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

doi:10.11843/j.issn.0366-6964.2022.07.031

摘要/Abstract

摘要： 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。

关键词: 青蒿琥酯, 庆大霉素, 犬, Keap1/Nrf2通路, 氧化应激, 急性肾损伤

Abstract: 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.

Key words: artesunate, gentamicin, dog, Keap1/Nrf2 signaling pathway, oxidative stress, acute kidney injury

中图分类号:

S856.5

陆江, 朱道仙, 刘莉, 郝福星, 吴植, 傅宏庆. 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[J]. 畜牧兽医学报, 2022, 53(7): 2343-2353.

LU Jiang, ZHU Daoxian, LIU Li, HAO Fuxing, WU Zhi, FU Hongqing. Artesunate Improves Acute Renal Injury in Dogs by Inhibiting Oxidative Stress Via Keap1/Nrf2 Signaling Pathway in vitro and in vivo[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2343-2353.

参考文献 30

[1]	罗俊崇,张梦丹,黎晓雯,等.一例犬急性肾衰的间歇性血液透析治疗报告[J].动物医学进展, 2018, 39(9):134-136.LUO J C, ZHANG M D, LI X W, et al. A case report of acute renal failure treated by intermittent hemodialysis[J]. Progress in Veterinary Medicine, 2018, 39(9):134-136.(in Chinese)
[2]	邓园园,董伟,李淼,等. 50例犬急性肾衰竭的临床调查分析[J].湖南畜牧兽医, 2017(2):26-28.DENG Y Y, DONG W, LI M, et al. Clinical investigation and analysis of acute renal failure in dogs by 50 cases[J]. Hunan Journal of Animal Science&Veterinary Medicine, 2017(2):26-28.(in Chinese)
[3]	GORI E, LIPPI I, GUIDI G, et al. Acute pancreatitis and acute kidney injury in dogs[J]. Vet J, 2019, 245:77-81.
[4]	GUIOT  G, GUIMAR ES-OKAMOTO P T C, CHACAR F C, et al. Reversal of acute renal injury after peritoneal dialysis in a dog[J]. Rev Bras Med Vet, 2015, 37(2):153-157.
[5]	CARCILLO J A, HALSTEAD E S, HALL M W, et al. Three hypothetical inflammation pathobiology phenotypes and pediatric sepsis-induced multiple organ failure outcome[J]. Pediatr Crit Care Med, 2017, 18(6):513-523.
[6]	PAVLAKOU P, LIAKOPOULOS V, ELEFTHERIADIS T, et al. Oxidative stress and acute kidney injury in critical Illness:pathophysiologic mechanisms-biomarkers-interventions, and future perspectives[J]. Oxid Med Cell Longev, 2017, 2017:6193694.
[7]	RADA P, ROJO A I, EVRARD-TODESCHI N, et al. Structural and functional characterization of Nrf2 degradation by the glycogen synthase kinase 3/β-Trcp axis[J]. Mol Cell Biol, 2012, 32(17):3486-3499.
[8]	KONG W W, FU J Q, LIU N, et al. Nrf2 deficiency promotes the progression from acute tubular damage to chronic renal fibrosis following unilateral ureteral obstruction[J]. Nephrol Dial Transplant, 2018, 33(5):771-783.
[9]	岑彦艳,赵祎博,李攀,等.青蒿琥酯的药代动力学以及相关药理作用研究进展[J].中国中药杂志, 2018, 43(19):3970-3978.CEN Y Y, ZHAO Y B, LI P, et al. Research progress on pharmacokinetics and pharmacological activities of artesunate[J]. China Journal of Chinese Materia Medica, 2018, 43(19):3970-3978.(in Chinese)
[10]	骆琼珍,林江涛,李鸿,等.青蒿琥酯对烟草烟雾暴露小鼠肺氧化损伤及Nrf2蛋白表达的影响[J].中华医学杂志, 2016, 96(12):960-965.LUO Q Z, LIN J T, LI H, et al. Effects of artesunate on cigarette smoke-induced lung oxidative damage in mice and the expression of Nrf2 and the possible mechanism[J]. National Medical Journal of China, 2016, 96(12):960-965.(in Chinese)
[11]	张萍,杜娟,沙聪,等.庆大霉素诱导的犬肾衰竭模型治疗期间的NGAL和Cys-C变化[J].动物医学进展, 2020, 41(2):74-79.ZHANG P, DU J, SHA C, et al. Changes and significance of NGAL and Cys-C during treatment of gentamicin-induced renal failure model in dogs[J]. Progress in Veterinary Medicine, 2020, 41(2):74-79.(in Chinese)
[12]	GENG R Q, REN Y Y, RAO R, et al. Titanium dioxide nanoparticles induced HeLa cell necrosis under UVA radiation through the ROS-mPTP pathway[J]. Nanomaterials, 2020, 10(10):2029.
[13]	DENG S, DAI G, CHEN S, et al. Dexamethasone induces osteoblast apoptosis through ROS-PI3K/AKT/GSK3β signaling pathway[J]. Biomed Pharmacother, 2019, 110:602-608.
[14]	ZHANG X L, YU L, XU H X. Lysosome calcium in ROS regulation of autophagy[J]. Autophagy, 2016, 12(10):1954-1955.
[15]	MA H J, ZHAO J J, MENG H B, et al. Carnosine-Modified fullerene as a highly enhanced ROS scavenger for mitigating acute oxidative stress[J]. ACS Appl Mater Interfaces, 2020, 12(14):16104-16113.
[16]	罗珊,宋立群,马晓鹏,等.肾间质纤维化氧化应激相关信号通路与中医药研究进展[J].中医药学报, 2020, 48(12):79-84.LUO S, SONG L Q, MA X P, et al. TCM research progress of oxidative stress-related signal pathways in renal interstitial fibrosis[J]. Acta Chinese Medicine and Pharmacology, 2020, 48(12):79-84.(in Chinese)
[17]	杨玲,严磊,张绘艳,等.依达拉奉在庆大霉素诱导MDCK细胞凋亡中的保护作用[J].中国兽医科学, 2019, 49(3):378-385.YANG L, YAN L, ZHANG H Y, et al. Protection of edaravone on apoptosis induced by gentamicin in MDCK cells[J]. Chinese Veterinary Science, 2019, 49(3):378-385.(in Chinese)
[18]	杨倩倩. PINK1在调控顺铂及庆大霉素耳毒性中的机制研究[D].济南:山东大学, 2019.YANG Q Q. Study on the mechanism of PINK1 in regulating cisplatin and gentamicin-induced ototoxity[D]. Ji'nan:Shandong University, 2019.(in Chinese)
[19]	OLVERA-POSADA D, DAYARATHNA T, DION M, et al. KIM-1 is a potential urinary biomarker of obstruction:results from a prospective cohort study[J]. J Endourol, 2017, 31(2):111-118.
[20]	YANG L, BROOKS C R, XIAO S, et al. KIM-1-mediated phagocytosis reduces acute injury to the kidney[J]. J Clin Invest, 2015, 125(4):1620-1636.
[21]	NGO V, KARUNATILLEKE N, SONG Z, et al. Oxidative stress-induced aggregation of Keap1 impairs Nrf2 regulation[J]. Free Radic Biol Med, 2020, 159(S1):S58.
[22]	LYAKHOVICH V V, VAVILIN V A, ZENKOV N K, et al. Active defense under oxidative stress. The antioxidant responsive element[J]. Biochemistry, 2006, 71(9):962-974.
[23]	SHELTON L M, PARK B K, COPPLE I M. Role of Nrf2 in protection against acute kidney injury[J]. Kidney Int, 2013, 84(6):1090-1095.
[24]	窦彩霞,王倩,安建勇,等. Keap1-Nrf2/ARE信号通路及其在畜禽抗氧化中的研究进展[J].中国畜牧兽医, 2019, 46(9):2567-2574.DOU C X, WANG Q, AN J Y, et al. The research progress of Keap1-Nrf2/ARE signaling pathway and its role in the antioxidation of livestock and poultry[J]. China Animal Husbandry&Veterinary Medicine, 2019, 46(9):2567-2574.(in Chinese)
[25]	LI C J, CHENG L, WU H T, et al. Activation of the Keap1-NRF2-ARE signaling pathway reduces oxidative stress in Hep2 cells[J]. Mol Med Rep, 2018, 18(3):2541-2550.
[26]	崔勤涛,王俊华,刘晓晨,等.大黄素激活Nrf2/ARE/HO-1信号通路对心肌缺血再灌注损伤大鼠心功能保护作用[J].安徽医科大学学报, 2020, 55(6):894-900.CUI Q T, WANG J H, LIU X C, et al. Protective effect of emodin on cardiac function in rats with myocardial ischemia reperfusion injury by activating Nrf2/ARE/HO-1 signaling pathway[J]. Acta Universitatis Medicinalis Anhui, 2020, 55(6):894-900.(in Chinese)
[27]	HASANVAND D, AMIRI I, ASL S S, et al. Effects of CeO2 nanoparticles on the HO-1, NQO1 and GCLC expression in the testes of diabetic rats[J]. Can J Physiol Pharm, 2018, 96(9):963-969.
[28]	裴若男,杨帆,廖建昭,等.高铜日粮对肉鸡肾氧化损伤和Nrf2信号通路相关基因表达的影响[J].畜牧兽医学报, 2019, 50(9):1912-1919.PEI R N, YANG F, LIAO J Z, et al. The effect of high dietary copper on oxidative damage and expression of Nrf2 signaling pathway related genes in the kidneys of broilers[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(9):1912-1919.(in Chinese)
[29]	谢志明,吴春梅,陈少元,等.柴胡多糖通过抑制氧化应激和炎症反应减轻铅诱导小鼠肝肾损伤的研究[J].畜牧兽医学报, 2021, 52(9):2660-2672.XIE Z M, WU C M, CHEN S Y, et al. Study on bupleurum polysaccharide reduces lead-induced liver and kidney injury in mice by inhibiting oxidative stress and inflammation[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9):2660-2672.(in Chinese)
[30]	AMINZADEH M A, REISMAN S A, VAZIRI N D, et al. The synthetic triterpenoid RTA dh404(CDDO-dhTFEA) restores Nrf2 activity and attenuates oxidative stress, inflammation, and fibrosis in rats with chronic kidney disease[J]. Xenobiotica, 2014, 44(6):570-578.