Nrf2/ARE信号通路在低氧条件下大鼠肾脏组织表达中的研究
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摘要
目的:研究在急、慢性低氧条件下Nrf2、HO-1、CTGF的表达水平,进一步了解Nrf2、HO-1、CTGF三者间的关系,通过Nrf2/ARE蛋白及基因水平在大鼠肾组织内的变化,从而证实Nrf2/ARE信号通路在低氧条件下对肾脏的保护作用,为低氧条件下肾脏纤维化的防治提供一定依据。方法:选用SPF级SD雄性大鼠为研究对象,研究组在低压氧舱(模拟海拔5000m低氧)环境中饲养,分别为7天、15天、30天组(每组均为8只大鼠),对照组为常氧条件下(海拔2300m)大鼠8只;分别检测不同缺氧时间段大鼠肾脏Nrf2 mRNA、HO-1、mRNA及CTGF mRNA的表达;分别检测不同缺氧时间段大鼠肾脏Nrf2、HO-1及CTGF蛋白水平。结果:大鼠肾脏组织HO-1、mRNA和HO-1蛋白表达水平在低氧7天组和低氧30天组的表达水平明显高于常氧7天组(P<0.05),低氧15天组与常氧7天组比较无明显差异(P>0.05),低氧组之间无明显差异(P>0.05)。CTGF、mRNA和CTGF蛋白表达在低氧7天组、低氧15天组及低氧30天组均明显高于常氧7天组(P<0.05),低氧组之间无明显差异(P>0.05)。Nrf2 mRNA和Nrf2蛋白表达在低氧15天组与常氧7天组及低氧7天组相比较均有统计学差异(P<0.05),与低氧30天组无明显差异(P>0.05)。结论:在缺氧环境下,Nrf2/ARE信号通路被激活,促化HO-1,使其催化血红素降解的作用增强,发挥较强的抗氧化作用,保护肾小管上皮细胞免受氧化损伤,7天后HO-1被大量消耗,蛋白水平表达逐渐下降,随着缺氧时间延长,HO-1再次呈现高表达状态,持续发挥对脏器的保护作用。Nrf2/ARE信号通路对CTGF的调节无明显变化。
Objective:The aim of this study is to explore the expression levels of Nrf2 factor in acute and chronic hypoxia and the relationship between the expression level of Nrf2 factor and HO-1 and CTGF. Through the changes of Nrf2/ARE signaling pathway in rat kidney tissue, the effect of the Nrf2/ARE signaling pathway on kidney protection under hypoxia was confirmed. This provides some basis for the prevention and treatment of renal fibrosis under hypoxia.Methods:SP-F SD male rats were selected for the study, which were raised in low-pressure oxygen tanks(simulated at an altitude of 5000 m low oxygen) in groups 7、15、and 30D(8 rats for each group). The control group was 8 rats under normal oxygen condition(2300 m above sea level). We detected the expression levels, distribution forms and distribution types of Nrf2 mRNA, HO-1 mRNA and CTGF mRNA in kidney of rats with different hypoxia periods. The relationship between Nrf2, HO-1 and CTGF was analyzed, and the expression level of Nrf2/ARE signaling pathway in rat kidney tissue under hypoxia was studied.Results:The expression levels of Nrf2 mRNA, HO-1 mRNA and CTGF mRNAHIF-2 αmRNA were significantly higher in rats with 7 and 30 days of hypoxia than in normal oxygen 7 days(P<0.05). There was no significant difference between the hypoxic groups(P>0.05); The expression of CTGF mRNA was significantly higher in 7 days of hypoxic group, 15 days of hypoxic group and 30 days of hypoxic group than that in 7 days of normal oxygen(P>0.05). Compared with 7 days group at the normal oxygen and 7 days group at the low oxygen, the expression of Nrf2 mRNA in the 15-day group at low oxygen had a statistical difference(P<0.05), which had no clear difference compared with 30 days of hypoxic group(P>0.05).Conclusion:In the hypoxic environment, the Nrf2/ARE signaling pathway is activated and HO-1 is catalyzed to enhance the catalytic effect of heme degradation, play a strong antioxidant effect, and protect renal tubules epithelial cells from oxidative damage. After 7 days, HO-1 was consumed in large quantities, and the protein level expression gradually decreased. With the extension of hypoxia time, HO-1 once again showed a high expression state and continued to exert its protective effect on organs. The Nrf2/ARE signal path has no significant changes in the adjustment of CTGF.
Objective:The aim of this study is to explore the expression levels of Nrf2 factor in acute and chronic hypoxia and the relationship between the expression level of Nrf2 factor and HO-1 and CTGF. Through the changes of Nrf2/ARE signaling pathway in rat kidney tissue, the effect of the Nrf2/ARE signaling pathway on kidney protection under hypoxia was confirmed. This provides some basis for the prevention and treatment of renal fibrosis under hypoxia.Methods:SP-F SD male rats were selected for the study, which were raised in low-pressure oxygen tanks(simulated at an altitude of 5000 m low oxygen) in groups 7、15、and 30D(8 rats for each group). The control group was 8 rats under normal oxygen condition(2300 m above sea level). We detected the expression levels, distribution forms and distribution types of Nrf2 mRNA, HO-1 mRNA and CTGF mRNA in kidney of rats with different hypoxia periods. The relationship between Nrf2, HO-1 and CTGF was analyzed, and the expression level of Nrf2/ARE signaling pathway in rat kidney tissue under hypoxia was studied.Results:The expression levels of Nrf2 mRNA, HO-1 mRNA and CTGF mRNAHIF-2 αmRNA were significantly higher in rats with 7 and 30 days of hypoxia than in normal oxygen 7 days(P<0.05). There was no significant difference between the hypoxic groups(P>0.05); The expression of CTGF mRNA was significantly higher in 7 days of hypoxic group, 15 days of hypoxic group and 30 days of hypoxic group than that in 7 days of normal oxygen(P>0.05). Compared with 7 days group at the normal oxygen and 7 days group at the low oxygen, the expression of Nrf2 mRNA in the 15-day group at low oxygen had a statistical difference(P<0.05), which had no clear difference compared with 30 days of hypoxic group(P>0.05).Conclusion:In the hypoxic environment, the Nrf2/ARE signaling pathway is activated and HO-1 is catalyzed to enhance the catalytic effect of heme degradation, play a strong antioxidant effect, and protect renal tubules epithelial cells from oxidative damage. After 7 days, HO-1 was consumed in large quantities, and the protein level expression gradually decreased. With the extension of hypoxia time, HO-1 once again showed a high expression state and continued to exert its protective effect on organs. The Nrf2/ARE signal path has no significant changes in the adjustment of CTGF.
引文
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7 Liu Xiaocheng,Yang Cheng,He Xiaofeng.Expression of HO-1 in Chonic Renal Insufficiency Rat Kidney and Implication.Journal of Huazhong university of Science and Technology(Med sci),2003,23(3):271～274.
8 朱丹,褚以德,胡文伯,等.高原低氧环境下大鼠肾脏血红氧合酶-1的表达及意义.青海医学院学报,2010,31(3):145～149.
9 Norman JT,Clark IM,Garcia PL.Hypoxia promotes fibrogenesis in human renal fibroblasts.Kidney Int,2000,5(6):2 351～2 366.
10 黄海长,于迎,梁燕,等.低氧刺激结缔组织生长因子表达与肾间质纤维化.中国病理生理杂志,2005,21(3):432～435.
11 Hirotsu Y,Katsuoka F,Funayama R,et al.Nrf2-MafG heterodimers contribute globally to antioxidant and metabolic networks.Nucleic Acids Res,2012,40(20):10 228～10 239.
12 Jo GH,Kim GY,Kim WJ,et al.Sulforaphane induces apoptosis in T24 human urinary bladder cancer cells through a reactive oxygen species-mediated mitochondrial pathway:The involvement of endoplasmic reticulum stress and Nrf2 signaling pathway.Int J Oncol,2014,45(4):1 497～1 506.
13 Ren J,Fan C,Chen N,et al.Resveratrol pretreatment attenuates cerebral ischemic injury byupregulating expression of transcription factor Nrf2 and HO-1 in rats.Neurochem Res,2011,36(12):2 352～2 362.
14 张志群,陈宏辉.核转录因子Nrf2与肝脏疾病的研究进展.中国现代医药杂志,2014,16(9):99～102.
2 Nioi P.The carboxy-terminal Neh3 domain of Nrf2 is required for transcriptional activation.Mol Cell Biol,2005,25(24):10 895～10 906.
3 Zhang J,Tomonori H,Maruyama A,et al.Nrf2 Neh5 domain is differentially untlized in the transactivation of cytoprotective genes.Biochem J,2007,404(3):459～466.
4 KosmiderB,Messier EM,Janssen WJ,et al.Nrf2 protects human alveolar epithelial cells against injury induced by influenza A virus.Respiratory research,2012,13(1):43.
5 靳国恩,韵海霞,马兰,等.高原红细胞增多症动物模型的建立及其生物功能反应.中国组织工程研究与临床康复,2009,13(24):4 713～4 716.
6 Shimizu H,Takahashi T,Suzuki T,et al.Protective effect of heme oxygenase induction in ischemic acute renal failure.Crit Care Med,2000,28(3):809～817.
7 Liu Xiaocheng,Yang Cheng,He Xiaofeng.Expression of HO-1 in Chonic Renal Insufficiency Rat Kidney and Implication.Journal of Huazhong university of Science and Technology(Med sci),2003,23(3):271～274.
8 朱丹,褚以德,胡文伯,等.高原低氧环境下大鼠肾脏血红氧合酶-1的表达及意义.青海医学院学报,2010,31(3):145～149.
9 Norman JT,Clark IM,Garcia PL.Hypoxia promotes fibrogenesis in human renal fibroblasts.Kidney Int,2000,5(6):2 351～2 366.
10 黄海长,于迎,梁燕,等.低氧刺激结缔组织生长因子表达与肾间质纤维化.中国病理生理杂志,2005,21(3):432～435.
11 Hirotsu Y,Katsuoka F,Funayama R,et al.Nrf2-MafG heterodimers contribute globally to antioxidant and metabolic networks.Nucleic Acids Res,2012,40(20):10 228～10 239.
12 Jo GH,Kim GY,Kim WJ,et al.Sulforaphane induces apoptosis in T24 human urinary bladder cancer cells through a reactive oxygen species-mediated mitochondrial pathway:The involvement of endoplasmic reticulum stress and Nrf2 signaling pathway.Int J Oncol,2014,45(4):1 497～1 506.
13 Ren J,Fan C,Chen N,et al.Resveratrol pretreatment attenuates cerebral ischemic injury byupregulating expression of transcription factor Nrf2 and HO-1 in rats.Neurochem Res,2011,36(12):2 352～2 362.
14 张志群,陈宏辉.核转录因子Nrf2与肝脏疾病的研究进展.中国现代医药杂志,2014,16(9):99～102.