The present study was performed to assess the protective effect of fluoxetine (FLX) on renal ischemia-reperfusion injury (IRI) via the regulation of miR-450b-5p/Nrf2 axis in male rats. In vivo, these male rats were randomly divided into different treatment groups. The rats were administered with FLX (20 mg/kg, intraperitoneally) once daily for 3 days before operation. The pathomorphological changes of renal tissues were assessed by histological examination and Masson staining. In vitro, HK-2 cells were used to detect the activity by CCK-8 assay in Hypoxia/Reoxygenation (H/R) group and Hypoxia/Reoxygenation+Fluoxetine (H/R+FLX) group. In addition, the oxidative stress biomarkers were evaluated. Subsequently, Nrf2, NF-κB, and Nrf2-dependent antioxidant enzymes, were detected by Western blot assay. In vivo, the pathological changes and serological renal function were significantly relieved in the rats with the pre-treatment of FLX, compared to IRI group. After FLX stimulation, the expression levels of oxidative stress indices significantly decreased, while tissue antioxidant indices significantly increased, compared to IRI group. The differently expressed miRNAs on renal IRI in male rats were screened out by miRNA microarray, especially showing that miR-450b-5p was selected as the target miRNA. Following miR-450b-5p agomir injection, the pathological changes and oxidative stress biomarkers significantly aggravated, whether in IRI group or IRI+FLX group. Bioinformatics analysis and double-luciferase reporter assay demonstrated that miR-450b-5p directly targeted Nrf2. The expression level of NF-κB significantly increased, while the expression levels of Nrf2 and Nrf2-dependent antioxidant enzymes significantly decreased after miR-450b-5p agomir injection. Furthermore, the expression levels of Nrf2 and it-dependent antioxidant enzymes were apparently increased in ischemic kidney after the transfection of miR-450b-5p mimic+recombination protein Nrf2, as well as the decreased expression levels of intracellular ROS and iNOS. In vitro, FLX significantly increased HK-2 cell viability, and relieved H/R HK-2 cell oxidative injury via down-regulating ROS and iNOS. In addition, H/R-induced oxidative damage was recovered with miR-450b-5p mimic and recombination protein Nrf2. Consequently, FLX played an important protective role in renal IRI-induced oxidative damage by promoting antioxidation via targeting miR-450b-5p/Nrf2 axis.

中文翻译：

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氟西汀通过调控miR-450b-5p/Nrf2轴对肾缺血再灌注损伤氧化应激的保护作用


本研究旨在评估氟西汀 (FLX) 通过调节 miR-450b-5p/Nrf2 轴对雄性大鼠肾缺血再灌注损伤 (IRI) 的保护作用。在体内，这些雄性大鼠被随机分为不同的治疗组。术前给大鼠给予FLX（20 mg/kg，腹腔注射），每天1次，连续3 d。通过组织学检查和Masson染色评估肾组织的病理形态学变化。体外采用HK-2细胞，采用CCK-8法检测缺氧/复氧（H/R）组和缺氧/复氧+氟西汀（H/R+FLX）组的活性。此外，还评估了氧化应激生物标志物。随后，通过蛋白质印迹法检测 Nrf2、NF-κB 和 Nrf2 依赖性抗氧化酶。体内实验结果显示，与IRI组相比，FLX预处理后大鼠的病理变化和血清学肾功能均明显减轻。 FLX刺激后，与IRI组相比，氧化应激指标的表达水平显着降低，而组织抗氧化指标显着升高。通过miRNA芯片筛选出雄性大鼠肾IRI上差异表达的miRNA，特别显示miR-450b-5p被选为目标miRNA。注射miR-450b-5p agomir后，无论IRI组还是IRI+FLX组，病理变化和氧化应激生物标志物均显着加重。生物信息学分析和双荧光素酶报告基因分析表明miR-450b-5p直接靶向Nrf2。 注射miR-450b-5p agomir后，NF-κB的表达水平显着升高，而Nrf2和Nrf2依赖性抗氧化酶的表达水平显着降低。此外，转染miR-450b-5p模拟+重组蛋白Nrf2后，缺血肾中Nrf2及其依赖的抗氧化酶的表达水平明显升高，细胞内ROS和iNOS的表达水平明显降低。在体外，FLX显着增加HK-2细胞活力，并通过下调ROS和iNOS减轻H/R HK-2细胞氧化损伤。此外，H/R 诱导的氧化损伤可通过 miR-450b-5p 模拟物和重组蛋白 Nrf2 恢复。因此，FLX 通过靶向 miR-450b-5p/Nrf2 轴促进抗氧化，在肾 IRI 诱导的氧化损伤中发挥重要的保护作用。