聚乙二醇-4000对暗纹东方鲀肠上皮电生理的影响
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摘要
以暗纹东方鲀(Takifugu obscurus)为研究对象,采用Ussing chamber技术研究了聚乙二醇-4000(PEG-4000)对肠上皮开路电压、短路电流(Isc)、短路电流变化("Isc)、电导(G)、跨膜电位(TEP)的影响。结果表明:PEG-4000在肠道中6 h残留浓度为1.83 g·L-1,显著高于其在水中浓度(P<0.05),残留率达184%; PEG处理组暗纹东方鲀离体肠上皮Isc为69.20μA·cm~(-2),显著低于对照组(P<0.05);ΔIsc显著低于对照组(P<0.05),仅为对照组的68.57%; PEG处理组暗纹东方鲀离体肠上皮电导为24.97 m S·cm~(-2),显著低于对照组(36.22 m S·cm~(-2))(P<0.05); PEG-4000在暗纹东方鲀肠道中极易残留、不易渗透,使得肠道上皮的转运蛋白或者离子通道受阻,从而显著影响肠上皮Isc、"Isc以及电导,降低了离子的主动流动量。本研究结果为鱼类肠道电生理平台的构建提供了技术参数,该平台不仅能用于评估PEG等污染物对鱼类肠道的影响,还能用于鱼类肠道上皮细胞离子转运机制的研究。
To evaluate the effects of polyethylene glycol( PEG) on fish intestinal epithelium,we examined the electrophysiological responses of intestinal tissue from the puffer fish( Takifugu obscurus) using Ussing chambers. We measured the open circuit voltage,short circuit current( Isc),variation of short-circuit current( "Isc),conductance( G),and transepithelial potential( TEP) of the intestinal epithelium. The results showed that the residual concentration of PEG-4000 in the intestine within 6 h was 1.83 g·L~(-1),which was significantly higher than that in the control( P<0.05). The residual rate was 184%. The intestine epithelial Isc of puffer fish in PEG group was 69. 20 μA · cm~(-2),which was significantly lower than that in the control( 117. 23μA cm~(-2))( P<0.05). The " Isc of the PEG group was significantly lower than that of the control,being only 68.57% of that in the control. The average conductance of the PEG group was around 24.97 m S·cm~(-2),which was significantly lower than that of the control( 36.22 m S·cm~(-2)). These results indicated that PEG-4000 was not permeable and had a high residual rate in the intestinal tract of puffer fish,which hindered ion channels and reduced the active flow of ions across the epithelium. Our results provide evidence that Ussing chamber is a valuable tool in assessing the effects of pollutants such as PEG on electrophysiological changes and ion transportation in the intestinal tract of fish.
To evaluate the effects of polyethylene glycol( PEG) on fish intestinal epithelium,we examined the electrophysiological responses of intestinal tissue from the puffer fish( Takifugu obscurus) using Ussing chambers. We measured the open circuit voltage,short circuit current( Isc),variation of short-circuit current( "Isc),conductance( G),and transepithelial potential( TEP) of the intestinal epithelium. The results showed that the residual concentration of PEG-4000 in the intestine within 6 h was 1.83 g·L~(-1),which was significantly higher than that in the control( P<0.05). The residual rate was 184%. The intestine epithelial Isc of puffer fish in PEG group was 69. 20 μA · cm~(-2),which was significantly lower than that in the control( 117. 23μA cm~(-2))( P<0.05). The " Isc of the PEG group was significantly lower than that of the control,being only 68.57% of that in the control. The average conductance of the PEG group was around 24.97 m S·cm~(-2),which was significantly lower than that of the control( 36.22 m S·cm~(-2)). These results indicated that PEG-4000 was not permeable and had a high residual rate in the intestinal tract of puffer fish,which hindered ion channels and reduced the active flow of ions across the epithelium. Our results provide evidence that Ussing chamber is a valuable tool in assessing the effects of pollutants such as PEG on electrophysiological changes and ion transportation in the intestinal tract of fish.
引文
陈锦芳,许孝良,李小年.2015.共轭烯酸与聚乙二醇缩合反应研究进展.有机化学,35(1):39-54.
郭兰,黄娟,欧阳玲玲,等.2015.应用Ussing chamber技术建立大鼠肠黏膜P-糖蛋白与药物相互作用实验模型的相关因素考察.中药药理与临床,31(2):116-120.
纪梁.2012.以PEG200为基体的线切割液基液的配制及其表面张力的测定(硕士学位论文).南京:南京理工大学.
林存侠,罗建平.2018.成人功能性便秘经聚乙二醇4000治疗效果及安全性分析.临床研究,26(1):108-110.
刘杰,周浩,黄郁芳,等.2018.聚乙二醇化学改性的大豆分离蛋白凝胶.高等学校化学学报,39(2):390-396.
刘晓雷,张莉,顾星,等.2015.Ussing chamber技术在药物肠吸收方面的应用进展.中国新药杂志,24(6):644-648.
路娟,刘清飞,罗国安,等.2009.药物的聚乙二醇修饰研究进展.有机化学,19(8):213-216.
沈旭明,赵清良.2011.温度、溶解氧对暗纹东方鲀幼鱼呼吸频率的影响.生态学杂志,20(4):13-15.
史建全,刘建虎,陈大庆,等.2004.青海湖裸鲤肠道组织学研究.淡水渔业,34(2):16-19.
王晓武,盛桂仙,姚杏荣.2012.快速肠道准备对结直肠术后肠道黏膜屏障功能的影响.河北医科大学学报,33(2):157-160.
吴福彪.2009.静息电位、动作电位的产生机制及影响其大小的主要因素.生物学教学,34(7):62-63.
杨翠娴,李清彪,廖鑫凯,等.2006.间歇式水解-好氧循环工艺处理聚乙二醇废水.化工学报,57(11):2731-2734.
臧传宝.2010.上皮细胞CFTR通道介导的阴离子转运机制(硕士学位论文).青岛:中国海洋大学.
湛江水产专科学校.1980.海洋饵料生物培养.北京:农业出版社.
张迪,徐缓,胡美娜,等.2015.脂质体面临的聚乙二醇“窘境”及其解决方法.药学学报,50(3):252-260.
张学军.2014.关于“静息电位和动作电位”的探究式教学过程.生物学通报,49(7):44-49.
张长青,张葵玲,张国伟,等.2003.聚乙二醇4000治疗功能性便秘疗效分析.中国实用内科杂志,23(12):748-750.
周丽雅,夏志伟,林三仁,等2001.聚乙二醇4000治疗成人慢性功能性便秘的多中心随机对照临床试验研究.中国临床药理学杂志,17(1):7-10.
左明雪.2006.动作电位形成的机制.生物学通报,41(6):12-13.
Buxton TB,Crockett JK,Moore WL,et al.1979.Protein precipitation by acetone for the analysis of polyethylene glycol in intestinal perfusion fluid.Gastroenterology,76:820-824.
Eddy FB.2007.Drinking in juvenile Atlantic salmon(Salmo salar L.)in response to feeding and activation of the endogenous renin-angiotensin system.Comparative Biochemistry&Physiology Part A:Molecular&Integrative Physiology,148:23-28.
Gilmour KM,Perry SF,Esbaugh AJ,et al.2012.Compensatory regulation of acid-base balance during salinity transfer in rainbow trout(Oncorhynchus mykiss).Journal of Comparative Physiology B:Biochemical Systemic&Environmental Physiology,182:259-274.
Grosell M,Genz J.2006.Ouabain-sensitive bicarbonate secretion and acid absorption by the marine teleost fish intestine play a role in osmoregulation.American Journal of Physiology Regulatory Integrative&Comparative Physiology,291:1145-1156.
Grosell M.2011.Intestinal anion exchange in marine teleosts is involved in osmoregulation and contributes to the oceanic inorganic carbon cycle.Acta Physiologica,202:421-434.
Guo N,Hu Z,Fan X,et al.2012.Simultaneous determination of salidroside and its aglycone metabolite p-tyrosol in rat plasma by liquid chromatography-tandem mass spectrometry.Molecules,17:4733-4754.
Heuer RM,Munley KM,Narsinghani N,et al.2016.Changes to intestinal transport physiology and carbonate production at various CO2levels in a marine teleost,the Gulf Toadfish(Opsanus beta).Physiological&Biochemical Zoology,89:402-416.
Lega YV,Chernitsky AG,Belkovsky NM.1992.Effect of low sea water temperature on water balance in the Atlantic salmon(Salmo salar L.).Fish Physiology&Biochemistry,10:145-148.
Parlesak A,Bode JC,Bode C.2009.Parallel determination of gut permeability in man with M(r)400,M(r)1500,M(r)4000 and M(r)10,000 polyethylene glycol.Clinical Chemistry&Laboratory Medicine,32:813-820.
Ruhr IM,Mager EM,Takei Y,et al.2015.The differential role of renoguanylin in osmoregulation and apical Cl-/HCO-exchange activity in the posterior intestine of the Gulf toadfish(Opsanus beta).American Journal of Physiology Regulatory Integrative&Comparative Physiology,309:399-499.
Ruizjarabo I,Baranyruiz A,Jerezcepa I,et al.2017.Intestinal response to salinity challenge in the Senegalese sole(Solea senegalensis).Comparative Biochemistry&Physiology Part A:Molecular&Integrative Physiology,204:57-64.
Ussing HH,Zerahn K.1951.Active transport of sodium as the source of electric current in the short-circuited isolated frog skin.Acta physiologica Scandinavica,23:110-127.
Volynets V,Reichold A,Brdos G,et al.2015.Assessment of the intestinal barrier with five different permeability tests in healthy C57BL/6J and BALB/c J mice.Digestive Diseases&Sciences,61:737-746.
Wilson RW,Grosell M.2003.Intestinal bicarbonate secretion in marine teleost fish-source of bicarbonate,p H sensitivity,and consequences for whole animal acid-base and calcium homeostasis.Biochimica et Biophysica Acta:Biomembranes,1618:163-174.
郭兰,黄娟,欧阳玲玲,等.2015.应用Ussing chamber技术建立大鼠肠黏膜P-糖蛋白与药物相互作用实验模型的相关因素考察.中药药理与临床,31(2):116-120.
纪梁.2012.以PEG200为基体的线切割液基液的配制及其表面张力的测定(硕士学位论文).南京:南京理工大学.
林存侠,罗建平.2018.成人功能性便秘经聚乙二醇4000治疗效果及安全性分析.临床研究,26(1):108-110.
刘杰,周浩,黄郁芳,等.2018.聚乙二醇化学改性的大豆分离蛋白凝胶.高等学校化学学报,39(2):390-396.
刘晓雷,张莉,顾星,等.2015.Ussing chamber技术在药物肠吸收方面的应用进展.中国新药杂志,24(6):644-648.
路娟,刘清飞,罗国安,等.2009.药物的聚乙二醇修饰研究进展.有机化学,19(8):213-216.
沈旭明,赵清良.2011.温度、溶解氧对暗纹东方鲀幼鱼呼吸频率的影响.生态学杂志,20(4):13-15.
史建全,刘建虎,陈大庆,等.2004.青海湖裸鲤肠道组织学研究.淡水渔业,34(2):16-19.
王晓武,盛桂仙,姚杏荣.2012.快速肠道准备对结直肠术后肠道黏膜屏障功能的影响.河北医科大学学报,33(2):157-160.
吴福彪.2009.静息电位、动作电位的产生机制及影响其大小的主要因素.生物学教学,34(7):62-63.
杨翠娴,李清彪,廖鑫凯,等.2006.间歇式水解-好氧循环工艺处理聚乙二醇废水.化工学报,57(11):2731-2734.
臧传宝.2010.上皮细胞CFTR通道介导的阴离子转运机制(硕士学位论文).青岛:中国海洋大学.
湛江水产专科学校.1980.海洋饵料生物培养.北京:农业出版社.
张迪,徐缓,胡美娜,等.2015.脂质体面临的聚乙二醇“窘境”及其解决方法.药学学报,50(3):252-260.
张学军.2014.关于“静息电位和动作电位”的探究式教学过程.生物学通报,49(7):44-49.
张长青,张葵玲,张国伟,等.2003.聚乙二醇4000治疗功能性便秘疗效分析.中国实用内科杂志,23(12):748-750.
周丽雅,夏志伟,林三仁,等2001.聚乙二醇4000治疗成人慢性功能性便秘的多中心随机对照临床试验研究.中国临床药理学杂志,17(1):7-10.
左明雪.2006.动作电位形成的机制.生物学通报,41(6):12-13.
Buxton TB,Crockett JK,Moore WL,et al.1979.Protein precipitation by acetone for the analysis of polyethylene glycol in intestinal perfusion fluid.Gastroenterology,76:820-824.
Eddy FB.2007.Drinking in juvenile Atlantic salmon(Salmo salar L.)in response to feeding and activation of the endogenous renin-angiotensin system.Comparative Biochemistry&Physiology Part A:Molecular&Integrative Physiology,148:23-28.
Gilmour KM,Perry SF,Esbaugh AJ,et al.2012.Compensatory regulation of acid-base balance during salinity transfer in rainbow trout(Oncorhynchus mykiss).Journal of Comparative Physiology B:Biochemical Systemic&Environmental Physiology,182:259-274.
Grosell M,Genz J.2006.Ouabain-sensitive bicarbonate secretion and acid absorption by the marine teleost fish intestine play a role in osmoregulation.American Journal of Physiology Regulatory Integrative&Comparative Physiology,291:1145-1156.
Grosell M.2011.Intestinal anion exchange in marine teleosts is involved in osmoregulation and contributes to the oceanic inorganic carbon cycle.Acta Physiologica,202:421-434.
Guo N,Hu Z,Fan X,et al.2012.Simultaneous determination of salidroside and its aglycone metabolite p-tyrosol in rat plasma by liquid chromatography-tandem mass spectrometry.Molecules,17:4733-4754.
Heuer RM,Munley KM,Narsinghani N,et al.2016.Changes to intestinal transport physiology and carbonate production at various CO2levels in a marine teleost,the Gulf Toadfish(Opsanus beta).Physiological&Biochemical Zoology,89:402-416.
Lega YV,Chernitsky AG,Belkovsky NM.1992.Effect of low sea water temperature on water balance in the Atlantic salmon(Salmo salar L.).Fish Physiology&Biochemistry,10:145-148.
Parlesak A,Bode JC,Bode C.2009.Parallel determination of gut permeability in man with M(r)400,M(r)1500,M(r)4000 and M(r)10,000 polyethylene glycol.Clinical Chemistry&Laboratory Medicine,32:813-820.
Ruhr IM,Mager EM,Takei Y,et al.2015.The differential role of renoguanylin in osmoregulation and apical Cl-/HCO-exchange activity in the posterior intestine of the Gulf toadfish(Opsanus beta).American Journal of Physiology Regulatory Integrative&Comparative Physiology,309:399-499.
Ruizjarabo I,Baranyruiz A,Jerezcepa I,et al.2017.Intestinal response to salinity challenge in the Senegalese sole(Solea senegalensis).Comparative Biochemistry&Physiology Part A:Molecular&Integrative Physiology,204:57-64.
Ussing HH,Zerahn K.1951.Active transport of sodium as the source of electric current in the short-circuited isolated frog skin.Acta physiologica Scandinavica,23:110-127.
Volynets V,Reichold A,Brdos G,et al.2015.Assessment of the intestinal barrier with five different permeability tests in healthy C57BL/6J and BALB/c J mice.Digestive Diseases&Sciences,61:737-746.
Wilson RW,Grosell M.2003.Intestinal bicarbonate secretion in marine teleost fish-source of bicarbonate,p H sensitivity,and consequences for whole animal acid-base and calcium homeostasis.Biochimica et Biophysica Acta:Biomembranes,1618:163-174.