肝豆汤联合青霉胺对Wilson病模型肝脏铜死亡抑制作用

作者	单位
徐乐文;董健健;高曼莉;徐陈陈;李波;程楠	1.安徽中医药大学研究生院（合肥 230012）,2.安徽中医药大学神经病学研究所（合肥 230038）

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中文摘要:

目的观察肝豆汤联合青霉胺对Wilson病（WD）模型肝脏铜死亡的抑制作用。方法以淡化致死基因（DL）小鼠为正常组，将毒牛奶（TX）小鼠随机分为模型组、肝豆汤组、青霉胺组和肝豆汤联合青霉胺组，每组8只。正常组及模型组予以0.9%氯化钠溶液0.2 mL/（10 g·d），肝豆汤组、青霉胺组和肝豆汤联合青霉胺组分别予以肝豆汤浓缩液0.2 mL/（10 g·d），青霉胺0.1 g/（kg·d），肝豆汤浓缩液 0.2 mL/（10 g·d）联合青霉胺0.1 g/（kg·d），连续灌胃30天。采用生化法、电感耦合等离子体-质谱法（ICP-MS）、HE染色、透射电镜、实时荧光定量PCR、Western Blot法和免疫组织化学法检测各组小鼠血清学指标和肝脏铜含量、病理改变、线粒体超微结构、铜死亡相关基因和蛋白以及硫辛酸（LA）的表达水平。结果与正常组比较，模型组血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）水平，乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）活性和肝组织铜含量升高（P＜0.05），肝脏局部碎片状坏死，炎性细胞数升高（P＜0.01），线粒体损伤显著，铜死亡相关基因mRNA，铁硫簇蛋白、硫辛酰化蛋白、LA表达水平下降（P＜0.05），二氢硫辛酸转乙酰基酶（DLAT）二聚体和热休克蛋白70（HSP70）表达水平升高（P＜0.01）。与模型组比较，各治疗组血清ALT、AST水平，LDH、SOD活性及肝组织铜含量下降（P＜0.05），肝脏病理损伤减轻，炎性细胞数降低（P＜0.01），线粒体结构恢复，铜死亡相关基因mRNA、铁硫簇蛋白、lipDLAT、 lipDBT、 lipDLST、LA表达水平升高（P＜0.05），DLAT二聚体下降（P＜0.05）。与青霉胺组比较，肝豆汤联合青霉胺组 ALT、 AST 水平下降（P＜0.05），肝组织铜含量降低（P＜0.01），PDHB mRNA 表达、铁硫簇蛋白、硫辛酰化蛋白表达升高（P＜0.01，P＜0.05），DLAT二聚体、HSP70 表达下降（P＜0.01）。结论肝豆汤联合青霉胺可促进铜排出并上调LA途径相关蛋白的表达，减轻WD模型肝脏铜死亡。肝豆汤对青霉胺治疗WD具有辅助增效作用。

英文摘要:

Objective To investigate the inhibitory effect of Gandou Decoction combined with penicillamine on cuproptosis in the liver of toxic milk （TX） mice，which serve as a model for Wilson's disease （WD）.Methods Using dilute lethal （DL）mice as the normal control group，and TX mice were randomly divided into the model group，Gandou Decoction group，penicillamine group，and Gandou Decoction combined with penicillamine group，with 8 mice in each group. The normal control group and the model group were given 0.2 mL/10 g-1· d-1 of 0.9% NaCl solution，while the Gandou Decoction group，penicillamine group and Gandou Decoction combined with penicillamine group were separately given 0.2 mL·10 g-1·d-1 of Gandou Decoction，penicillamine 0.1 g·kg-1·d-1 and Gandou Decoction 0.2 mL·10 g-1·d-1 combined with penicillamine 0.1 g·kg-1·d-1 by gavage for 30 consecutive days. Biochemical methods， inductively coupled plasma mass spectrometry（ICP-MS），HE staining，transmission electron microscopy，real-time quantitative PCR，Western Blot and immunohistochemistry were used to determine the serological indexes and hepatic copper content，pathological changes，mitochondrial ultrastructure，cuproptosis-related genes and proteins，and the expression level of lipoic acid （LA） in each group of mice. Results Compared with the control group，the model group exhibited elevated serum ALT and AST levels，LDH and SOD activities，and hepatic tissue copper content （P<0.05）. Additionally，the model group showed localized fragmentary necrosis of the liver，an increased number of inflammatory cells （P<0.01），significant mitochondrial damage，decreased expression levels of cuproptosis-related genes mRNA，iron-sulfur cluster proteins，thioctanoylated proteins，and LA （P<0.05）. Furthermore，the expression levels of dihydrolipoic acid transacetylase （DLAT） dimer and heat shock protein 70 （HSP70） increased （P<0.01）. Compared with the model group，the treatment groups exhibited a decrease in serum alanine aminotransferase（ALT） and aspartate aminotransferase（AST） levels，lactate dehydrogenase（LDH） and superoxide dismutase（SOD） activities，as well as hepatic tissue copper content （P<0.05）. Additionally，there was a reduction in hepatic pathological injury and a decrease in the number of inflammatory cells （P<0.01）. Moreover，the mitochondrial structure was restored，and there was an elevation in the expression levels of cuproptosis-related genes，lipDLAT， lipDBT， lipDLST，thioredoxin，and LA mRNA （P<0.05）. Conversely，the expression levels of DLAT dimer decreased （P<0.05）.Compared with the penicillamine group，the levels of ALT and AST decreased in the Gandou Decoction combined with penicillamine group （P<0.05）， the copper content in liver tissue decreased（P<0.01），the mRNA expression of PDHB, iron-sulfur cluster protein and lipoylated protein increased（P<0.01，P<0.05），and the expressions of DLAT dimer and HSP70 decreased （P<0.01）. Conclusions The combination of Gandou Decoction and penicillamine demonstrates enhanced copper excretion and increased expression of LA pathway-related proteins，leading to the mitigation of hepatic cuproptosis in a WD model. Furthermore，Gandou Decoction exhibits an auxiliary potentiating effect when used in conjunction with penicillamine for the treatment of WD.

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