柠檬酸对纳米细菌致人肾小管上皮细胞损伤的保护作用
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摘要
目的:体外观察柠檬酸对纳米细菌(NB)致人肾小管上皮细胞损伤的影响,并探讨其可能机制。方法:取对数生长期人肾小管上皮细胞并随机分为五组:对照组、损伤组、干预组A、干预组B及干预组C。检测各组细胞中丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性和一氧化氮(NO)水平,实时荧光定量PCR检测ICAM-1mRNA相对表达量,Western blot检测ICAM-1、NF-κB p65、p-IκBα蛋白的表达。结果:与对照组比较,损伤组MDA含量、NO水平显著增加,SOD活性显著下降,柠檬酸干预后,MDA含量、NO水平降低,SOD活性升高,而且上述改变随柠檬酸浓度增加而明显,差异有统计学意义(P<0.05)。RT-PCR法检测结果显示对照组、损伤组、干预组A、干预组B和干预组C ICAM-1mRNA相对表达量分别为1、36.56±2.65、17.36±1.51、6.76±0.95和2.77±0.46;Western blot法检测结果显示各组细胞ICAM-1蛋白相对表达量分别为0.35±0.05、0.93±0.03、0.86±0.04、0.81±0.04和0.70±0.03;NF-κB p65蛋白相对表达量分别为0.41±0.03、0.99±0.02、0.91±0.03、0.82±0.03和0.55±0.05;p-IκBα蛋白表达量分别为0.21±0.04、0.61±0.05、0.52±0.03、0.44±0.03和0.32±0.02。结论:NB作用于体外培养的人肾小管上皮细胞后可通过激活NF-κB通路来刺激细胞表达ICAM-1,而柠檬酸可显著抑制此过程,且具有剂量依赖性。
Objective:To observe the effect of citric acid on the injury of human renal tubular epithelial cells induced by nanobacteria in vitro and to explore its possible mechanism.Method:Human renal tubular epithelial cells were randomly divided into five groups:control group,injury group,intervention group A,intervention group B and intervention group C.The levels of malondialdehyde(MDA),superoxide dismutase(SOD)and nitric oxide(NO)in the cells were detected.The relative expression of ICAM-1 mRNA was detected by real-time quantitative PCR.The expression of ICAM-1,NF-κB p65 and p-IκBαprotein was detected by western blot.Result:Compared with the control group,the MDA content,NO level increased and SOD activity decreased in the injury group.After citric acid intervention,MDA content,NO level decreased,and SOD activity increased,and these changes increased significantly with increasing concentration of citric acid.The results of RT-PCR showed that the relative expression of ICAM-1 mRNA in the control group,injury group,intervention group A,intervention group B and intervention group C were respectively 1,36.56±2.65,17.36±1.51,6.76±0.95 and 2.77±0.46.Western blot results showed that the relative expression of ICAM-1 protein in each group were 0.35±0.05,0.93±0.03,0.86±0.04,0.81±0.04 and 0.70±0.03.The relative expression of NF-κB p65 protein was 0.41±0.03,0.99±0.02,0.91±0.03,0.82±0.03 and 0.55±0.05 respectively.The expression of p-IκBαprotein was0.21±0.04,0.61±0.05,0.52±0.03,0.44±0.03 and 0.32±0.02.Conclusion:Nanobacteria can stimulate the expression of ICAM-1 by activating NF-κB pathway in human renal tubular epithelial cells cultured in vitro,while citric acid can significantly inhibit this process and is dose-dependent.
Objective:To observe the effect of citric acid on the injury of human renal tubular epithelial cells induced by nanobacteria in vitro and to explore its possible mechanism.Method:Human renal tubular epithelial cells were randomly divided into five groups:control group,injury group,intervention group A,intervention group B and intervention group C.The levels of malondialdehyde(MDA),superoxide dismutase(SOD)and nitric oxide(NO)in the cells were detected.The relative expression of ICAM-1 mRNA was detected by real-time quantitative PCR.The expression of ICAM-1,NF-κB p65 and p-IκBαprotein was detected by western blot.Result:Compared with the control group,the MDA content,NO level increased and SOD activity decreased in the injury group.After citric acid intervention,MDA content,NO level decreased,and SOD activity increased,and these changes increased significantly with increasing concentration of citric acid.The results of RT-PCR showed that the relative expression of ICAM-1 mRNA in the control group,injury group,intervention group A,intervention group B and intervention group C were respectively 1,36.56±2.65,17.36±1.51,6.76±0.95 and 2.77±0.46.Western blot results showed that the relative expression of ICAM-1 protein in each group were 0.35±0.05,0.93±0.03,0.86±0.04,0.81±0.04 and 0.70±0.03.The relative expression of NF-κB p65 protein was 0.41±0.03,0.99±0.02,0.91±0.03,0.82±0.03 and 0.55±0.05 respectively.The expression of p-IκBαprotein was0.21±0.04,0.61±0.05,0.52±0.03,0.44±0.03 and 0.32±0.02.Conclusion:Nanobacteria can stimulate the expression of ICAM-1 by activating NF-κB pathway in human renal tubular epithelial cells cultured in vitro,while citric acid can significantly inhibit this process and is dose-dependent.
引文
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14 Malu R G,Nagoba B S,Jaju C R,et al.Topical use of citric acid for wound bed preparation[J].Int Wound J,2016,13(5):709-712.
15梁茜茜.柠檬酸预防小鼠糖尿病心脏损害的实验研究[C].郑州大学,2016.
16常翠青,王香生.有机酸对人血管内皮细胞的保护作用[J].中华预防医学杂志,2001,35(2):79-82.
2 Tabuchi T,Igoshi K.Regulation of enzyme synthesis of the glyoxylate,the citric acid,and the methylcitric acid cycles in Candida lipolytica[J].Agric Biol Chem,2014,42(12):2381-2386.
3邓耀良,陶芝伟,王翔.含钙肾结石复发的危险因素及个体化防治策略[J].临床泌尿外科杂志,2018,33(2):85-88.
4íft9íoglu N,Miller-Hjelle M A,Hjelle J T,et al.Inhibition of Nanobacteria by Antimicrobial Drugs as Measured by a Modified Microdilution Method[J].Antimicrob Agents Chemother,2002,46(7):2077-2086.
5 Jeremy J Y,Yim A P,Wan S,et al.Oxidative stress,nitric oxide,and vascular disease[J].J Card Surg,2002,17(4):324-327.
6 Wei L F,Zhang H M,Wang S S,et al.Changes of MDAand SOD in Brain Tissue after Secondary Brain Injury with Seawater Immersion in Rats[J].Turk Neurosurg,2016,26(3):384-388.
7于澄钒,黄晓波,陈亮,等.纳米细菌对肾小管上皮细胞的损伤及晶体滞留的影响[J].北京大学学报(医学版)医学版,2010,42(4):436-442.
8雷小康,宋一萌,马潞林.肾细胞癌相关单核苷酸多态性的研究进展[J].临床泌尿外科杂志,2016,31(11):1040-1045.
9 Huang J,Wan D,Li J,et al.Histone acetyltransferase PCAF regulates inflammatory molecules in the development of renal injury[J].Epigenetics,2015,10(1):62-72.
10 Yan S,Zhang X,Zheng H,et al.Clematichinenoside inhibits VCAM-1and ICAM-1expression in TNF-α-treated endothelial cells via NADPH oxidase-dependent IκBkinase/NF-κB pathway[J].Free Radic Biol Med,2015,78:190-201.
11 Huang W,Lan X,Li X,et al.Long non-coding RNAPVT1promote LPS-induced septic acute kidney injury by regulating TNFa and JNK/NF-κB pathways in HK-2cells[J].Int Immunopharmacol,2017,47:134-140.
12 Lee A S,Jung Y J,Thanh T N,et al.Paricalcitol attenuates lipopolysaccharide-induced myocardial inflammation by regulating the NF-κB signaling pathway[J].Int JMol Med,2016,37(4):1023-1029.
13李成文,孙光,邓耀良,等.草酸钙晶体对大鼠肾小管上皮细胞损伤作用的实验研究[J].现代泌尿外科杂志,2006,11(2):198-199.
14 Malu R G,Nagoba B S,Jaju C R,et al.Topical use of citric acid for wound bed preparation[J].Int Wound J,2016,13(5):709-712.
15梁茜茜.柠檬酸预防小鼠糖尿病心脏损害的实验研究[C].郑州大学,2016.
16常翠青,王香生.有机酸对人血管内皮细胞的保护作用[J].中华预防医学杂志,2001,35(2):79-82.