The Protective Function And Metabolomics Study On HLD Model Cells Injured By Copper

Objective:Gandou Decoction is a classic Chinese medicine treatment of Hepatolenticular Degeneration（HLD）.Clinical studies have shown that Gandou Decoction can not only increase urinary copper excretion,but also promote the recovery of liver function in patients.The previous study of the research group found that Gandou Decoction had a significant protective effect on the liver injury of copper-loaded HLD model rats.However,the liver-protecting mechanism of this prescription is still unclear.Cell metabolomics is a newly developing discipline that reflects the cell phenotype and changes in a dynamic system.Metabolomics explores the relationship and dynamics of metabolites,pathological changes,and drug intervention through the qualitative and quantitative description of the final metabolites in the cell.The pathogenesis of the disease and the mechanism of action of drugs are systematically explained from the metabolic level,which systematically explain the pathogenesis of disease and the mechanism of drugs from the metabolic level.In this study,the protective effect of Gandou Decoction on HLD hepatocytes was investigated by constructing a copper-damaged HLD hepatocytes model.At the same time,the cell metabolomics technology was used to explore the mechanism of the prescription to play a role in the protection of hepatocytes.This will provide a reliable theoretical basis for Gandou Decoction in the clinical treatment of HLD.Methods:ATP7B sh RNA plasmid transient transfection technology was used to construct HLD hepatocyte model,Cu SO₄ was used to damage the model cells,cell viability was detected by MTT method.The best modeling conditions were determined,and the copper-loaded HLD hepatocytes injury model was established.In this study,the protective effect of Gandou Decoction on copper-loaded HLD hepatocytes by observing the cell morphology,measuring LDH release rate,MDA,SOD,GSH/GSSG and ROS levels,combined with transmission electron microscopy to observe the ultrastructure of hepatocytes.At the same time,the influence of Gandou Decoction on copper content in HLD hepatocytes was investigated,and the driving effect of Gandou Decoction on copper was evaluated.It is established that HILIC-Q-TOF/MS and RPLC-Q-TOF/MS analysis methods for endogenous metabolites in cell samples.Multivariate statistical analyses including principal component analysis（PCA）and orthogonal partial least squares discriminant analysis（OPLS-DA）are performed on metabolomics data to identify differential metabolites.On this basis,the key metabolites affected by GDD intervention in HLD was quantified,meanwhile combing with Western blot technology to detect the expression of key enzymes in the biosynthetic pathway,the change level of metabolites were verified.Results:1.300μM Cu SO₄ treatment for 24 hours is the best modeling condition for HLD hepatocyte model;Gandou Decoction can significantly improve the cell survival rate after intervention.Compared with the model group,Gandou Decoction can significantly improve the cell survival rate and restore damaged cell morphology,decrease MDA,ROS content and LDH release rate in the model group,increase SOD level and GSH/GSSG ratio,and improve the ultramicroscopic lesion structure of hepatocyte,indicating that Gandou Decoction can reduce HLD hepatocyte damage caused by copper load.Using PP-Cu probe and atomic absorption method to detect intracellular copper levels,the results show that Gandou Decoction can significantly promote the copper excretion of HLD hepatocytes.2.The cell metabolomics research results show that HILIC-Q-TOF/MS screened and identified a total of 22 differential metabolites.There were 10 metabolites decreased and12 metabolites were up-regulated in copper-loaded HLD hepatocytes.RPLC-Q-TOF/MS detected a total of 25 differential metabolites.Among them,17 metabolites were decreased and 8 metabolites showed an increasing trend copper-loaded HLD hepatocytes.The 47 differential metabolites are mainly involved in D-glutamate and D-glutamine,alanine,aspartic acid and glutamate metabolism,glycerophospholipid metabolism,pyruvate metabolism and other metabolic pathways.By administering Gandou Decoction,the related metabolites can be adjusted to the normal metabolic level.The results of glutamate quantitative analysis and D-glutamate and D-glutamine metabolic pathway verification showed that after Cu SO₄treatment,the glutamate content in copper-loaded HLD hepatocytes was significantly increased,and Gandou Decoction could significantly reduce the glutamate content in model cells.This mechanism may be related to promoting the protein expression of GS and inhibiting the protein expression of GLS1 and GLS2.Conclusion:Gandou Decoction can promote copper excretion in copper-loaded HLD hepatocytes and effectively protect hepatocytes damage.At the same time,Gandou decoction can exert therapeutic effect by intervening in the metabolism of D-glutamate and glutamine,the metabolism of alanine,aspartate and glutamate,the metabolism of glycerophospholipids,and the metabolism of pyruvate.This study provides a new scientific basis for the clinical treatment of HLD with Gandou Decoction.