| Objective:Hepatolenticular Degeneration(HLD)is an autosomal recessive inherited copper metabolic disorder disease.Due to ATP7B gene mutation,ATP-dependent copper transporter cannot be normally expressed.As a consequence,copper accumulation in the body first involving the liver,followed by major organs such as brain and kidney,resulting in different degrees of damage.HLD is one of the few treatable genetic diseases.The Institute of Neurology of Anhui University of Traditional Chinese Medicine has established Gandou Decoction(GDD)for the treatment of HLD in clinical practice and achieved good results.However,the treatment mechanism of this side is still unclear.The chronic accumulation of copper in HLD patients causes liver metabolic disorders.Whether the disease is related to metabolism and whether GDD can improve HLD by improving metabolic therapy is worthy of further study.In this study,the HLD copper-laden model was established.UPLC-Q-TOF-MS~E analysis technique combined with multivariate statistical analysis method was used to find biomarkers in HLD copper-laden model rats.Further explore how GDD affects the metabolism of HLD biomarkers in vivo.From the perspective of metabolomics,the mechanism of action of GDD in the treatment of HLD was clarified,which provided a theoretical basis for clinical treatment.Method:Firstly,the rat model of HLD copper-laden was constructed,and the content of copper in serum,liver and urine was detected by atomic absorption spectrophotometry.The model was further verified by combined with pathological sections of liver tissue,and the intervention effect of GDD on HLD was also investigated.Secondly,the UPLC-Q-TOF-MS~E method for the endogenous metabolites of three biological samples was established by collecting the urine,serum and liver tissues of normal rats,HLD copper load model rats and liver bean soup administration rats.The metabolic profile and raw data of the biological sample are available.In order to further obtain the metabolic difference of biological samples combined with multivariate statistical analysis methods,the metabolites in the online database were matched to initially identify endogenous metabolites in the biological samples.On this basis,the MS~E model secondary verification was used to verify the structure of metabolites,and the differential metabolites in the copper-laden model rats and normal rats were screened to explore the mechanism of the copper-laden model.At the same time,the same metabolites changes after the intervention of GDD was identified,and how the GDD affected the endogenous metabolites and metabolic pathways in the copper-laden model rats.Results:1.The results of copper-laden model rat showed that serum copper content,urinary copper content and hepatic copper content in the copper-laden model group were significantly higher than those in the normal group(P?0.05).After treatment with GDD,serum copper and hepatic copper level were decreased,and urinary copper content was increased(P?0.05).Combined with pathological section results,it was confirmed that the copper-laden model rat was successfully established,and GDD can effectively intervene in the copper-laden model rat,which has a protective effect on liver tissue.2.The results of liver metabolomics study showed that 19 metabolites related to the liver of the copper-loaded model group were screened.Among them,7 metabolites were detected in negative ion mode and 12 metabolites were detected in positive ion mode.According to the normalized abundance ratio,there were 5 metabolite levels in the liver of the copper-laden model group,and 14 metabolite levels were up-regulated.Metabolites such as Ursodeoxycholic acid,Alpha-Linolenic acid,Taurochenodesoxycholic acid,and PC(18:3/22:6)are closely related to fatty acid metabolism and phospholipid metabolism in the body.After taking GDD,the copper-laden model group can be effectively adjusted and the level tended to the normal group.3.Serum metabolomics studies showed that 25 differentially expressed metabolites were identified as potential biomarkers in the copper-laden model group,12 metabolites were detected by negative ions,and 13 metabolites were detected by positive ion mode.According to the standardized abundance comparison,it was found that there were 15metabolite levels down-regulated in the serum of the copper load model,and 9metabolite levels were up-regulated.Metabolites such as Taurine,L-Phenylalanine,Gamma-Linoleic acid,Linoleic acid,Arachidonic acid and PC(22:5/16:0)are closely related to amino acid metabolism and fatty acid metabolism.By administering GDD,the relevant endogenous metabolites are adjusted to normal metabolic levels.4.The results of the urine metabolomics study showed that 13 differentially expressed metabolites were identified as potential biomarkers in the copper-laden model group,one in the negative ion mode and 12 in the positive ion mode.According to the standardized abundance comparison,there were 6 metabolites levels were down-regulated and 7 metabolite levels were up-regulated in the urine of the copper-laden model group.Among them,Sphinganine,Phytosphingosine and PC(15:0/24:0)affect pathways such as sphingolipid metabolism and fatty acid metabolism.The intervention of GDD promoted the metabolite level callback,and actively explored the mechanism of action of traditional Chinese medicine compound GDD in the treatment of HLD.Conclusion:In this study,the HLD copper-laden model was successfully established.GDD intervention has a protective effect on the liver,which indicates that GDD has a certain effect on the treatment of HLD.Using the UPLC-Q-TOF/MS~E technique and multivariate statistical analysis of metabolomics research methods to screen and identify57 biomarkers associated with copper-laden model rat.The results indicate that they may be related to lipid metabolism and amino acid metabolism.The mechanism of action of GDD in copper-laden model rats is preliminarily demonstrated,which provides a scientific basis for better clinical treatment of HLD patients. |
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