阻断β-catenin基因对亚砷酸钠诱导大鼠肺组织氧化应激的影响
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摘要
目的探讨阻断β-catenin基因对亚砷酸钠致大鼠肺组织氧化应激的影响。方法将48只健康SPF(specific pathogen free)级SD大鼠按性别和体质量随机分为对照组(超纯水)、低剂量组(0.45 mg/kg)、中剂量组(2.25 mg/kg)、空白对照高剂量组(11.25 mg/kg)、转染β-catenin siRNA高剂量组(11.25 mg/kg)和转染阴性对照高剂量组(11.25 mg/kg),自由经口饮水染毒16周,β-catenin siRNA转染1周。HE染色法观察大鼠肺组织病理学;石墨炉原子吸收法测定大鼠血液、尿液和肺组织中砷水平;化学荧光法测定ROS含量;ELISA法测定SOD活性、GSH和MDA含量。结果低剂量组、中剂量组和空白对照高剂量组大鼠肺组织随着染砷剂量和染砷时间的增加而呈现不同程度的炎症细胞浸润,肺泡间隔增宽,肺泡融合;空白对照高剂量组大鼠肺组织病理损伤较严重,出现部分肺组织纤维化。低剂量组、中剂量组和空白对照高剂量组大鼠血砷、尿砷和肺组织砷水平随着染砷剂量增加和染砷时间延长呈上升趋势(P<0.05)。空白对照高剂量组大鼠肺组织中ROS荧光值较对照组、低剂量组和中剂量组增加(P<0.05);MDA含量较对照组增加(P<0.05)。空白对照高剂量组大鼠肺组织中SOD和GSH含量较对照组和低剂量组减少(P<0.05)。阻断β-catenin基因后,转染β-catenin siRNA高剂量组大鼠肺组织病理损伤较空白对照高剂量组和转染阴性对照高剂量组严重,出现大部分肺组织纤维化。转染β-catenin siRNA高剂量组和转染阴性对照高剂量组大鼠血砷、尿砷和肺组织水平较对照组、低剂量组和中剂量组增加明显(P<0.05)。转染β-catenin siRNA高剂量组和转染阴性对照高剂量组大鼠肺组织中ROS荧光值较对照组、低剂量组和中剂量组增加(P<0.05);MDA含量较对照组增加(P<0.05)。而转染β-catenin siRNA高剂量组和转染阴性对照高剂量组大鼠肺组织SOD活性和GSH含量较对照组和低剂量组减少(P<0.05)。大鼠肺组织砷含量和ROS荧光值呈显著正相关(r_s=0.655,P<0.001)。大鼠肺组织中ROS荧光值与SOD活性呈低度负相关(r_s=-0.441,P<0.002);与GSH含量呈显著负相关(r_s=-0.599,P<0.001);与MDA含量呈低度正相关(r_s=0.395,P<0.006)。结论长期砷暴露使砷在大鼠体内持续性蓄积,诱导大鼠肺组织氧化应激,导致大鼠肺组织损伤。阻断β-catenin基因,对大鼠肺组织氧化应激影响较小。
Objective To investigate the effects of blocking the β-catenin gene on oxidative stress induced by sodium arsenite in rat lung. Methods 48 healthy SPF(Specific Pathogen Free) SD rats were randomly divided into control group(ultra-pure water), low dose group(0.45 mg/kg), medium dose group(2.25 mg/kg), blank control high dose group(11.25 mg/kg), high dose group transfected with β-catenin siRNA(11.25 mg/kg) and negative control high dose group(11.25 mg/kg) according to gender and body weight, free oral drinking water for 16 weeks, and β-catenin siRNA transfection for 1 week. The lung histopathology was observed by Hematoxylin-eosin(H&E) staining. The levels of arsenic in blood, urine and lung tissue of rats were determined by graphite furnace atomic absorption spectrometry. The content of ROS was determined by chemical fluorescence method. SOD activity, GSH and MDA content were determined by ELISA. Results The lung tissue of rats in low, medium and blank control high dose groups showed different degrees of inflammatory cell infiltration, alveolar septum widening and alveolar fusion with the increase of arsenic dosage. The lung tissue of rats in blank control high dose group was severely damaged and some pulmonary fibrogenesis appeared. The arsenic levels in blood, urine and lung tissues of rats in low, medium and blank control high dose groups were increased with the increase of arsenic dosage and time(P<0.05). The fluorescence values of ROS in blank control high dose group of lung tissue in rats was significantly higher than those in control, low and medium groups(P<0.05), and the content of MDA was remarkably higher than that in control group(P<0.05). SOD and GSH content in blank control high dose group was markedly lower than those in control and low dose groups(P<0.05). After blocking the β-catenin gene, most of the pulmonary fibrosis appeared in the lung tissue of rats in high dose group transfected with β-catenin siRNA, was shown to be more serious than that in blank control and negative control high dose groups. The levels of arsenic in blood, urine and lung tissue of rats in high dose group transfected with β-catenin siRNA and negative control high dose group were significantly higher than those in control, low and medium dose groups(P<0.05). The fluorescence value of ROS in lung tissue of rats in high dose group transfected with β-catenin siRNA and negative control high dose group were higher than that control, low and medium groups(P<0.05), and the content of MDA was higher than that control group(P<0.05). However, SOD and GSH contents in lung tissue of rats in high dose group transfected with β-catenin siRNA and negative control high dose group were lower than those in control and low dose groups(P<0.05). There was a significant positive correlation between arsenic content and ROS fluorescence in rat lung tissue(r_s=0.655, P<0.001). Lung tissue of ROS fluorescence was negatively correlated with SOD activity(r_s=-0.441, P<0.002), negatively correlated with GSH content(r_s=-0.599, P<0.001), and positively correlated with MDA content(r_s=0.395, P<0.006). Conclusion Long-term exposure to arsenic could cause persistent accumulation of arsenic in rats, which may result in oxidative stress in rat lung tissue and may lead to lung injury. Blocking the β-catenin gene had little effect on oxidative stress in rat lung tissue.
Objective To investigate the effects of blocking the β-catenin gene on oxidative stress induced by sodium arsenite in rat lung. Methods 48 healthy SPF(Specific Pathogen Free) SD rats were randomly divided into control group(ultra-pure water), low dose group(0.45 mg/kg), medium dose group(2.25 mg/kg), blank control high dose group(11.25 mg/kg), high dose group transfected with β-catenin siRNA(11.25 mg/kg) and negative control high dose group(11.25 mg/kg) according to gender and body weight, free oral drinking water for 16 weeks, and β-catenin siRNA transfection for 1 week. The lung histopathology was observed by Hematoxylin-eosin(H&E) staining. The levels of arsenic in blood, urine and lung tissue of rats were determined by graphite furnace atomic absorption spectrometry. The content of ROS was determined by chemical fluorescence method. SOD activity, GSH and MDA content were determined by ELISA. Results The lung tissue of rats in low, medium and blank control high dose groups showed different degrees of inflammatory cell infiltration, alveolar septum widening and alveolar fusion with the increase of arsenic dosage. The lung tissue of rats in blank control high dose group was severely damaged and some pulmonary fibrogenesis appeared. The arsenic levels in blood, urine and lung tissues of rats in low, medium and blank control high dose groups were increased with the increase of arsenic dosage and time(P<0.05). The fluorescence values of ROS in blank control high dose group of lung tissue in rats was significantly higher than those in control, low and medium groups(P<0.05), and the content of MDA was remarkably higher than that in control group(P<0.05). SOD and GSH content in blank control high dose group was markedly lower than those in control and low dose groups(P<0.05). After blocking the β-catenin gene, most of the pulmonary fibrosis appeared in the lung tissue of rats in high dose group transfected with β-catenin siRNA, was shown to be more serious than that in blank control and negative control high dose groups. The levels of arsenic in blood, urine and lung tissue of rats in high dose group transfected with β-catenin siRNA and negative control high dose group were significantly higher than those in control, low and medium dose groups(P<0.05). The fluorescence value of ROS in lung tissue of rats in high dose group transfected with β-catenin siRNA and negative control high dose group were higher than that control, low and medium groups(P<0.05), and the content of MDA was higher than that control group(P<0.05). However, SOD and GSH contents in lung tissue of rats in high dose group transfected with β-catenin siRNA and negative control high dose group were lower than those in control and low dose groups(P<0.05). There was a significant positive correlation between arsenic content and ROS fluorescence in rat lung tissue(r_s=0.655, P<0.001). Lung tissue of ROS fluorescence was negatively correlated with SOD activity(r_s=-0.441, P<0.002), negatively correlated with GSH content(r_s=-0.599, P<0.001), and positively correlated with MDA content(r_s=0.395, P<0.006). Conclusion Long-term exposure to arsenic could cause persistent accumulation of arsenic in rats, which may result in oxidative stress in rat lung tissue and may lead to lung injury. Blocking the β-catenin gene had little effect on oxidative stress in rat lung tissue.
引文
[1] JOMOVA K,JENISOVA Z,FESZTEROVA M,et al.Arsenic:toxicity,oxidative stress and human disease[J].J Appl Toxicol,2015,31(2):95-107.
[2] 杨克敌,郑玉健.环境卫生学[M].北京:人民卫生出版社,2015:242-243.
[3] PARVEZ F,CHEN Y,YUNUS M,et al.Arsenic exposure and impaired lung function.findings from a large population-based prospective cohort study[J].Am J Res Crit Care Med,2013,188(7):813-819.
[4] SMITH A H,MARSHALL G,YUAN Y,et al.Increased mortality from lung cancer and bronchiectasis in young adults after exposure to arsenic in utero and in early childhood[J].Environ Health Persp,2006,114(8):1293-1296.
[5] PARVEZ G,FARUQUE M,CHEN A,et al.A prospective study of arsenic induced respiratory symptoms and chronic obstructive pulmonary disease (COPD):findings from health effects of arsenic exposure longitudinal study (HEALS) in Bangladesh[J].Epidemiology,2009,20(6):S82-S83.
[6] LI M,CAI J F,CHIU J F.Arsenic induces oxidative stress and activates stress gene expressions in cultured lung epithelial cells[J].J Cell Biochem,2002,87(1):29-38.
[7] LI G,LEE L S,LI M,et al.Molecular changes during arsenic-induced cell transformation[J].J Cell Physiol,2011,226(12):3225-3232.
[8] SUSHWETA M,SUKANYA S,SAYANTA D,et al.Mangiferin alleviates arsenic induced oxidative lung injury via upregulation of the Nrf2-HO1 axis[J].Food Chem Toxicol,2019,12(2):41-55.
[9] RODRíGUEZLADO L,SUN G,BERG M,et al.Groundwater arsenic contamination throughout China[J].Science,2013,341(6148):866-868.
[10] DAS D,BINDHANI B,MUKHERJEE B,et al.Chronic low-level arsenic exposure reduces lung function in male population without skin lesions[J].Int J Pub He,2014,59(4):655-663.
[11] LAMM S H,HAMID F,DISSEN E K,et al.A systematic review and meta-regression analysis of lung cancer risk and inorganic arsenic in drinking water[J].Int J Env Res Pub He,2015,12(12):15498-15515.
[12] 徐梦伟,任冬燕,孙高峰,等.亚砷酸钠对SD大鼠脏器损伤的实验研究[J].新疆医科大学学报,2018,41(2):193-198.
[13] KENYON E M,HUGHES M F,ADAIR B M,et al.Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in C57BL6 mice following subchronic exposure to arsenate in drinking water[J].Toxicol Appl Pharm,2008,232(3):448-455.
[14] 成振江,张玲慧,曹永智.砷暴露对肺损伤的机制的研究进展[J].中国社区医师,2018,34(10):7-8.
[15] FLORA S J.Arsenic and dichlorvos:Possible interaction between two environmental contaminants[J].J Trace Elem Med Biol,2016,35:43-60.
[16] JOMOVA K,JENISOVA Z,FESZTEROVA M,et al.Arsenic:toxicity,oxidative stress and human disease[J].J Appl Toxicol,2015,31(2):95-107.
[17] 董娟娟.Wnt/β-catenin信号通路在亚砷酸钠致HELF细胞损伤中的作用[D].乌鲁木齐:新疆医科大学,2016.
[18] ACF S,MARCHESI S C,GD DAL,et al.Effects of arsenic compounds on microminerals content and antioxidant enzyme activities in rat liver[J].Biol Trace Elem Res,2017,183(2):1-9.
[19] NIRANKARI S,KAMAL R.Neuroprotective role of quercetin against arsenic induced oxidative stress in rat brain[J].J Anal Toxicol,2016,6(2):1-6.
[20] NIMSE S B,PAL D.Free radicals,natural antioxidants,and their reaction mechanisms[J].Rsc Adv,2015,5(35):27986-28006.
[21] KUMAR S,YEDJOU C G,TCHOUNWOU P B.Arsenic trioxide induces oxidative stress,DNA damage,and mitochondrial pathway of apoptosis in human leukemia (HL-60) cells[J].J Exp Clin Canc Res,2014,33(1):42.
[22] AYALA A,MU?OZ M F,ARGüELLES S.Lipid peroxidation:production,metabolism,and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal[J].Oxid Med Cell Longev,2014(6):360-438.
[23] 杨全军,胡以利,望晓波,等.shRNA 靶向沉默 ZEB2 表达通过 Wnt/β-catenin 信号通路降低肺癌细胞增殖活性的实验研究[J].现代肿瘤医学,2019,27(4):541-546.
[2] 杨克敌,郑玉健.环境卫生学[M].北京:人民卫生出版社,2015:242-243.
[3] PARVEZ F,CHEN Y,YUNUS M,et al.Arsenic exposure and impaired lung function.findings from a large population-based prospective cohort study[J].Am J Res Crit Care Med,2013,188(7):813-819.
[4] SMITH A H,MARSHALL G,YUAN Y,et al.Increased mortality from lung cancer and bronchiectasis in young adults after exposure to arsenic in utero and in early childhood[J].Environ Health Persp,2006,114(8):1293-1296.
[5] PARVEZ G,FARUQUE M,CHEN A,et al.A prospective study of arsenic induced respiratory symptoms and chronic obstructive pulmonary disease (COPD):findings from health effects of arsenic exposure longitudinal study (HEALS) in Bangladesh[J].Epidemiology,2009,20(6):S82-S83.
[6] LI M,CAI J F,CHIU J F.Arsenic induces oxidative stress and activates stress gene expressions in cultured lung epithelial cells[J].J Cell Biochem,2002,87(1):29-38.
[7] LI G,LEE L S,LI M,et al.Molecular changes during arsenic-induced cell transformation[J].J Cell Physiol,2011,226(12):3225-3232.
[8] SUSHWETA M,SUKANYA S,SAYANTA D,et al.Mangiferin alleviates arsenic induced oxidative lung injury via upregulation of the Nrf2-HO1 axis[J].Food Chem Toxicol,2019,12(2):41-55.
[9] RODRíGUEZLADO L,SUN G,BERG M,et al.Groundwater arsenic contamination throughout China[J].Science,2013,341(6148):866-868.
[10] DAS D,BINDHANI B,MUKHERJEE B,et al.Chronic low-level arsenic exposure reduces lung function in male population without skin lesions[J].Int J Pub He,2014,59(4):655-663.
[11] LAMM S H,HAMID F,DISSEN E K,et al.A systematic review and meta-regression analysis of lung cancer risk and inorganic arsenic in drinking water[J].Int J Env Res Pub He,2015,12(12):15498-15515.
[12] 徐梦伟,任冬燕,孙高峰,等.亚砷酸钠对SD大鼠脏器损伤的实验研究[J].新疆医科大学学报,2018,41(2):193-198.
[13] KENYON E M,HUGHES M F,ADAIR B M,et al.Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in C57BL6 mice following subchronic exposure to arsenate in drinking water[J].Toxicol Appl Pharm,2008,232(3):448-455.
[14] 成振江,张玲慧,曹永智.砷暴露对肺损伤的机制的研究进展[J].中国社区医师,2018,34(10):7-8.
[15] FLORA S J.Arsenic and dichlorvos:Possible interaction between two environmental contaminants[J].J Trace Elem Med Biol,2016,35:43-60.
[16] JOMOVA K,JENISOVA Z,FESZTEROVA M,et al.Arsenic:toxicity,oxidative stress and human disease[J].J Appl Toxicol,2015,31(2):95-107.
[17] 董娟娟.Wnt/β-catenin信号通路在亚砷酸钠致HELF细胞损伤中的作用[D].乌鲁木齐:新疆医科大学,2016.
[18] ACF S,MARCHESI S C,GD DAL,et al.Effects of arsenic compounds on microminerals content and antioxidant enzyme activities in rat liver[J].Biol Trace Elem Res,2017,183(2):1-9.
[19] NIRANKARI S,KAMAL R.Neuroprotective role of quercetin against arsenic induced oxidative stress in rat brain[J].J Anal Toxicol,2016,6(2):1-6.
[20] NIMSE S B,PAL D.Free radicals,natural antioxidants,and their reaction mechanisms[J].Rsc Adv,2015,5(35):27986-28006.
[21] KUMAR S,YEDJOU C G,TCHOUNWOU P B.Arsenic trioxide induces oxidative stress,DNA damage,and mitochondrial pathway of apoptosis in human leukemia (HL-60) cells[J].J Exp Clin Canc Res,2014,33(1):42.
[22] AYALA A,MU?OZ M F,ARGüELLES S.Lipid peroxidation:production,metabolism,and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal[J].Oxid Med Cell Longev,2014(6):360-438.
[23] 杨全军,胡以利,望晓波,等.shRNA 靶向沉默 ZEB2 表达通过 Wnt/β-catenin 信号通路降低肺癌细胞增殖活性的实验研究[J].现代肿瘤医学,2019,27(4):541-546.