青蒿琥酯通过Keap1/Nrf2通路抑制氧化应激改善犬急性肾损伤的体内外分析

本研究旨在观察青蒿琥酯对庆大霉素诱导犬急性肾损伤的抗氧化调节作用及其机制。将20只犬随机等分成4组：对照组(Control)、庆大霉素模型组(GM)、青蒿琥酯治疗组(GM+ART)、青蒿琥酯+ML385干预组(GM+ART+ML385)。除对照组，其他组犬采用注射GM建立AKI模型。成功造模后，GM+ART组给与青蒿琥酯，ART+ML385组给与ART和ML385，对照组和GM组给予生理盐水，试验期12 d。用不同浓度GM与MDCK细胞共培养，确定最佳浓度为4.0 mmol·L^-1，用相同方法确定ART最佳浓度为50.0 μmol·L^-1。将体外培养MDCK细胞、过表达Kelch样ECH相关蛋白1(Keap1)的MDCK细胞(M-K)和敲减核因子E2相关因子2(Nrf2)表达的MDCK细胞(M-SiNrf2)分别分成3组：健康对照组、GM对照组和GM+ART干预组，共培养24 h后用于检测。结果显示：1)在动物试验中，GM+ART组肌酐(Cr)、尿素氮(UN)及肾损伤因子1(KIM-1)水平显著低于GM组，GM+ART+ML385组Cr、UN及KIM-1水平显著高于GM+ART组。2)在动物试验中，与GM组比较，GM+ART组Nrf2和谷胱甘肽半胱氨酸连接酶催化亚基(GCLC)蛋白表达上调，Keap1蛋白表达下调，肾组织匀浆中总超氧化物歧化酶(T-SOD)和谷胱甘肽(GSH)水平升高，丙二醛(MDA)水平降低。与GM+ART组比较，给与ML385后Nrf2和GCLC蛋白表达下调，T-SOD和GSH水平降低。3)在细胞试验中，与4.0 mmol·L^-1 GM共培养的MDCK细胞比较，加入50.0 μmol·L^-1ART能显著提高细胞增殖率，降低ROS水平，下调Keap1表达，上调Nrf2、血红素氧合酶1(HO1)及GCLC表达。4)在细胞试验中，与MDCK细胞比较，在GM+ART相同处理下，M-K细胞和M-SiNrf2细胞Keap1蛋白表达上调，Nrf2、HO1及GCLC蛋白表达下调，细胞增殖率降低，ROS含量升高(P＜0.05或P＜0.01)。综上所述，青蒿琥酯对庆大霉素诱导犬急性肾损伤具有保护作用，其作用机制与青蒿琥酯通过Keap1/Nrf2信号通路抑制氧化应激反应有关。

Artesunate Improves Acute Renal Injury in Dogs by Inhibiting Oxidative Stress Via Keap1/Nrf2 Signaling Pathway in vitro and in vivo

The aim of this study was to investigate the antioxidant effect of artesunate (ART) on acute kidney injury induced by gentamicin (GM) in dogs and its mechanism. Twenty dogs were randomly divided into 4 groups:control group (Control), gentamicin model group (GM), artesunate group (GM+ART), artesunate + ML385 group (GM+ART + ML385). In addition to the control group, other groups were injected with GM to establish AKI model. After successful modeling, Dogs in GM+ART group were given ART, and GM+ART+ML385 group were given ART and ML385 at the same time. The control group and GM group were given the same volume of normal saline. The test period is 12 days. Different concentrations of GM were co-cultured with MDCK cells, and the optimal concentration was 4.0 mmol·L^-1. The optimum concentration of ART was determined to be 50.0 μ mol·L^-1 by the same method. The MDCK cells, MDCK cells overexpressing kelch like ECH related protein 1 (Keap1) (M-K) and MDCK cells knockdown nuclear factor E2 related factor 2 (Nrf2) (M-SiNrf2) were divided into three groups respectively:Healthy control group, GM control group and GM + ART intervention group, co-culturing for 24 hours. The results show as follows:1) In animal experiments, the levels of Cr, UN and KIM-1 in GM + ART group were significantly lower than those in GM Group, and the levels of Cr, UN and KIM-1 in GM + ART + ML385 group were significantly higher than those in GM + ART group. 2) In animal experiments, compared with GM Group, the expression of Nrf2 and glutathione cysteine ligase catalytic subunit (GCLC) protein was up-regulated, Keap1 protein was down-regulated, the levels of total superoxide dismutase (T-SOD) and glutathione (GSH) in renal homogenate were increased, and the level of malondialdehyde (MDA) was decreased in GM + ARTgroup. Compared with GM + ART group, the expression of Nrf2 and GCLC decreased, and the levels of T-SOD and GSH decreased after intervening by ML385. 3) In the cell test, compared with MDCK cells co cultured with 4.0 mmol·L^-1 GM, the dose of 50.0 μmol·L^-1 ART added could significantly increase the cell proliferation rate, reduce the level of ROS, down regulate the expression of Keap1 and up regulate the expression of Nrf2, heme oxygenase 1 (HO1) and GCLC. 4) In the cell test, compared with MDCK cells, the expression of Keap1 was up-regulated, the expression of Nrf2, HO1 and GCLC were down regulated, the cell proliferation rate was decreased, and the content of ROS was increased both in M-K cells and M-SiNrf2 cells under the same treatment of GM + ART(P<0.05 or P<0.01). In conclusion, artesunate has a protective effect on acute renal injury induced by gentamicin in dogs, and its mechanism is related to inhibit oxidative stress via Keap1/Nrf2 signaling pathway.