肝豆汤效应中药对TX小鼠铜代谢通路分子调控及内脏保护作用机制的研究

WD is a treatable inherited disorder of copper metabolism.It's very important for the prognosis to have a high compliance as well as safe and effective therapeutic schedule.Gandou decoction,which can clear away heat and toxic materials parallel with pure fu-organs to removing dampness was treated WD patients by the project applicant. long-term clinical practices showed that Gandou decoction was of good efficacy and less side effects.For the reason that the effect of Chinese herbs in Gandou decoction is not clear and WD is systemic disease with complex clinical phenotype,the formula for patients with different clinical phenotype is not optimal compatibility and the optimum dose. The project make assumption about Gandou decoction have different main effect of Chinese herbs with different pathways for different clinical phenotypes WD patients,WD model TX mouse as research objects,by the method of uniform design,application of cell and molecular biology techniques to screen out the formula of intervention of copper metabolism and main effect of Chinese herbs with protection of liver, brain, kidney damage.Further using Real-PCR,west blot and laser scanning confocal microscope technology, to observe effect of Chinese herbs regulating function of protein involved in metabolic pathways and protective functions of viscera and reveal mechanism of curative effect at the molecular level.In order to establish high adherence and high pertinency theoretical basis for standardized WD treatment.

Wilson病(WD)是一种可治性铜代谢障碍性遗传病，依从性高且安全有效的治疗方案对其预后至关重要。项目申请者创用清热解毒、通腑利尿法组成肝豆汤治疗WD患者，长期临床实践表明其疗效好，不良反应少。但其效应中药并不明确，且WD系临床表型复杂的全身性疾病，该方对于不同临床表型的患者并非是最优配伍和最适剂量。本项目假设肝豆汤对不同临床表型的WD患者存在不同作用途径的主效应中药，以WD模型TX小鼠为研究对象，采用均匀设计法，应用细胞和分子生物学等技术筛选出该方中干预铜代谢及对肝、脑、肾损害具有保护等作用途径的主效应中药，进一步运用Real-time PCR、West blot和激光扫描共聚焦显微镜等技术，观察效应中药对TX小鼠铜代谢通路相关蛋白的调控作用和对其内脏的保护作用，在分子水平揭示其疗效机理。为建立依从性高、针对性强的规范化WD治疗方案提供理论依据，并为中医药防治其它神经遗传病提供新思路。

Wilson病（WD）是一种常染色体隐性遗传铜代谢障碍性的可治性罕见病，是中医药防治的优势病种。经验方肝豆汤在长期治疗WD过程中疗效肯定，未见明显毒副作用，且价格低廉，但是该复方对于不同临床表型患者的疗效不一，其最佳配伍和最适剂量尚未可知。本研究结果表明：（1）中药肝豆汤可干预TX小鼠的铜锌代谢，其效应中药是：大黄和姜黄；最佳药物剂量为大黄15g，姜黄15g。（2）中药肝豆汤可抑制TX小鼠的神经元细胞凋亡过程，其抑制凋亡的机理是通过多途径、多靶点来实现，其效应中药是：姜黄、黄连、泽泻和金钱草;最佳药物剂量为姜黄15g、金钱草18g、黄连14g和泽泻2g。（3）姜黄可升高TX小鼠血清铜、锌含量，且升高血清锌的作用较大；参三七可降低TX小鼠肝组织内铜、锌含量，且降低铜的作用更显著。（4）大黄、参三七可抑制P38的表达，参三七可抑制ERK的表达。（5）泽泻可促进BCL-2的表达，参三七可抑制BAX的表达。（6）肝豆汤治疗WD肾损害效应中药最佳配伍为：大黄0.059g、姜黄0.059g、黄连0.059g、泽泻0.072g、金钱草0.072g、三七0.009g。（7）肝豆汤中与TX小鼠肾组织凋亡蛋白Bax、Bcl-2、Caspase-3表达密切相关的效应中药有黄连、泽泻、金钱草、三七；肝豆汤中与TX小鼠肾组织纤维化相关蛋白α-SMA、CIV表达密切相关的效应中药有大黄、黄连、泽泻。另外，肝豆汤尚具有调节TX小鼠细胞因子相关免疫紊乱的作用。本研究以WD模型TX小鼠为研究对象，采用均匀设计法，筛选出了肝豆汤方中干预铜代谢及对肝、脑、肾损害具有保护等作用途径的主效应中药，观察了其对TX小鼠铜代谢通路相关蛋白的调控作用，用现代科技语言诠释了中药配伍科学的内涵，进一步规范了WD治疗方案，也为中医药防治其他神经遗传病提供了治法思路，有助于中医药的现代化和国际化。