Screening Of Copper Chelating Capacity Of Anti-HLD Drugs And Evaluation Of Its Decoppering Activity

Hepatolenticular degeneration（HLD）,also known as Wilson’s disease（WD）,is an autosomal recessive disorder of copper metabolism.The aetiology is ATP7B mutation in the patient’s gene,which leads to abnormal synthesis of ceruloplasmin and disorder of copper excretion in bile.Excessive copper is deposited in the body,causing damage to multiple major tissues and organs such as the liver,brain,kidney,and cornea.The clinical treatment of HLD is mainly to decopper from the body through chelation with metal chelating agents such as penicillamine,trientine,and tetrathiomolybdate;In recent years,traditional Chinese medicine（TCM）has also achieved good clinical efficacy in treating HLD.However,due to the lack of evaluation methods of copper chelating capacity,the development of anti-HLD drugs is hindered.At present,the methods to determine the chelating capacity of compounds with metal ions mainly include ultraviolet and fluorescence,etc.These methods have relatively poor repeatability,many interference factors,and only the determination of the chelating capacity of monomeric compounds,which can not meet the needs of anti-HLD drug screening.There is an urgent need to establish a screening method for decoppering activity of anti-HLD drugs,which is used to earch for copper chelating agents with high efficiency and low toxicity,and provide scientific basis for research and development of innovative anti-HLD drugs.Objective:To establish and evaluate a rapid and effective screening method for decoppering activity of anti-HLD drugs.The copper chelating capacity of existing chemical drugs and TCM was determined by a screening method for establishing the chelating capacity of compounds with Cu²⁺by ion meter;A HLD hepatocyte model was constructed using plasmid transfection technique to study the biological activity of compounds at the cellular level,and the compounds screened by this method were evaluated for decoppering effect of intracellular Cu²⁺.Methods:An ion meter screening method was established for the chelating capacity of compounds with Cu²⁺.The optimal conditions were obtained by investigating the chelating capacity under different p H values,ionic strength,Cu²⁺concentration,and compound concentration.This method was used to evaluate the copper chelating capacity of Gandou Decoction,single herbs,7 TCM monomers and 6 chemical monomers.By measuring the chelation degree of free Cu²⁺after chelation and calculating the chelating rate,the compounds with strong chelating capacity to Cu²⁺were screened out.Four plasmids were transiently transfected into BRL-3A cells using ATP7B sh RNA interference technology.The fluorescence transfection efficiency,ATP7B m RNA and protein expression levels were detected by fluorescence microscope,RT-PCR and Western blot after 48 h transfection.sh ATP7B plasmid with the best inhibitory effect was selected to construct HLD hepatic cells.A copper-laden HLD hepatocyte injury model was established by incubation with Cu SO₄,and Maximum proliferation rate(P_max)was used as an index to further evaluate the protective capacity of TCM and chemical monomers.Based on the established method,combined with the effect of compound intervention on cell morphology and Cu²⁺content,the decoppering effect of monomeric compounds screened was evaluated.Results:1.The establishment of a method for measuring the chelating capacity of anti-HLD drugs with Cu²⁺by ion meter（1）Optimized determination conditions and methodological study:Cu²⁺ concentration 0.1 m M,drug concentration 0.1 m M,ionic strength 0.01 M,buffer concentration 10 m M;The methodological investigation of precision,stability and repeatability showed that the established method was accurate and reliable,and could be used to determine the chelating capacity of compounds with Cu²⁺.（2）The results of screening by the established method showed that:Gandou Decoction,single medicine（rhubarb,turmeric）,TCM monomers（rhein,quercetin）and chemical monomers（trientine hydrochloride,ammonium tetrathiomolybdate）had strong chelating capacity to Cu²⁺（70.15%～96.44%）.2.Evaluation of protective capacity of monomeric compounds based on copper-laden HLD hepatocyte model（1）The fluorescence microscope showed that no GFP green fluorescence expression was observed in normal BRL-3A cells,while the sh NC negative control group and four sh ATP7B plasmid groups showed GFP-labeled green fluorescence after 48 h transfection,and the transfection efficiency was the highest in sh ATP7B-4group,about 70%.The results of RT-PCR and Western blot confirmed that the ATP7B m RNA and protein levels in four plasmid transfected groups were significantly lower than those in the control and sh NC groups（P<0.01）.Among them,the ATP7B m RNA and protein expression in sh ATP7B-4 group decreased most obviously,which could be used as a good HLD hepatocyte model.（2）By comparing copper toxicity of BRL-3A cells before and after transfection,it was found that the cells in sh ATP7B group had more obvious copper damage than those in control and sh NC groups.At the same time,the optimal modeling conditions of HLD hepatocytes at 300μM Cu SO₄for 24 h were determined.（3）The cytoprotective efficacy of 13 monomeric compounds were screened,which showed that TCM and chemical monomers could improve the cell survival rate to varying degrees,and reduce HLD hepatocytes injury caused by copper load.Among them,the P_maxvalues of emodin,rhein,trientine hydrochloride,and ammonium tetrathiomolybdate were larger and more than 50%,which had a strong protective effect on copper-laden HLD hepatocyte injury（P<0.001）.The corresponding P_maxconcentrations were 20μM,20μM,1 m M,80μM,respectively.3.Evaluation of decoppering effect of four anti-HLD compounds obtained by screening（1）Cell morphology was observed by inverted microscope.BRL-3A cells in normal group showed flat or polygonal shape with adherent growth and intact cell membrane;The cells in the copper-laden sh NC group clustered and some cells split into fragments;Half of the cells in sh ATP7B model group turned round and died,and the cell membrane broke;However,the number of dead cells and the degree of damage decreased after the intervention of 4 monomeric compounds.（2）The PP-Cu probe was used for Cu²⁺fluorescent labeling,and the red fluorescence intensity was proportional to the intracellular copper concentration.Fluorescence microscopy showed that cells in the normal group had almost no red fluorescence;The red fluorescence of sh NC group and sh ATP7B group incubated with 300μM Cu SO₄was significantly enhanced;After pretreatment with each monomeric compound,the fluorescence was weakened compared with the sh ATP7B model group,indicating tha the intracellular Cu²⁺concentration decreased.（3）The Cu²⁺content inside and outside the cells was determined by atomic absorption spectrometry（AAS）.Compared with the sh NC group,the intracellular Cu²⁺content in sh ATP7B model group was significantly higher（P<0.01）,while the extracellular Cu²⁺content was lower（P<0.01）;After the intervention of 4monomeric compounds,the intracellular Cu²⁺content decreased significantly compared with those of the model group（P<0.05,P<0.01）,and the extracellular Cu²⁺content increased inversely（P<0.01）.The order of decoppering effect was:ammonium tetrathiomolybdate>rhein>trientine hydrochloride>emodin.The results showed that the four monomeric compounds could promote copper excretion and reduce the content of Cu²⁺in HLD hepatocytes.Conclusion:In this study,based on the evaluation of in vitro Cu²⁺chelating capacity by ion meter and the copper-laden HLD hepatocyte model,a systematic screening method for decoppering activity of anti-HLD drugs was established,which provides an effective screening pathway for potential anti-HLD drugs.It is of great significance and application value for the research and development of anti-HLD drugs.