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

发布时间：2019-05-10 09:28

【摘要】：目的:探讨肝豆汤调控毒牛奶(TX)小鼠肝细胞内铜转运通路的分子靶点并观察其相应的调控机制。方法:20只家兔适应性喂养3 d后,随机分为空白组(生理盐水)和肝豆汤组(4.5 g·kg-1·d~(-1)),每日2次ig,连续10 d,制得含药血清;采用2步灌注法分离提取DL,TX小鼠肝细胞,采用原子吸收法检测加含不同百分含量(5%,10%,15%,20%)含肝豆汤兔血清作用24 h后,对TX小鼠肝细胞内微量元素的影响,另设空白组;蛋白质免疫印迹(Western blot)法检测经含肝豆汤兔血清作用后腺嘌呤核苷三磷酸7b(adenosine triphosphate7b,ATP7b),抗氧化蛋白1(anti-oxidant1,ATOX1),铜转运体1(copper transporter1,CTR1),金属巯蛋白(metallothionein,MT),超氧化剂物歧化酶铜伴侣蛋白(copper chaperone of superoxide dismutase,CCS),细胞色素氧化酶17(cytochrome-coxidase17,COX17)蛋白的表达。结果:与空白组比较,模型组TX小鼠肝细胞内铜含量升高,锌含量降低,铁含量升高,ATP7b,ATOX1,CCS,COX17蛋白表达明显降低,CTR1,MT蛋白表达显著升高(P0.01);与模型组比较,含肝豆汤兔血清可显著降低TX小鼠肝细胞内铜含量,增加锌含量,降低铁含量(P0.05,P0.01);Western blot检测结果显示,含肝豆汤兔血清可明显增加ATP7b,ATOX1,CCS,COX17蛋白表达(P0.05,P0.01),降低CTR1,MT蛋白表达(P0.01)。结论:肝豆汤可能是通过调控细胞内铜代谢相关蛋白,上调ATP7b,ATOX1,CCS,COX17蛋白表达,抑制CTR1,MT表达,从而发挥细胞内排铜作用。肝豆汤可通过多靶点、多途径调控铜代谢通路达到降低肝细胞内铜含量的治疗效果。
[Abstract]:Aim: to investigate the molecular target of Gantou decoction regulating copper transport pathway in hepatocytes of toxic milk (TX) mice and to observe its corresponding regulatory mechanism. Methods: after 3 days of adaptive feeding, 20 rabbits were randomly divided into blank group (saline) and Gantou decoction group (4.5g kg- 1 路d ~ (- 1), twice a day for 10 days) to prepare drug-containing serum. The hepatocytes of DL,TX mice were isolated and extracted by two-step perfusion method. The contents of rabbit serum containing different percentage (5%, 10%, 15%, 20%) were detected by atomic absorption spectrometry (AAS) for 24 hours. The effect of trace elements in hepatocytes of TX mice was set up in the blank group. Western blot (Western blot) assay for detection of adenine nucleoside triphosphate 7b (adenosine triphosphate7b,ATP7b), antioxidant protein 1 (anti-oxidant1,ATOX1), copper transporter 1 (copper transporter1,CTR1) and metallomercapto protein (metallothionein,) in rabbit serum containing Gantou decoction Expression of MT), superoxidant SOD copper chaperone (copper chaperone of superoxide dismutase,CCS and cytochrome oxidase 17 (cytochrome-coxidase17,COX17) protein. Results: compared with the blank group, the content of Cu, Zn, Fe, ATP7b,ATOX1,CCS,COX17 protein and CTR1,MT protein in the model group of TX mice were higher than those in the control group (P 0.01), while the content of iron in the liver cells of the model group was lower than that of the control group (P0.01). Compared with the model group, the serum of rabbits containing Gandou decoction could significantly decrease the content of copper, increase the content of zinc and decrease the content of iron in the hepatocytes of TX mice (P0.05, P01). The results of Western blot showed that the serum containing Gantou decoction could significantly increase the expression of ATP7b,ATOX1,CCS,COX17 protein (P0.05, P01) and decrease the expression of CTR1,MT protein (P0.01). Conclusion: Gantou decoction may up-regulate the expression of ATP7b,ATOX1,CCS,COX17 protein and inhibit the expression of CTR1,MT by regulating intracellular copper metabolism related proteins, so as to play the role of intracellular copper excretion. Gantou decoction can reduce the content of copper in hepatocytes through multi-target and multi-channel regulation of copper metabolism pathway.
【作者单位】：安徽中医药大学神经病学研究所附属医院;
【基金】：国家自然科学基金项目(81473535,81173212)
【分类号】：R-332;R277.7

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