Apocynin和DPI通过抑制NOD1信号通路在缺血性AKI中的保护作用

The protective effect of apocynin and DPI in ischemic AKI by inhibiting NOD1 signaling pathway

目的 NOD样受体可促发炎症反应,夹竹桃麻素(apocynin)和二苯基碘鎓(DPI)均为氧化酶抑制剂。本研究观察在缺血性急性肾损伤中抑制氧化应激产生是否能通过NOD1信号通路减轻肾间质炎症反应与细胞凋亡。方法将雄性Wistar大鼠随机分为4组:假手术(Sham)组、肾脏缺血再灌注(I/R)组、I/R+夹竹桃麻素(apocynin)组、I/R+二苯基碘鎓(DPI)组。通过Western印记法分别对肾组织核苷酸结合寡聚域样受体1(NOD1)、半胱天冬酶(caspase-1)及细胞核因子-κB(NF-κB)蛋白的表达进行检测;实时定量PCR法对NOD1mRNA的表达进行检测; HE染色法观察肾脏组织学改变;免疫组织化学法检测肾组织肿瘤坏死因子(TNF-α)的表达; TUNEL法检测肾组织细胞凋亡。采用SPSS 22. 0统计软包对实验数据进行统计学处理。结果与Sham组比较,I/R组大鼠肾组织NOD1、caspase-1、NF-κB、TNF-α蛋白表达增加(t=16. 81,t=7. 28,t=11. 08,t=10. 11; P<0. 05); NOD1mRNA表达增加(t=-7. 93,P<0. 05); HE染色表现为急性肾小管坏死,肾小管损伤评分明显增加(t=-11. 0,P<0. 05); TUNEL染色显示缺血区凋亡细胞数目增加(t=-18. 38,P<0. 05)。与I/R组比较,I/R+apocynin组的NOD1、caspase-1、NF-κB、TNF-ɑ蛋白表达减少(t=-10. 9,t=-7. 6,t=-4. 9,t=-9. 7; P<0. 05); NOD1mRNA表达减少(t=8. 49,P<0. 05); HE染色后者较前者急性肾小管坏死减轻,肾小管损伤评分减低(t=-12,P<0. 05); TUNEL染色显示缺血区凋亡细胞数目减少(t=-11. 3,P<0. 05)。与I/R组比较,I/R+DPI组的NOD1、caspase-1、NF-κB、TNF-α蛋白表达减少(t=-11. 4,t=-6. 8,t=-5. 4,t=-10. 6,P<0. 05); NOD1mRNA表达减少(t=7. 5,P<0. 05); HE染色后者较前者急性肾小管坏死减轻,肾小管损伤评分减低(t=-11,P<0. 05); TUNEL染色显示缺血区凋亡细胞数目减少(t=-10. 8,P<0. 05)。结论抑制氧化应激可阻断NOD1样受体依赖的炎症途径与细胞凋亡,从而减轻肾缺血再灌注损伤。

Objective NOD-like receptors can promote inflammation,and apocynin and diphenylene iodonium(DPI)are both oxidase inhibitors.The aim of this study was to investigate whether inhibition of oxidative stress in ischemic acute kidney injury could attenuate renal interstitial inflammatory responses and apoptosis through the NOD1 signaling pathway.Methods Male Wistar rats were randomly divided into 4 groups:sham operation group,renal ischemia-reperfusion(I/R)group,I/R+apocynin group,and I/R+DPI group.Western blotting was used to detect the renal protein expression of nucleotidebinding oligomeric domain like receptor 1(NOD1),caspase-1,and nuclear factor-κB(NF-κB).Realtime quantitative PCR was used to detect NOD1 mRNA.The histological changes of the kidney were observed by HE staining.The expression of tumor necrosis factor-α(TNF-α)in renal tissue was detected by immunohistochemistry.The apoptosis of renal cells was detected by TUNEL method.Statistical analysis of experimental data was made with the SPSS 22.0 statistical soft pack.Results Compared with the sham group,the I/R group had higher protein expression of NOD1,caspase-1,NF-κB,and TNF-αin the kidney(t=16.81,t=7.28,t=11.08,t=10.11;P<0.05),as well as higher mRNA expression of NOD1(t=-7.93,P<0.05);HE staining showed renal lesions of acute tubular necrosis,and the renal tubular injury score increased significantly(t=-11.0,P<0.05);TUNEL staining showed that apoptotic cells increased significantly in the renal ischemia zone(t=-18.38,P<0.05).Compared with the I/R group,the I/R+apocynin group had lower protein expression of NOD1,caspase-1,NF-κB,and TNF-αin the kidney(t=-10.9,t=-7.6,t=-4.9,t=-9.7;P<0.05),as well as lower mRNA expression of NOD1(t=8.49,P<0.05);HE staining showed lower renal lesions of acute tubular necrosis,and the renal tubular injury score reduced significantly(t=-12,P<0.05);TUNEL staining showed that apoptotic cells reduced significantly in the renal ischemia zone(t=-11.3,P<0.05).Compared with the I/R group,the I/R+DPI group had lower protein expression of NOD1,caspase-1,NF-κB,and TNF-αin the kidney(t=-11.4,t=-6.8,t=-5.4,t=-10.6,P<0.05),as well as lower mRNA expression of NOD1(t=7.5,P<0.05);HE staining showed lower renal lesions of acute tubular necrosis,and the renal tubular injury score reduced significantly(t=-11,P<0.05);TUNEL staining showed that apoptotic cells reduced significantly in the renal ischemia zone(t=-10.8,P<0.05).Conclusion Inhibition of oxidative stress might block NOD1-like receptor-dependent inflammatory pathways and cellular apoptosis,thereby reducing the renal ischemia-reperfusion injury.