.Results Compared with the sham group,the protein expressions of Nrf2 and HO-1 in renal tissues of rats in I/R group were significantly increased(P<0.01).The contents of ROS,MDA,Scr,BUN were increased and SOD activity was decreased(P<0.01).Renal tubular injury score and positive rate of apoptotic cells were higher(P<0.05),among them,Nrf2 protein and HO-1 protein were 0.52±0.09 and 0.37±0.79,respectively,which were higher than those of Sham group 0.06±0.01 and 0.02±0.01.Compared with the I/R group,the expressions of Nrf2 and HO-1 protein in kidney tissue of ATRA group was significantly decreased(P<0.01).The con-tents of ROS,MDA,Scr and BUN were significantly increased and the SOD activity was reduced(P<0.01).The renal tubular injury score and positive rate of apoptotic cells were significantly increased(P<0.05).Among them,The protein levels of Nrf2 and HO-1 in ATRA group were 0.29±0.04 and 0.15±0.03,respectively,which were significantly lower than those in I/R group.Conclusion Nrf2/HO-1 signaling pathway is involved in the process of renal I/R injury.ATRA may further aggravate renal I/R injury by inhibiting Nrf2/HO-1 pathway and aggravating oxidative stress.