Lepr~(db/db)小鼠肾损伤机制探讨
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摘要
目的探讨瘦素受体完全缺陷的Lepr~(db/db)小鼠肾损伤机制。方法选取28周龄瘦素受体杂合缺陷的Lepr~(db/+)(作为对照)与Lepr~(db/db)雄性小鼠各10只,禁食8 h后,分别测量两组小鼠体质量、空腹血糖(FBG)和糖化血红蛋白(HbA1c)水平。小鼠经股动脉采血后处死。采用试剂盒检测血清中肌酐(CRE)、尿素氮(BUN)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)、丙二醛(MDA)的水平。酶联免疫吸附法(ELISA)检测血清中炎性细胞因子白细胞介素-1β(IL-1β)、单核细胞趋化因子-1(MCP-1)、肿瘤坏死因子-α(TNF-α)的表达水平。取肾进行病理观察。Western blot检测肾组织中核因子E2相关因子2(Nrf2)及核因子-κB(NF-κB)蛋白表达。提取肾组织细胞线粒体,Western blot检测线粒体中硫辛酸合成酶(lipoic acid synthase, LIAS)蛋白的表达水平。结果与Lepr~(db/+)小鼠相比,Lepr~(db/db)小鼠体质量、FPG、HbA1c、CRE、BUN水平均升高,差异有统计学意义(P<0.05)。病理观察发现,Lepr~(db/+)小鼠肾细胞结构完整,而Lepr~(db/db)小鼠肾小球体积增大,基底膜及毛细血管壁增厚,系膜细胞和系膜基质增多。与Lepr~(db/+)小鼠相比,Lepr~(db/db)小鼠血清中GSH水平降低,MDA和炎性因子MCP-1、IL-1β、TNF-α水平均升高,差异均有统计学意义(P<0.05);Lepr~(db/db)组小鼠肾脏线粒体内LIAS和肾组织中Nrf2蛋白表达量均降低,而NF-κB蛋白水平升高,差异均有统计学意义(P<0.05)。结论 28周龄Lepr~(db/db)小鼠存在氧化应激和炎症反应,肾损伤机制可能与LIAS调控Nrf2和NF-κB有关。
Objective To study the mechanism of renal injury in Lepr~(db/db) mice with the leptin receptor homozygous deficiency. Methods Ten male of 28-week-old Lepr~(db/+) mice with leptin receptor heterozygous deficiency were selected as control group and ten male Lepr~(db/db) mice with leptin receptor homozygous deficiency were used in this study. After fasting for 8 hours, the body mass, fasting blood glucose(FBG) and glycosylated hemoglobulin(HbA1c) of the mice were measured. Blood of the mice was obtained from femoral artery before euthanasia. Serum creatinine(CRE), blood urea nitrogen(BUN), superoxide dismutase(SOD), glutathione(GSH) and malonaldehyde(MDA) were detected by corresponding kits, and serum interleukin-1β(IL-1β), monocyte chemotactic protein-1(MCP-1) and tumor necrosis factor-α(TNF-α) were measured using enzyme-linked immunosorbent assay(ELISA) method. The kidney was taken for pathological observation. The expression levels of nuclear factor E2-related factor 2(Nrf2) and nuclear factor kappa B(NF-κB) in renal were analyzed by Western blotting. The mitochondria of renal was isolated by the corresponding kit. Meanwhile, the expression level of lipoic acid synthase(LIAS) in renal mitochondria was measured by Western blotting. Results The body mass, FPG, HbA1 c, CRE and BUN levels of the Lepr~(db/db) mice were significantly increased in comparison with the Lepr~(db/+) mice(P<0.05). Compared with the Lepr~(db/+) mice, the Lepr~(db/db) mice renal exhibited glomerular hypertrophy, thickened basement membrane and capillary wall, the mesangial matrix expansion and mesangial cell hyperplasia. Compared with the Lepr~(db/+) mice, the serum level of GSH in the Lepr~(db/db) mice was decreased significantly(P<0.05). The levels of MDA and concentrations of MCP-1, IL-1β and TNF-α in serum of the Lepr~(db/db) mice were higher than those of the Lepr~(db/+) mice(P<0.05). Compared with the Lepr~(db/+) mice, the expression of LIAS and Nrf2 protein in the Lepr~(db/db) mice renal were decreased(P<0.05), while the expression of NF-κB protein was increased(P<0.05). Conclusion LIAS, Nrf2 and NF-κB might play significant roles through regulation of oxidative stress and inflammation in the renal injury of Lepr~(db/db) mice.
Objective To study the mechanism of renal injury in Lepr~(db/db) mice with the leptin receptor homozygous deficiency. Methods Ten male of 28-week-old Lepr~(db/+) mice with leptin receptor heterozygous deficiency were selected as control group and ten male Lepr~(db/db) mice with leptin receptor homozygous deficiency were used in this study. After fasting for 8 hours, the body mass, fasting blood glucose(FBG) and glycosylated hemoglobulin(HbA1c) of the mice were measured. Blood of the mice was obtained from femoral artery before euthanasia. Serum creatinine(CRE), blood urea nitrogen(BUN), superoxide dismutase(SOD), glutathione(GSH) and malonaldehyde(MDA) were detected by corresponding kits, and serum interleukin-1β(IL-1β), monocyte chemotactic protein-1(MCP-1) and tumor necrosis factor-α(TNF-α) were measured using enzyme-linked immunosorbent assay(ELISA) method. The kidney was taken for pathological observation. The expression levels of nuclear factor E2-related factor 2(Nrf2) and nuclear factor kappa B(NF-κB) in renal were analyzed by Western blotting. The mitochondria of renal was isolated by the corresponding kit. Meanwhile, the expression level of lipoic acid synthase(LIAS) in renal mitochondria was measured by Western blotting. Results The body mass, FPG, HbA1 c, CRE and BUN levels of the Lepr~(db/db) mice were significantly increased in comparison with the Lepr~(db/+) mice(P<0.05). Compared with the Lepr~(db/+) mice, the Lepr~(db/db) mice renal exhibited glomerular hypertrophy, thickened basement membrane and capillary wall, the mesangial matrix expansion and mesangial cell hyperplasia. Compared with the Lepr~(db/+) mice, the serum level of GSH in the Lepr~(db/db) mice was decreased significantly(P<0.05). The levels of MDA and concentrations of MCP-1, IL-1β and TNF-α in serum of the Lepr~(db/db) mice were higher than those of the Lepr~(db/+) mice(P<0.05). Compared with the Lepr~(db/+) mice, the expression of LIAS and Nrf2 protein in the Lepr~(db/db) mice renal were decreased(P<0.05), while the expression of NF-κB protein was increased(P<0.05). Conclusion LIAS, Nrf2 and NF-κB might play significant roles through regulation of oxidative stress and inflammation in the renal injury of Lepr~(db/db) mice.
引文
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[17] 徐光标,陈德君,陈伟珍.雷公藤多苷片对糖尿病肾病患者临床疗效及炎症因子水平影响研究.中华中医药学刊,2017,35(8):2206-2208.
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[2] HOU X,LU J,WENG J,et a1.Impact of waist circumference and body mass index on risk of eardiometabolie disorder and cardiovascular disease in Chinese adults:a national diabetes and metabolic disorders survey.PLoS One,2013,8(3):e57319[2018-06-11].https://doi.org/10.1371/journal.pone.0057319.
[3] NAVARRO-GONZALEZ JF,MORA-FERNANDEZ C.The role of inflame- matory cyto-kines in diabetic nephropathy.J Am Soc Nephrol,2008,19(3):433 -442.
[4] ELMARAKBY AA,SULLIVAN JC.Relationship between oxidative stress and inflammatory cytokines in diabetic nephropathy.Cardiovasc Ther,2012,30(1):49-59.
[5] NAVARRO-GONZALEZ JF,MORA-FERNANDEZ C,MUROSD FM,et al.Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy.Nat Rev Nephrol,2011,7 (6 ):327- 340.
[6] SUH JH,WANG H,LIU RM,et al.(R)-alpha-lipoic acid reverses the age-related loss in tissues:evidence for increased cysteine requirement for GSH synthesis.Arch Biochem Biophys,2004,42(3):126-135.
[7] LIU J.The effects and mechanisms of mitochondrial nutrient alpha-lipoic acid on improving age-associated mitochondrial and cognitive dysfunction:an overview.Neurochem Res,2008,33(1):194-203.
[8] PACKER L,KRAEMER K,RIMBACH G.Molecular aspects of lipoic acid in the prevention of diabetes complications.Nutrition,2001,17(10):888-895.
[9] 彭强,赵英政,闫婷婷,等.硫辛酸合成酶在Leprdb/db小鼠肝肾中的表达.中国实验动物学报,2018,26(2):145-149.
[10] AZUSHIMA K,GURLEY SB,COFFMAN TM.Modelling diabetic nephropathy in mice.Nat Rev Nephrol,2018,14(1):48-56.
[11] JIANG Z,LU W,ZENG Q,et al.High glucose-induced excessive reactive oxygen species promote apoptosis through mitochondrial damage in rat cartilage endplate cells.J Orthop Res,2018[2018-06-11].https://doi.org/10.1002/jor.24016.
[12] SAKAI N,WADA T.Revisiting inflammation in diabetic nephropathy:the role of the Nlrp3 inflammasome in glomerular resident cells.Kidney Int,2015,87(1):12-14.
[13] JEONG WS,JUN M,KONG AN.Nrf2:a potential molecular target for cancer chemoprevention by natural compounds.Antioxid Redox Signal,2006,8(1-2):99-106.
[14] 黄婉冰,叶增纯,李吟,等.线粒体功能障碍参与糖尿病肾脏疾病足细胞损伤的初步研究.新医学,2017,48(5):308-312.
[15] LV C,MAHARJAN S,WANG Q,et al.α-lipoic acid promotes neurological recovery after ischemic stroke by activating the Nrf2/HO-1 pathway to attenuate oxidative damage.Cell Physiol Biochem,2017,43(3):1273-1287.
[16] ZHANG J,MCCULLOUGH PA.Lipoic acid in the prevention of acute kidney injury.Nephron,2016,134(3):133-140.
[17] 徐光标,陈德君,陈伟珍.雷公藤多苷片对糖尿病肾病患者临床疗效及炎症因子水平影响研究.中华中医药学刊,2017,35(8):2206-2208.
[18] TUTTLE KR.Linking metabolism and immunology:diabetic nephropathy is an inflammatory disease.J Am Soc Nephrol,2005,16(6):1537-1538.
[19] RASHID S,NAFEES S,SIDDIQI A,et al.Partial protection by 18β Glycrrhetinic acid against Cisplatin induced oxidative intestinal damage in wistar rats:possible role of NF-κB and caspases.Pharmacol Rep,2017,69(5):1007-1013.
[20] ZHANG WJ,FREI B.Alpha-lipoic acid inhibits TNF-alpha-induced NF-kappaB activation and adhesion molecule expression in human aortic endothelial cells.FASEB J ,2001,15(13):2423-2432.