盐酸纳美芬缺血后处理通过Sirt1/Nrf2/HO-1轴抑制铁死亡途径减轻大鼠肺缺血-再灌注损伤

Post-ischemic treatment of nalmefene hydrochloride attenuated lung ischemia-reperfusion injury in rats via the Sirt1/Nrf2/HO-1 pathway with inhibition of ferroptosis

目的研究纳美芬缺血后处理通过激活Sirt1/Nrf2/HO-1轴抑制铁死亡途径减轻肺缺血-再灌注损伤的作用及其机制。方法将60只大鼠随机均分为假手术组、模型组(I/R)、纳美芬组、纳美芬+EX527组、纳美芬+ML385组、纳美芬+Fe-柠檬酸盐组共6组,每组10只。假手术组大鼠不行缺血再灌注处理,未予药物治疗。I/R组大鼠采用阻断左肺门法建立肺缺血-再灌注模型,未给予药物治疗。纳美芬组大鼠于肺循环再灌注前5 min予尾静脉注射纳美芬(15μg/kg)。纳美芬+EX527组、纳美芬+ML385组、纳美芬+Fe-柠檬酸盐组分别于造模前2 h腹腔注射EX527(5 mg/kg)、ML385(30 mg/kg)、Fe-柠檬酸盐(15 mg/kg),同时在肺循环再灌注前5 min时尾静脉注射纳美芬(15μg/kg)。各组大鼠于再灌注3 h末留取处死后留取左肺上叶组织,检测肺组织湿/干重比值,评估各组大鼠肺组织损伤程度,检测肺组织Fe^(2+)、MDA和TNF-α、IL-6含量、GSH活性以及Sirt1、Nrf2、HO-1、ACSL4、GPX4表达水平。结果与假手术组比较,模型组湿/干重比值、肺组织损伤评分、ACSL4表达水平、Fe^(2+)、TNF-α、IL-6和MDA含量、Sirt1、Nrf2、HO-1信使RNA及蛋白表达水平显著增高(P<0.01),GPX4表达水平及GSH活性显著下降(P<0.01)。与模型组比较,纳美芬组、纳美芬+EX527组湿/干重比值、肺组织损伤评分、ACSL4表达水平、Fe^(2+)、TNF-α、IL-6和MDA含量显著下降(P<0.01),Nrf2、HO-1信使RNA及蛋白表达水平、GPX4表达水平及GSH活性显著增高(P<0.01);其中纳美芬组Sirt1信使RNA及蛋白表达水平显著增高(P<0.01)而纳美芬+EX527组无显著变化(P>0.05)。纳美芬+ML385组湿/干重比值、肺组织损伤评分、TNF-α、IL-6含量显著下降(P<0.01),Sirt1信使RNA及蛋白表达水平显著增高(P<0.01),Nrf2、HO-1信使RNA及蛋白表达水平、ACSL4和GPX4表达水平、Fe^(2+)、MDA含量和GSH活性无显著变化(P>0.05)。纳美芬+Fe-柠檬酸盐组湿/干重比值、肺组织损伤评分、TNF-α、IL-6、MDA含量显著下降(P<0.01);Sirt1、Nrf2、HO-1信使RNA及蛋白表达水平、GSH活性显著增高(P<0.01);Fe^(2+)含量、ACSL4和GPX4表达水平无显著变化(P>0.05)。与纳美芬组比较,纳美芬+EX527组、纳美芬+ML385组、纳美芬+Fe-柠檬酸盐组湿/干重比值、肺组织损伤评分、ACSL4表达水平、Fe^(2+)、TNF-α、IL-6和MDA含量显著增高(P<0.01)、GPX4表达水平及GSH活性显著下降(P<0.01);纳美芬+EX527组Sirt1、Nrf2、HO-1信使RNA及蛋白表达水平显著下降(P<0.01);纳美芬+ML385组Nrf2、HO-1信使RNA及蛋白表达水平显著下降(P<0.01),Sirt1信使RNA及蛋白表达水平无显著变化(P>0.05);纳美芬+Fe-柠檬酸盐组Sirt1、Nrf2、HO-1信使RNA及蛋白表达水平无显著变化(P>0.05)。结论盐酸纳美芬缺血后处理可通过激活Sirt1/Nrf2/HO-1轴抑制铁死亡途径减轻肺缺血-再灌注损伤。

ObjectiveTo study the effect and mechanism of post-ischemic treatment of nalmefene in alleviating the lung ischemia-reperfusion injury by inhibiting ferroptosis through activation of the Sirt 1/Nrf 2/HO-1 axis.MethodsA total of 60 rats were randomly divided into six groups equally(n=10):the sham group,the model group(I/R),the nalmefene group,the nalmefene+EX527 group,the nalmefene+ML385 group,the nalmefene+Fe-citrate group(nalmefene+Fe group).The sham group without drug treatment was not treated with ischemia-reperfusion.The pulmonary ischemia-reperfusion model was established by occlusion of the left pulmonary hilum in the model group without drug treatment.After ischemic treatment,the nalmefene group was injected with nalmefene(15μg/kg)via the tail vein at 5 minutes before reperfusion.The nalmefene+EX527 group,the nalmefene+ML385 group,and the nalmefene+Fe group were injected intraperitoneally with EX527(5 mg/kg),ML385(30 mg/kg),Fe-citrate(15 mg/kg),respectively,2 h before moulding and then injected with nalmefene(15μg/kg)via the tail vein at 5 minutes before reperfusion.All rats were sacrificed three hours after reperfusion,and the specimens from the upper lobe of the left lung tissue were preserved.The degree of lung tissue injury and the wet/dry weight ratio were assessed in each group of rats.Fe^(2+),MDA,TNF-α,and IL-6 content,GSH activity and the expression levels of Sirt1,Nrf2,HO-1,ACSL4 and GPX4 were determined.ResultsCompared with the sham group,the wet/dry weight ratio,lung tissue injury score,ACSL 4 expression level,Fe^(2+),TNF-α,IL-6 and MDA content,Sirt 1,Nrf 2,HO-1 messenger RNA and protein expression levels were significantly increased(P<0.01),while GPX 4 expression level and GSH activity were significantly decreased in the model group(P<0.01).Compared with the model group,wet/dry weight ratio,lung tissue injury score,ACSL 4 expression level,Fe^(2+),TNF-α,IL-6,and MDA content decreased significantly(P<0.01),Nrf 2,HO-1 messenger RNA and protein,GPX 4 expression,and GSH activity were significantly increased in the nalmefene group and the nalmefene+EX527 group(P<0.01).Sirt 1 messenger RNA and protein expression increased significantly in the nalmefene(P<0.01)and the nalmefene+EX527 groups(P>0.05).In the nalmefene+ML385 group,the wet/dry weight ratio,lung tissue injury score,TNF-αand IL-6 content were decreased significantly(P<0.01),while Sirt 1 messenger RNA and protein expression levels were significantly increased(P<0.01),but there were no significant changes in Nrf 2,HO-1 messenger RNA and protein expression levels,ACSL 4 and GPX 4 expression levels,Fe^(2+),MDA content,and GSH activity(P>0.05).In the nalmefene+Fe group,wet/dry weight ratio,lung-injury score,TNF-α,IL-6,MDA content were decreased significantly(P<0.01),messenger RNA and protein expression levels of Sirt 1,Nrf 2,HO-1,and GSH activity were increased significantly(P<0.01),but there were no significant changes in Fe^(2+)content,ACSL 4 and GPX 4 expression levels(P>0.05).Compared with the nalmefene group,in the nalmefene+EX527 group,the nalmefene+ML385 group and the nalmefene+Fe group,wet/dry weight ratio,lung tissue damage score,ACSL 4 expression level,TNF-α,IL-6 and MDA content were significantly increased(P<0.01),the expression level of GPX 4 and GSH activity were significantly decreased(P<0.01).The expression levels of Sirt 1,Nrf 2,HO-1 messenger RNA and protein were significantly decreased in the nalmefene+EX527 group(P<0.01).The expression levels of Nrf 2,HO-1 messenger RNA and protein decreased significantly in the namemefene+ML385 group(P<0.01),but there was no significant change in Sirt 1 messenger RNA and protein expression level(P>0.05).Sirt 1,Nrf 2,HO-1 messenger RNA-protein expression levels did not change significantly in the nalmefene+Fe group(P>0.05).ConclusionPost-ischemic treatment with nalmefene hydrochloride may alleviate pulmonary ischemia-reperfusion injury by inhibiting ferroptosis through activation of the Sirt 1/Nrf 2/HO-1 axis.