肝豆汤对Wilson病模型铜负荷大鼠肝脏Wnt/β-catenin信号通路的调控作用
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摘要
目的:探讨肝豆汤对铜负荷大鼠肝脏中分泌型糖蛋白(Wnt)/β-连环蛋白(β-catenin)信号通路的调控作用及机制。方法:SD大鼠115只随机分为正常组(20只)、造模组。造模组予以1 g·kg~(-1)·d~(-1)的硫酸铜饲料及0. 02 m L·g-1·d-1的0. 185%硫酸铜溶液连续喂养12周;造模成功大鼠随机分为模型组(45只),肝豆汤组(25只)和青霉胺(PCA)组(25只);模型组、肝豆汤组和PCA组分别予以0. 02 m L·g-1·d-1生理盐水,0. 4 g·kg~(-1)·d~(-1)的中药肝豆汤和0. 09 g·kg~(-1)·d~(-1)青霉胺灌胃,连续4周,末次灌胃后取各组大鼠肝脏组织检测:电感耦合等离子体原子发射光谱法(ICP-AES)检测各组大鼠肝铜含量;苏木素-伊红(HE)染色观察各组大鼠肝脏组织病理改变,免疫组织化学法检测肝脏组织氧化应激相关蛋白的表达,蛋白免疫印迹法(Western blot)检测肝组织中Wnt/β-catenin信号通路相关蛋白的表达水平。结果:ICP-AES法结果显示,与正常组比较,模型组大鼠肝脏铜含量显著升高(P <0. 01);肝豆汤可降低铜负荷大鼠肝脏铜含量(P <0. 05),PCA可显著降低模型组大鼠肝脏铜含量(P <0. 01); HE染色结果显示,与正常组比较,模型组大鼠肝脏组织镜下可见肝细胞大小不一、排列紊乱,部分肝索丧失;肝豆汤组和PCA组细胞损伤程度较模型组明显降低;免疫组化结果显示:肝豆汤和PCA可显著降低模型组8-羟基脱氧鸟苷(8-Hydroxyguanosine),硝基酪氨酸(Nitrotyrosine)蛋白表达量(P <0. 01); Western blot结果显示,肝豆汤和PCA可使铜负荷大鼠肝脏内Wnt通路相关蛋白β-连环蛋白(β-catenin),磷酸化糖原合成激酶3β(glycogen synthase kinase-3,p-GSK3β),原癌基因(cellular myelocytomatosis oncogene,c-Myc)蛋白表达量显著增加(P <0. 01)。结论:肝豆汤可通过促进过量铜排出减轻高铜诱导的肝脏损伤,并通过激活肝脏Wnt/β-catenin信号通路促进损伤的肝组织代偿性自愈以达到减轻高铜诱导氧化应激损伤的治疗效果。
Objective: To investigate the regulatory effect of Gandou decoction( GDD) on Wnt/β-catenin signaling pathway in hepatic tissue of Wilson disease model copper-loaded rats and its potential mechanism.Method: One hundred and fifteen SD rats were randomly divided into the normal group( n = 20) and modeling group. Modeling group was given copper sulfate feed( 1 g·kg~(-1)·d~(-1)) and 0. 185% copper sulfate solution( 0. 02 m L·g~(-1)·d~(-1)) for 12 weeks after one week's adaptive feeding,so as to build the copper loaded rats model.After modeling,95 model rats were randomly divided into model group( n = 45),which were fed by modeling method for continuously four weeks; GDD group and penicillamine( PCA) group( n = 25 per group). GDD group and PCA group were given GDD( 0. 4 g·kg~(-1)·d~(-1)) and PCA( 0. 09 g·kg~(-1)·d~(-1)) by gavage for four weeks. The hepatic tissues of rats in each group were removed after final medication for further research: inductively coupled plasma-atomic emission spectrometry( ICP-AES) was used to detect the content of Cu element in rat livers.Htoxylin eosin( HE) staining was used to detect the pathological changes of rat liver. Immunohistochemistry was used to detect expression of oxidative stress. Western blot was used to detect protein expressions in Wnt/β-catenin of rat livers. Result: Compared with model group,content of Cu element in GDD group was less( P < 0. 05);compared with model group,content of Cu element in PCA group was less( P < 0. 01). The hepatic cells of model group showed different sizes,disordered arrangement and partial loss of hepatic cord compared with normal group;the hepatic injuries of GDD and PCA group were significantly lower than model group. Compared with model group,8-Hydroxyguanosine,Nitrotyrosine protein positive expression in GDD and PCA group were significantly less( P < 0. 01). Compared with model group,the expression quantities of β-catenin,p-glycogen synthase kinase-3β( p-GSK3β),cellular myelocytomatosis oncogene( c-Myc) in GDD and PCA group increased,while p-β-catenin,Dishevelled3,GSK3β protein expressions reduced( P < 0. 01). Conclusion: GDD can relieve liver damage by promoting excessive copper discharge. GDD decoction can promote the compensatory self-healing of the injured liver tissue by activating Wnt/β-catenin signaling pathway in the hepatic tissue of Wilson disease model copperloaded rats,so as to reduce the therapeutic effect of hepatocellular injury induced by high copper.
Objective: To investigate the regulatory effect of Gandou decoction( GDD) on Wnt/β-catenin signaling pathway in hepatic tissue of Wilson disease model copper-loaded rats and its potential mechanism.Method: One hundred and fifteen SD rats were randomly divided into the normal group( n = 20) and modeling group. Modeling group was given copper sulfate feed( 1 g·kg~(-1)·d~(-1)) and 0. 185% copper sulfate solution( 0. 02 m L·g~(-1)·d~(-1)) for 12 weeks after one week's adaptive feeding,so as to build the copper loaded rats model.After modeling,95 model rats were randomly divided into model group( n = 45),which were fed by modeling method for continuously four weeks; GDD group and penicillamine( PCA) group( n = 25 per group). GDD group and PCA group were given GDD( 0. 4 g·kg~(-1)·d~(-1)) and PCA( 0. 09 g·kg~(-1)·d~(-1)) by gavage for four weeks. The hepatic tissues of rats in each group were removed after final medication for further research: inductively coupled plasma-atomic emission spectrometry( ICP-AES) was used to detect the content of Cu element in rat livers.Htoxylin eosin( HE) staining was used to detect the pathological changes of rat liver. Immunohistochemistry was used to detect expression of oxidative stress. Western blot was used to detect protein expressions in Wnt/β-catenin of rat livers. Result: Compared with model group,content of Cu element in GDD group was less( P < 0. 05);compared with model group,content of Cu element in PCA group was less( P < 0. 01). The hepatic cells of model group showed different sizes,disordered arrangement and partial loss of hepatic cord compared with normal group;the hepatic injuries of GDD and PCA group were significantly lower than model group. Compared with model group,8-Hydroxyguanosine,Nitrotyrosine protein positive expression in GDD and PCA group were significantly less( P < 0. 01). Compared with model group,the expression quantities of β-catenin,p-glycogen synthase kinase-3β( p-GSK3β),cellular myelocytomatosis oncogene( c-Myc) in GDD and PCA group increased,while p-β-catenin,Dishevelled3,GSK3β protein expressions reduced( P < 0. 01). Conclusion: GDD can relieve liver damage by promoting excessive copper discharge. GDD decoction can promote the compensatory self-healing of the injured liver tissue by activating Wnt/β-catenin signaling pathway in the hepatic tissue of Wilson disease model copperloaded rats,so as to reduce the therapeutic effect of hepatocellular injury induced by high copper.
引文
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[12]张杰,胡纪源,马心锋,等.肝豆汤合驱铜疗法对肝豆状核变性患者生活质量影响的前瞻性研究[J].安徽中医药大学学报,2015,34(1):14-16.
[13]杨任民,韩咏竹,任明山,等.中药治疗肝豆状核变性107例疗效观察[J].中医杂志,1993(11):676-677,644.
[14]张静,陈怀珍,艾文龙,等.肝豆汤联合DMPS驱铜治疗对Wilson病湿热内蕴型认知功能障碍的影响[J].中国实验方剂学杂志,2018,24(15):210-215.
[15]陈林,艾文龙,程楠,等.肝豆汤对tx小鼠肝脏微量元素含量及ATP7B蛋白表达的影响[J].中国临床研究,2015,28(2):137-140.
[16]董健健,韩咏竹,程楠.肝豆汤对Wilson病模型TX小鼠肝细胞内铜代谢通路的分子调控机制[J].中国实验方剂学杂志,2016,22(24):128-133.
[17]徐陈陈,董健健,程楠,等.肝豆汤改良方对Wilson's病模型TX乳鼠神经元内Cyt C/Caspase信号通路的分子调控机制[J].中国实验方剂学杂志,2017,23(6):143-148.
[18] Riveiro-Naveira R R,Valcárcel-Ares M N,AlmonteBecerril M, et al. Resveratrol lowers synovial hyperplasia,inflammatory markers and oxidative damage in an acute antigen-induced arthritis model[J].Rheumatology,2016,55(10):1889-1900.
[19] Stanicka J,Russell E G,Woolley J F,et al. NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells[J].J Biol Chem,2015,290(15):9348-9361.
[20] Tabak O,Gelisgen R,Erman H,et al. Oxidative lipid,protein,and DNA damage as oxidative stress markers in vascular complications of diabetes mellitus[J]. Clin Invest Med,2011,34(3):163-171.
[21] Uversky V N,LI J,Fink A L. Metal-triggered structural transformations,aggregation,and fibrillation of human alpha-synuclein. a possible molecular NK between Parkinson's disease and heavy metal exposure[J]. J Biol Chem,2001,276(47):44284-44296.
[22] Willert K,Nusse R.β-catenin:a key mediator of Wnt signaling[J]. Curr Opin Genet Dev,1998,8(1):95-102.
[23] Stamos J L, Weis W I. Theβ-catenin destruction complex[J]. Cold Spring Harb Perspect Biol,2013,5(1):a007898.
[24] LI V S,Ng S S,Boersema P J,et al. Wnt signaling through inhibition of beta-catenin degradation in an intact Axin1 complex[J]. Cell, 2012,149(6):1245-1256.
[25] Hur E M,ZHOU F Q. GSK3 signalling in neural development[J]. Nat Rev Neurosci,2010,11(8):539-551.
[26] Gardner L B,Com P G. Hypoxic regulation of mRNA expression[J]. Cell Cycle,2008,7(13):1916-1924.
[27] Schmidt E V. The role of c-myc in regulation of translation initiation[J]. Oncogene,2004,23(18):3217-3221.
[28] MAO B,WU W,Davidson G,et al. Kremen proteins are Dickkopf receptors that regulate Wnt/β-catenin signalling[J]. Nature,2002,417(6889):664-667.
[29] Niida A,Hiroko T,Kasai M,et al. DKK1,a negative regulator of Wnt signaling, is a target of the betacatenin/TCF pathway[J]. Oncogene,2004,23(52):8520-8526.
[30] De Marco P,Merello E,Consales A,et al. Genetic analysis of disheveled 2 and disheveled 3 in human neural tube defects[J]. J Mol Neurosci,2013,49(3):582-588.
[31] Petersen C P,Reddien P W. Wnt signaling and the polarity of the primary body axis[J]. Cell,2009,139(6):1056-1068.
[32]冯柯. Eaf2通过Wnt信号通路抑制氧化应激诱导的人晶状体上皮细胞凋亡的研究[D].广州:南方医科大学,2016.
[33]罗欣,庄白妹,饶海英,等.经典Wnt/β-catenin信号通路介导子痫前期滋养细胞氧化应激损伤的机制研究[J].重庆医科大学学报,2015,40(6):801-805.
[34]李静.人参皂苷Rg1调控经典Wnt信号通路延缓造血干/祖细胞衰老的机制研究[D].重庆:重庆医科大学,2017.
[2]叶群荣,胡纪源,王共强,等. Wilson病临床症状特征和中医证型分析[J].安徽中医学院学报,2013,32(4):34-37.
[3]徐陈陈,董健健,程楠,等.中药肝豆汤含药血清对Wilson病模型TX小鼠肝细胞内ATP7b蛋白亚细胞定位和功能表达的影响[J].中华中医药杂志,2017,32(1):250-253.
[4]杨任民,韩咏竹,任明山,等.中药治疗肝豆状核变性107例疗效观察[J].中医杂志,1993,34(11):676-677.
[5]汤其强,杨任民,韩咏竹,等.肝豆汤对肝豆状核变性皮肤成纤维细胞模型铜代谢的影响[J].中国中西医结合杂志,2000,20(1):37-39.
[6]董健健.中药肝豆汤对Wilson病模型TX小鼠肝细胞内铜转运通路的分子调控机制研究[D].合肥:安徽中医药大学,2015.
[7]尹定子,宋海云. Wnt信号通路:调控机理和生物学意义[J].中国细胞生物学学报,2011,33(2):103-111.
[8] ZHANG D Y,PAN Y,ZHANG C,et al. Wnt/betacatenin signaling induces the aging of mesenchymal stem cells through promoting the ROS production[J]. Mol Cell Biochem,2013,374(12):13-20.
[9]黄雅楠,李海,董健健,等.高铜对于肝细胞Wnt/β-catenin信号通路的影响[J].中国神经精神疾病杂志,2017,45(11):646-650.
[10]王训,杨任民.二巯丁二钠对大鼠铜负荷模型铜代谢的影响[J].中国新药杂志,1999,8(7):458-460.
[11]朱世殊,董漪,徐志强,等. 2001—2010年儿童非病毒性肝病谱分析[J].传染病信息,2011,24(5):279-281,285.
[12]张杰,胡纪源,马心锋,等.肝豆汤合驱铜疗法对肝豆状核变性患者生活质量影响的前瞻性研究[J].安徽中医药大学学报,2015,34(1):14-16.
[13]杨任民,韩咏竹,任明山,等.中药治疗肝豆状核变性107例疗效观察[J].中医杂志,1993(11):676-677,644.
[14]张静,陈怀珍,艾文龙,等.肝豆汤联合DMPS驱铜治疗对Wilson病湿热内蕴型认知功能障碍的影响[J].中国实验方剂学杂志,2018,24(15):210-215.
[15]陈林,艾文龙,程楠,等.肝豆汤对tx小鼠肝脏微量元素含量及ATP7B蛋白表达的影响[J].中国临床研究,2015,28(2):137-140.
[16]董健健,韩咏竹,程楠.肝豆汤对Wilson病模型TX小鼠肝细胞内铜代谢通路的分子调控机制[J].中国实验方剂学杂志,2016,22(24):128-133.
[17]徐陈陈,董健健,程楠,等.肝豆汤改良方对Wilson's病模型TX乳鼠神经元内Cyt C/Caspase信号通路的分子调控机制[J].中国实验方剂学杂志,2017,23(6):143-148.
[18] Riveiro-Naveira R R,Valcárcel-Ares M N,AlmonteBecerril M, et al. Resveratrol lowers synovial hyperplasia,inflammatory markers and oxidative damage in an acute antigen-induced arthritis model[J].Rheumatology,2016,55(10):1889-1900.
[19] Stanicka J,Russell E G,Woolley J F,et al. NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells[J].J Biol Chem,2015,290(15):9348-9361.
[20] Tabak O,Gelisgen R,Erman H,et al. Oxidative lipid,protein,and DNA damage as oxidative stress markers in vascular complications of diabetes mellitus[J]. Clin Invest Med,2011,34(3):163-171.
[21] Uversky V N,LI J,Fink A L. Metal-triggered structural transformations,aggregation,and fibrillation of human alpha-synuclein. a possible molecular NK between Parkinson's disease and heavy metal exposure[J]. J Biol Chem,2001,276(47):44284-44296.
[22] Willert K,Nusse R.β-catenin:a key mediator of Wnt signaling[J]. Curr Opin Genet Dev,1998,8(1):95-102.
[23] Stamos J L, Weis W I. Theβ-catenin destruction complex[J]. Cold Spring Harb Perspect Biol,2013,5(1):a007898.
[24] LI V S,Ng S S,Boersema P J,et al. Wnt signaling through inhibition of beta-catenin degradation in an intact Axin1 complex[J]. Cell, 2012,149(6):1245-1256.
[25] Hur E M,ZHOU F Q. GSK3 signalling in neural development[J]. Nat Rev Neurosci,2010,11(8):539-551.
[26] Gardner L B,Com P G. Hypoxic regulation of mRNA expression[J]. Cell Cycle,2008,7(13):1916-1924.
[27] Schmidt E V. The role of c-myc in regulation of translation initiation[J]. Oncogene,2004,23(18):3217-3221.
[28] MAO B,WU W,Davidson G,et al. Kremen proteins are Dickkopf receptors that regulate Wnt/β-catenin signalling[J]. Nature,2002,417(6889):664-667.
[29] Niida A,Hiroko T,Kasai M,et al. DKK1,a negative regulator of Wnt signaling, is a target of the betacatenin/TCF pathway[J]. Oncogene,2004,23(52):8520-8526.
[30] De Marco P,Merello E,Consales A,et al. Genetic analysis of disheveled 2 and disheveled 3 in human neural tube defects[J]. J Mol Neurosci,2013,49(3):582-588.
[31] Petersen C P,Reddien P W. Wnt signaling and the polarity of the primary body axis[J]. Cell,2009,139(6):1056-1068.
[32]冯柯. Eaf2通过Wnt信号通路抑制氧化应激诱导的人晶状体上皮细胞凋亡的研究[D].广州:南方医科大学,2016.
[33]罗欣,庄白妹,饶海英,等.经典Wnt/β-catenin信号通路介导子痫前期滋养细胞氧化应激损伤的机制研究[J].重庆医科大学学报,2015,40(6):801-805.
[34]李静.人参皂苷Rg1调控经典Wnt信号通路延缓造血干/祖细胞衰老的机制研究[D].重庆:重庆医科大学,2017.