银杏内酯对急性肾损伤大鼠的保护作用及机制研究

    Protective effect of ginkgolide on rats with acute kidney injury and its mechanism

    • 摘要: 目的 探讨银杏内酯对急性肾损伤(acute kidney injury,AKI)大鼠的保护作用及对肾组织中腺苷酸激活蛋白激酶(AMP-activated protein kinase,AMPK)及沉默信息调节因子(silent information regulator 1,SIRT1)通路的影响。方法 SD大鼠随机分为正常对照组(Control组)、AKI组和银杏内酯低(2.5 mg/kg)、中(5 mg/kg)、高(7.5 mg/kg)剂量组,每组10只。除Control组经皮下注射生理盐水外,其余各组大鼠均经皮下注射庆大霉素80 mg/kg,6 d,1次/d,建立AKI模型后,Control组、AKI组经腹腔给予生理盐水,银杏内酯各剂量组经腹腔给予相应剂量药物,1次/d,共给药14 d。末次给药1 h后,收集各组大鼠血液、尿液后处死大鼠,取肾组织标本,用酶联免疫吸附法检测肾功能指标血清肌酐(Scr)、血清尿素氮(BUN)、血清炎性因子白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)及尿液中中性粒细胞明胶酶相关脂质运载蛋白(NGAL)和肾损伤因子1(KIM-1)含量;用苏木精-伊红染色(HE)检测各组大鼠肾组织病理变化;以试剂盒检测肾组织氧化应激反应物超氧化物歧化酶(SOD)、丙二醛(MDA)含量;凝胶电泳迁移率转变分析法检测肾脏组织NF-κB活性;蛋白免疫印迹法检测肾组织pAMPK/AMPK、SIRT1蛋白表达情况。结果 与Control组相比,AKI组大鼠可见肾小管上皮细胞崩解脱落、空泡变性等病理损伤,Scr、BUN、NGAL、KIM-1含量、肾组织TNF-α、IL-6、MDA含量及NF-κB活性均明显升高(P<0.05),肾组织SOD活性、pAMPK/AMPK、SIRT1蛋白表达量明显下降(P<0.05)。与AKI组相比,银杏内酯高、中、低剂量组肾小管病理损伤减轻,Scr、BUN、NGAL、KIM-1、TNF-α、IL-6、MDA含量及NF-κB活性均明显降低(P<0.05),SOD活性、pAMPK/AMPK、SIRT1蛋白表达量明显升高(P<0.05)。与银杏内酯低剂量组比较,中、高剂量组肾小管病理损伤减轻,Scr、BUN、NGAL、KIM-1、TNF-α、IL-6、MDA含量及NF-κB活性均明显降低(P<0.05),SOD活性、pAMPK/AMPK、SIRT1蛋白表达量明显升高(P<0.05)。结论 银杏内酯可能通过激活AMPK/SIRT1通路,减轻肾脏氧化应激和炎性损伤,改善AKI大鼠病理损伤。

       

      Abstract: Objective To investigate the protective effect of ginkgolide on acute kidney injury(AKI)rats and its effect on the pathway of AMP activated protein kinase(AMPK)and silent information regulator 1(SIRT1)in kidney tissue. Methods SD rats were randomly divided into control group,AKI model group,low(2.5 mg/kg),medium(5 mg/kg)and high(7.5 mg/kg)dose ginkgolidegroups,with 10 rats in each group.In addition to the control group injected with saline subcutaneously,rats in other groups were injected with gentamicin 80 mg/kg subcutaneously,once a day for 6 days.After establishing the AKI model,rats in the control group and AKI model group were injected intraperitoneally with normal saline,and rats in the ginkgolide groups were injected intraperitoneally with ginkgolide at the corresponding doses,once a day,for a total of 14 days.At one hour after the last administration,the blood and urine of rats in each group were collected,then the rats were killed,and the kidney tissue samples were taken.Enzyme-linked immunosorbent assay was adopted to detect the contents of serum creatinine(Scr),serum urea nitrogen(BUN),serum inflammatory factor interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and neutrophil gelatinase associated lipid transporter(NGAL)and kidney injury factor(KIM-1)in urine;hematoxylin eosin staining(HE)was used to detect the kidney pathological changes in each group;and the contents of superoxide dismutase(SOD)and malondialdehyde(MDA)in kidney tissue were detected withthe kit.Gel electrophoresis mobility shift assay(EMSA)was used to determine the activity of NF-κB in kidney tissue;and Western blot was used to detect the expressions of AMPK/AMPK and SIRT1 in kidney tissue. Results Compared with the Control group,the AKI group showed pathological damages such as disintegration,abscission,vacuolation and degeneration of kidney tubular epithelial cells;the contents of Scr,BUN,NGAL and KIM-1,the contents of TNF-α,IL-6 and MDA in kidney tissues,and the activity of NF-κB in kidney tissue were significantly higher(P<0.05);and the activity of SOD,the protein expressions of pAMPK/AMPK and SIRT1 were significantly lower(P<0.05).Compared with the AKI group,the pathological damage of kidney tubules in the high,middle and low dose ginkgolide groups was alleviated;the contents of Scr,BUN,NGAL,KIM-1,TNF-α,IL-6 and MDA,and the activity of NF-κB in kidney tissue were significantly lower(P<0.05);and the activity of SOD,the protein expressions of pAMPK/AMPK and SIRT1 were significantly higher(P<0.05).Compared with the low dose ginkgolide group,the pathological damage of kidney tubules in the middle and high dose ginkgolide groups was alleviated;the contents of Scr,BUN,NGAL,KIM-1,TNF-α,IL-6 and MDA,and the activity of NF-κB in kidney tissue were significantly lower(P<0.05);and the activity of SOD,the protein expressions of pAMPK/AMPK and SIRT1 were significantly higher(P<0.05). Conclusions By activating AMPK/SIRT1 pathway,ginkgolide may reduce oxidative stress and inflammatory damages of kidney and improve pathological damages of AKI rats.

       

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