肝豆汤对Wilson病模型铜负荷大鼠肝脏Wnt/<italic style="font-style: italic">β</italic>-catenin信号通路的调控作用

耿昊; 李海; 徐陈陈; 董健健; 陈燕; 韩咏竹; 韩永升; 杨任民; 程楠

doi:10.13422/j.cnki.syfjx.20190403

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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

肝豆汤治疗Wilson病的实验研究专题 | 更新时间：2021-02-09

- 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
- Regulatory Effect of Gandou Decoction on Wnt/β-catenin Signaling Pathway in Liver of Wilson Model Copper-loaded Rats
- 中国实验方剂学杂志 2019年25卷第7期页码：75-81
- 作者机构：
  
  1.安徽中医药大学　研究生院，合肥　 230038；
  2.安徽中医药大学　神经病学研究所　附属医院，合肥　 230061；
  3.武警安徽总队医院，合肥　 230041；
  4.中国科技大学医院，合肥　 230000
- 作者简介：
  
  耿昊，在读硕士，医师，从事神经系统疾病研究， E-mail：1466755694@qq.com
  程楠，副教授，从事神经系统疾病研究， E-mail:neurodoc120@163.com
- 基金信息：
  
  国家自然科学基金项目(81673948;81603596);安徽省自然科学青年基金项目(1608085MH171)
- DOI：10.13422/j.cnki.syfjx.20190403
  中图分类号： R2-0;R22;R285.5;R289
- 收稿日期：2018-07-05，
  
  网络出版日期：2018-06-11，
  
  纸质出版日期：2019-04-05
- 稿件说明：
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耿昊, 李海, 徐陈陈, 等. 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[J]. 中国实验方剂学杂志, 2019,25(7):75-81.

Hao GENG, Hai LI, Chen-chen XU, et al. Regulatory Effect of Gandou Decoction on Wnt/β-catenin Signaling Pathway in Liver of Wilson Model Copper-loaded Rats[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(7): 75-81.
耿昊, 李海, 徐陈陈, 等. 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[J]. 中国实验方剂学杂志, 2019,25(7):75-81. DOI： 10.13422/j.cnki.syfjx.20190403.

Hao GENG, Hai LI, Chen-chen XU, et al. Regulatory Effect of Gandou Decoction on Wnt/β-catenin Signaling Pathway in Liver of Wilson Model Copper-loaded Rats[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(7): 75-81. DOI： 10.13422/j.cnki.syfjx.20190403.

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)/

-连环蛋白(

-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 mL·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 mL·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/

-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（

0.01）；肝豆汤可降低铜负荷大鼠肝脏铜含量（

0.05），PCA可显著降低模型组大鼠肝脏铜含量（

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（

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

-连环蛋白（

-catenin），磷酸化糖原合成激酶3

（glycogen synthase kinase-3

p-GSK3

），原癌基因（cellular myelocytomatosis oncogene，c-Myc）蛋白表达量显著增加（

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/

-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。

Abstract

Objective:

To investigate the regulatory effect of Gandou decoction (GDD) on Wnt/

-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 (

=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 mL·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 (

=45)

which were fed by modeling method for continuously four weeks; GDD group and penicillamine (PCA) group (

=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/

-catenin of rat livers.

Result:

Compared with model group

content of Cu element in GDD group was less (

0.05); compared with model group

content of Cu element in PCA group was less (

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 (

0.01). Compared with model group

the expression quantities of

-catenin

p-glycogen synthase kinase-3

(p-GSK3

)

cellular myelocytomatosis oncogene(c-Myc) in GDD and PCA group increased

while p-

-catenin

Dishevelled3

GSK3

protein expressions reduced (

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/

-catenin signaling pathway in the hepatic tissue of Wilson disease model copper-loaded rats

so as to reduce the therapeutic effect of hepatocellular injury induced by high copper.

关键词

Keywords

references

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DOI：10.13422/j.cnki.syfjx.20190403

摘要

Abstract

关键词

Keywords

references