运脾化浊方对Wilson病铜稳态影响及机制

Clinical Efficacy and Mechanism of Yunpi Huazhuo Prescription in Regulating Copper Homeostasis in Wilson's Disease

目的:观察运脾化浊方对Wilson病(WD)铜稳态影响的临床作用特点,分析验证其作用机制,为临床有效方药的应用提供进一步的证据支持。方法:将2023年01月至2023年12月安徽中医药大学第一附属医院符合入组标准的60例WD患者分为观察组(30例)和对照组(30例),对照组予常规排铜保肝治疗,观察组在常规治疗的基础上加用运脾化浊方治疗,共治疗4个疗程(32 d)。观察治疗前后两组患者铜生化指标(血清游离铜、铜蓝蛋白、铜氧化酶、血清铜、血清铜锌比、24 h尿铜、24 h尿锌、24 h尿铜锌比)、中医证候积分、临床有效率水平。利用网络药理学及分子对接技术,构建“疾病-中药-活性成分-核心靶点”相互作用网络图和PyMOL可视化分析图,分析验证其作用机制。结果:①与本组治疗前比较,治疗后观察组患者中医证候积分明显下降(P<0.05),且观察组中医证候积分下降水平明显低于对照组(P<0.05)。对照组总有效率为40.00%(12/30),观察组总有效率为66.67%(20/30),观察组总有效率高于对照组(χ^(2)=4.286,P<0.05)。与本组治疗前比较,治疗后观察组患者24 h尿铜水平显著升高、血清游离铜和24 h尿铜锌比水平明显下降(P<0.05),且治疗后观察组24 h尿铜水平明显高于对照组(P<0.05)、血清游离铜和24 h尿铜锌比水平明显低于对照组(P<0.05)。两组患者治疗后血清铜水平均显著降低(P<0.01)、铜蓝蛋白水平明显下降(P<0.05),治疗后观察组血清铜水平明显低于对照组(P<0.05)。与本组治疗前比较,两组治疗后铜氧化酶、血清铜锌比、24 h尿锌差异无统计学意义。②共得到运脾化浊方治疗WD铜稳态的有效成分83个、交集靶点共341个,主要有效成分有槲皮素(quercetin)、异鼠李素(isorhamnetin)、谷甾醇(sitosterol),关键靶点有蛋白激酶B1(Akt1)、肿瘤坏死因子(TNF)、胱天蛋白酶-3(Caspase-3);基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析显示核转录因子-κB(NF-κB)信号通路、长寿蛋白信号通路、癌变途径、脂质与动脉粥样硬化途径为显著富集的通路。分子对接结果表明关键活性成分与核心靶点之间存在较强的结合活性。结论:运脾化浊方能够显著改善WD患者中医证候积分、提高临床有效率,通过作用于多个关键靶点、多条信号通路发挥调节WD铜稳态的作用。

Objective:To evaluate the clinical efficacy and decipher the potential mechanism of Yunpi Huazhuo prescription in regulating copper homeostasis of Wilson's disease(WD)and thus provide evidence for the clinical application of this prescription.Method:Sixty patients with WD treated in the First Affiliated Hospital of Anhui University of Chinese Medicine from January to December in 2023 and meeting the inclusion criteria were enrolled in this study and assigned into an observation group(30 cases)and a control group(30 cases).The control group was treated with the routine therapy for copper excretion and liver protection,and the observation group with Yunpi Huazhuo prescription in addition to routine therapy.The treatment lasted for four courses(32 days).The serum levels of non-ceruloplasmin-bound copper,ceruloplasmin,copper,copper oxidase,and ratio of copper to zine,24-hour urinary copper excretion,24-hour urinary zinc excretion,ratio of 24-hour urinary copper excretion to zinc excretion,and traditional Chinese medicine(TCM)syndrome score were determined before and after treatment,and the response rate was calculated for both groups.Furthermore,network pharmacology and molecular docking were employed to construct the"disease-Yunpi Huazhuo prescription-active ingredients-core targets"network,which was visualized by PyMOL,on the basis of which the potential treatment mechanism of Yunpi Huazhuo Prescription was explored.Result:①The treatment in both groups decreased the TCM syndrome scores(P<0.05),and the decrease in the observation group was larger than that in the control group(P<0.05).The total response rate in the observation group was 66.67%(20/30),which was higher than that(40.00%,12/30)in the control group(χ^(2)=4.286,P<0.05).The treatment in both groups elevated the 24-hour urinary copper excretion and lowered the serum levels of non-ceruloplasmin-bound copper and ratio of 24-hour urinary copper excretion to zine excretion(P<0.05),and the changes were larger in the observation group than in the control group(P<0.05).The serum levels of copper(P<0.01)and ceruloplasmin(P<0.05)declined in both groups after treatment,and the observation group had lower serum level of copper than the control group after treatment(P<0.05).There was no significant change in the serum level of copper oxidase,ratio of serum copper to zine or 24-hour urinary zinc excretion in the two groups after treatment.②There were 83 active ingredients in Yunpi Huazhuo prescription,which shared 341 common targets with copper homeostasis in WD.The active ingredients included quercetin,isorhamnetin,and sitosterol and the core targets included protein kinase B1(Akt1),tumor necrosis factor(TNF),and Caspase-3.The gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses predicted nuclear factor kappa B signaling pathway,sirtuin signaling pathway,pathway in cancer,and lipid and atherosclerosis pathway were significantly enriched.Molecular docking results showed strong binding affinity between key active ingredients and core targets.Conclusion:Yunpi Huazhuo prescription could significantly reduce the TCM syndrome score and improve the clinical efficacy in treating for WD.It may regulate the copper homeostasis of WD by acting on multiple key targets and signaling pathways.