白藜芦醇调控Keap1-Nrf2信号通路对大鼠运动性肾损伤的保护作用

周海涛; 胡戈; 张静; 周绮云; 秦菲

doi:10.13386/j.issn1002-0306.2024060169

白藜芦醇调控Keap1-Nrf2信号通路对大鼠运动性肾损伤的保护作用

Protective Effect of Resveratrol on Exercise-Induced Kidney Injury in Rats by Regulating Keap1-Nrf2 Signaling Pathway

摘要

摘要: 目的：探究白藜芦醇对大鼠运动性肾损伤的保护机制。方法：选取32只7周龄雄性SD大鼠分为安静组（CONT组，8只）、白藜芦醇组（RESV组，8只）、运动性肾损伤模型组（EIKIM组，8只）和白藜芦醇干预的运动性肾损伤模型组（REIKIM组，8只）。EIKIM和REIKIM组大鼠进行跑台训练建立运动性肾损伤模型。4周训练期间，RESV、REIKIM组大鼠在每次训练前1 h根据体重给予剂量和体积分别为150 mg/kg·bw和5 mL/kg的白藜芦醇溶液灌胃，其余大鼠给予等体积溶剂灌胃。末次训练24 h后采集各组大鼠血液和肾样本。通过HE染色评估大鼠肾组织形态。测定大鼠血液中肌酐（creatinine，Cr）和尿素氮（urea nitrogen，UN）水平及肾组织中Kelch样环氧氯丙烷相关蛋白-1（kelch-like ECH-associated protein-1，Keap1）、核因子E2相关因子2（nuclear factor-E2-related factor 2，Nrf2）、NAD(P)H醌脱氢酶1（NAD(P)H: quinone oxidoreductase 1，NQO1）、血红素氧合酶1（heme oxygenase-1，HO-1）mRNA和蛋白质表达水平，磷酸化（phosphorylated nuclear factor erythroid 2-related factor 2，p-Nrf2）蛋白质表达水平，超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）和谷胱甘肽S移换酶（glutathione S-transferase，GSH-ST）活性，丙二醛（malondialdehyde，MDA）和活性氧簇（reactive oxygen species，ROS）含量，细胞凋亡及B淋巴细胞瘤因子-2（B cell lymphoma-2 protein，Bcl-2）、Bcl-2相关X蛋白（bcl-2-associated X protein，Bax）、含半胱氨酸的天冬氨酸蛋白水解酶-9（cysteinyl aspartate specific proteinase-9，Caspase-9）、含半胱氨酸的天冬氨酸蛋白水解酶-3（cysteinyl aspartate specific proteinase-3，Caspase-3）蛋白质表达水平。结果：经4周大强度跑台训练，与CONT组大鼠比较，EIKIM组大鼠肾组织病理改变明显；血液中Cr和UN水平、肾组织中Keap1 mRNA和蛋白质表达水平、MDA和ROS含量、细胞凋亡及Bax、Caspase-9和Caspase-3蛋白质表达水平均显著升高（P<0.01）；肾组织中Nrf2、NQO1、HO-1 mRNA和蛋白质表达水平、SOD、CAT和GSH-ST活性、Bcl-2蛋白质表达水平和Bcl-2/Bax比值均显著下降（P<0.01）。经4周白藜芦醇干预，与EIKIM组大鼠比较，REIKIM组大鼠肾组织病理改变有效改善；血液中Cr和UN水平、肾组织中Keap1 mRNA和蛋白质表达水平、MDA和ROS含量、细胞凋亡、Bax、Caspase-9、Caspase-3蛋白质表达水平均显著下降（P<0.05或P<0.01）；肾组织中Nrf2、NQO1、HO-1 mRNA和蛋白质表达水平、SOD、CAT和GSH-ST活性、Bcl-2蛋白质表达水平和Bcl-2/Bax比值均显著升高（P<0.05或P<0.01）。结论：白藜芦醇可有效激活大强度运动大鼠肾组织中Keap1-Nrf2信号通路，抑制氧化应激和细胞凋亡，保护肾结构和功能完整性。

Abstract: Objective: To investigate the protective mechanism of resveratrol on exercise-induced kidney injury in rats. Methods: Thirty-two male SD rats, age 7 weeks, were randomly assigned to four groups: control group (CONT, n=8), resveratrol group (RESV, n=8), exercise-induced kidney injury model group (EIKIM, n=8), and resveratrol-treated exercise-induced kidney injury group (REIKIM, n=8). Rats in groups EIKIM and R EIKIM underwent high-intensity treadmill training to establish a model of exercise-induced kidney injury. During the four-week training, rats in groups RESV and REIKIM were administered resveratrol solution intragastrically at a dosage of 150 mg/kg·bw and a volume of 5 mL/kg, 1 hour prior to each training session, and the remaining rats received an equal volume of solvent. Blood and kidney samples were obtained 24 hours post the final training session. Kidney morphology was assessed using HE staining. Levels of serum creatinine (Cr) and urea nitrogen (UN), renal kelch-like ECH-associated protein-1 (Keap1), nuclear factor-E2-related factor 2 (Nrf2), NAD(P)H: quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1) mRNA and protein expression levels, phosphorylated nuclear factor erythroid 2-related factor 2 (p-Nrf2) protein expression level, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GSH-ST) activity, levels of malondialdehyde (MDA), reactive oxygen species (ROS), apoptosis, B cell lymphoma-2 protein (Bcl-2), bcl-2-associated X protein (Bax), cysteinyl aspartate specific proteinase-9 (Caspase-9), and cysteinyl aspartate specific proteinase-3 (Caspase-3) protein expression were detected. Results: After 4 weeks of high-intensity treadmill training, significant renal pathological changes of rats were observed in group EIKIM compared to group CONT, with increased levels of serum Cr and UN, renal Keap1 mRNA and protein expression, MDA, ROS, apoptosis, Bax, Caspase-9 and Caspase-3 protein expression (P<0.01), and decreased levels of renal Nrf2, NQO1, HO-1 mRNA and protein expression, SOD CAT and GSH-ST activity, Bcl-2 protein expression and Bcl-2/Bax ratio (P<0.01). In contrast, after 4 weeks of resveratrol treatment, rats in group REIKIM exhibited significant improvements in renal pathology compared to group EIKIM, with decreased levels of serum Cr and UN, renal Keap1 mRNA and protein expression, MDA, ROS, apoptosis, Bax, Caspase-9 and Caspase-3 protein expression (P<0.05 or P<0.01), and increased levels of renal Nrf2, NQO1, HO-1 mRNA and protein expression, SOD, CAT and GSH-ST activity, Bcl-2 protein expression and Bcl-2/Bax ratio (P<0.05 or P<0.01). Conclusions: Resveratrol can activate the Keap1-Nrf2 signaling pathway of rats subjected to high-intensity exercise effectively, suppress oxidative stress and apoptosis, thereby protecting the integrity of renal structure and function.