β-胡萝卜素对H_2O_2诱导的斑马鱼肝损伤的保护作用
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摘要
研究β-胡萝卜素对H_2O_2诱导的斑马鱼肝损伤的保护作用。选择适宜浓度的H_2O_2诱导斑马鱼肝脏氧化应激损伤,分别以5、10、20 μg/mL的β-胡萝卜素对其进行保护。给药48 h后检测组织匀浆中谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)、超氧化物歧化酶(superoxide dismutase,SOD)活力和丙二醛(malondialdelyde,MDA)含量;苏木精-伊红染色观察斑马鱼肝组织病理变化情况;实时荧光定量聚合酶链式反应技术检测SOD、CAT和GSTP2以及核转录因子Nrf2 mRNA的相对表达量。结果表明:H_2O_2处理斑马鱼仔鱼48 h后,组织匀浆液中ALT和AST活力升高,SOD活力降低,MDA含量升高;与模型组比较,β-胡萝卜素能够抑制ALT和AST的升高,提高SOD活力,降低MDA含量;病理切片结果显示β-胡萝卜素组能显著改善斑马鱼肝脏组织病理形态;提高Nrf2 mRNA的相对表达量,上调抗氧化酶基因的相对表达量。β-胡萝卜素对H_2O_2诱导的斑马鱼肝损伤具有保护作用。
The protective effect of β-carotene on H_2O_2-induced liver injury in zebrafish was investigated. Liver injury model was induced by H_2O_2 at the proper concentration, and β-carotene at doses of 5, 10 and 20 μg/mL was used to suppress H_2O_2-induced damage. After 48 h of incubation, the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST) and superoxide dismutase(SOD) activities and malondialdelyde(MDA) content were measured. Pathological changes in the liver were observed by hematoxylin-eosin(HE) staining. The relative mRNA expression levels of SOD, CAT, GSTP2 and Nrf2 were assessed by real-time quantitative polymerase chain reaction(qPCR). The results showed that H_2O_2 elevated the levels of ALT, AST and MDA and reduced the level of SOD. Compared with the model group, β-carotene decreased the levels of ALT, AST, and MDA and improved the activity of SOD. Moreover, β-carotene significantly ameliorated hepatic pathological changes and upregulated the relative mRNA expression levels of SOD, CAT, GSTP2 and Nrf2. Finally we conclude that β-carotene has a protective effect on liver injury induced by H_2O_2 in zebrafish.
The protective effect of β-carotene on H_2O_2-induced liver injury in zebrafish was investigated. Liver injury model was induced by H_2O_2 at the proper concentration, and β-carotene at doses of 5, 10 and 20 μg/mL was used to suppress H_2O_2-induced damage. After 48 h of incubation, the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST) and superoxide dismutase(SOD) activities and malondialdelyde(MDA) content were measured. Pathological changes in the liver were observed by hematoxylin-eosin(HE) staining. The relative mRNA expression levels of SOD, CAT, GSTP2 and Nrf2 were assessed by real-time quantitative polymerase chain reaction(qPCR). The results showed that H_2O_2 elevated the levels of ALT, AST and MDA and reduced the level of SOD. Compared with the model group, β-carotene decreased the levels of ALT, AST, and MDA and improved the activity of SOD. Moreover, β-carotene significantly ameliorated hepatic pathological changes and upregulated the relative mRNA expression levels of SOD, CAT, GSTP2 and Nrf2. Finally we conclude that β-carotene has a protective effect on liver injury induced by H_2O_2 in zebrafish.
引文
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[27]陈光海,刘晓平.Keap1-Nrf2信号通路与细胞氧化应激反应相关性研究进展[J].医学理论与实践, 2016, 29(15): 2012-2015. DOI:10.19381/j.issn.1001-7585.2016.15.011.
[28]赵慧慧,王道艳,王春波.胶原蛋白肽经由Nrf2信号通路对H2O2诱导的肝细胞氧化损伤的保护作用[J].现代生物医学进展, 2014(3): 4434-4439.DOI:10.13241/j.cnki.pmb.2014.23.009.
[29] KAZIM S, CEMAL O, HASAN Y, et al. Lycopene improves activation of antioxidant system and Nrf2/HO-1 pathway of muscle in rainbow trout (Oncorhynchus mykiss) with different stocking densities[J].Aquaculture,2014,430:133-138.DOI:10.1016/j.aquaculture.2014.04.007.
[30] NING J L, MO L W, LAI X N. Low- and high-dose hydrogen peroxide regulation of transcription factor NF-E2-related factor 2[J]. Chinese Medical Journal, 2010, 123(8): 1063-1069. DOI:10.3760/cma.j.issn.0366-6999.2010.08.016.
[2] CUCCO M, GUASCO B, MALACARNE G, et al. Effects of β-carotene on adult immune condition and antibacterial activity in the eggs of the Grey Partridge, Perdix perdix[J]. Comparative Biochemistry and Physiology, 2007, 147: 1038-1046. DOI:10.1016/j.cbpa.2007.03.014.
[3] MATOS H R, MARQUES S A, GOMES O F, et al. Lycopene and beta-carotene protect in vivo iron-induced oxidative stress damage in rat prostate[J]. Brazilian Journal of Medical and Biological Research, 2006, 39(2): 203-210.
[4]尹学哲,金延华,何鑫,等.大豆异黄酮对过氧化氢诱导的肝细胞损伤的保护作用[J].大豆科学, 2016, 35(4): 687-690. DOI:10.11861/j.issn.1000-9841.2016.04.0687.
[5]刘红亮,胡磊,王靖凯,等.槲皮素对H2O2损伤PC12细胞的保护效果与机制[J].中国药理学通报, 2014, 30(3): 373-377. DOI:10.3969/j.issn.1001-1978.2014.03.017.
[6] EIMON P M, ASHKENAZI A. The zebrafish as a model organism for the study of apoptosis[J]. Apoptosis, 2010, 15(3): 331-349. DOI:10.1007/s10495-009-0432-9.
[7]刘晓金,陈华利,高燕,等.斑马鱼模式研究现状及应用情况进展[J].辽宁中医药大学学报, 2016, 18(4): 125-127. DOI:10.13194/j.issn.1673-842x.2016.04.039.
[8] WESTERFIELD M. The zebrafish book[M]. Eugene: University of Oregon Press, 1995: 16-21.
[9] INGHAM P W. The power of the zebrafish for disease analysis[J]. Human Molecular Genetics, 2009, 18(R1): 107-112. DOI:10.1093/hmg/ddp091.
[10] KAFINA M D, PAW B H. Methods in molecular biology[M]. New York: Humana Press, 2018: 11-36. DOI:10.1007/978-1-4939-7428-3_2.
[11] MESHALKINA D A, KIZLYK M N, KYSIL E V, et al. Zebrafish models of autism spectrum disorder[J]. Experimental Neurology, 2018, 299:207-216.DOI:10.1016/j.expneurol.2017.02.004.
[12] CHU J, SADLER K C. New school in liver development: lessons from zebrafish[J]. Hepatology, 2009, 50(5): 1656-1663. DOI:10.1002/hep.23157.
[13]韩建,彭维兵,韩利文,等.铜、汞和铅对斑马鱼仔鱼肝脏发育的影响[J].山东科学, 2016, 29(5), 54-59; 75. DOI:10.3976/j.issn.1002-4026.2016.05.009.
[14]付远妨,赵尉丹,蒙西南,等.鸡血藤黄酮乙酸乙酯部位对H2O2诱导RAW264.7细胞氧化应激的调节作用[J].黑龙江畜牧兽医, 2017(12): 145-147; 296.
[15] RYTER S W, KIM H P, HOETZEL A, et al. Mechanisms of cell death in oxidative stress[J]. Antioxidants and Redox Signaling, 2007, 9(1) : 49-89.DOI:10.1089/ars.2007.9.49.
[16]王秋举.L-肉碱对H2O2诱导氧化应激的两种鱼细胞抗氧化功能影响及其机理研究[D].吉林:吉林农业大学,2015:1-117.
[17]冯燕,张波,王少兰,等.H2O2诱导SD大鼠视网膜细胞凋亡过程中细胞内钙离子浓度的变化[J].西安交通大学学报(医学版), 2015, 36(5): 580-586. DOI:10.7652/jdyxb201505002.
[18]金明,王玉娇,金梅花,等.两种细胞建立肝细胞氧化损伤模型比较[J].中国公共卫生, 2015, 31(3): 324-326. DOI:10.11847/zgggws2015-31-03-21.
[19] ZHAO X Y, DOU M M, ZHANG Z H, et al. Protective effect of Dendrobium officinale polysaccharides on H2O2-induced injury in H9c2 cardiomyocytes[J]. Biomedicine and Pharmacotherapy, 2017, 94:72-78.DOI:10.1016/j.biopha.2017.07.096.
[20]尹学哲,王玉娇,尹基峰,等.草苁蓉提取物对HepG2细胞氧化应激损伤的保护作用[J].食品科学, 2015, 36(15): 173-178. DOI:10.7506/spkx1002-6630-201515032.
[21]吴豪,宋捷,仲青香,等.基于斑马鱼模型研究补骨脂肝损伤效应及作用机制[J].南京中医药大学学报, 2017, 33(3): 263-267. DOI:10.14148/j.issn.1672-0482.2017.0263.
[22]黄勇,廖彪.超氧化物歧化酶在斑马鱼早期胚胎中的表达特点[J].陕西理工学院学报(自然科学版), 2008, 24(3): 91-94.
[23]陈汉,王慧君,李学峰,等. 甲基苯丙胺对大鼠脑组织中NO、SOD和MDA的影响[J].中国药物依赖性杂志, 2007, 16(2): 102-104. DOI:10.13936/j.cnki.cjdd1992.2007.02.006.
[24]肖琳,喻剑华.3种维生素联用对四氯化碳所致大鼠慢性肝损伤的防护作用[J].中西医结合肝病杂志, 2001, 11(5): 292-293.
[25]苏伟,王纪宁,刘韬.胡萝卜素的提取及其护肝作用的研究[J].食品科学, 2006, 27(11): 486-488.
[26] CUI Bokang, LIU Su, WANG Qibo, et al. Effect of β-carotene on immunity function and tumour growth in hepatocellular carcinoma rats[J]. Molecules, 2012, 17(7): 8595-8603. DOI:10.3390/molecules17078595.
[27]陈光海,刘晓平.Keap1-Nrf2信号通路与细胞氧化应激反应相关性研究进展[J].医学理论与实践, 2016, 29(15): 2012-2015. DOI:10.19381/j.issn.1001-7585.2016.15.011.
[28]赵慧慧,王道艳,王春波.胶原蛋白肽经由Nrf2信号通路对H2O2诱导的肝细胞氧化损伤的保护作用[J].现代生物医学进展, 2014(3): 4434-4439.DOI:10.13241/j.cnki.pmb.2014.23.009.
[29] KAZIM S, CEMAL O, HASAN Y, et al. Lycopene improves activation of antioxidant system and Nrf2/HO-1 pathway of muscle in rainbow trout (Oncorhynchus mykiss) with different stocking densities[J].Aquaculture,2014,430:133-138.DOI:10.1016/j.aquaculture.2014.04.007.
[30] NING J L, MO L W, LAI X N. Low- and high-dose hydrogen peroxide regulation of transcription factor NF-E2-related factor 2[J]. Chinese Medical Journal, 2010, 123(8): 1063-1069. DOI:10.3760/cma.j.issn.0366-6999.2010.08.016.