Methodological Investigation Of High-content Liver Toxicity Screening And Its Application In Traditional Chinese Medicine Injection

The so-called Drug toxicity is by the dose is too large,the medication is too long or the body of the Drug sensitivity is too high when the harmful response.The liver is the most important organism that contains Drugs in vivo metabolism and transformation.It is particularly vulnerable to Drug damage and is an important target organ for Drug toxicity.Therefore,the evaluation of Drug hepatotoxicity plays an important role in the toxicological evaluation of Drug discovery and Drug safety risk assessment.However,the experimental animals in the liver toxicity evaluation cycle is long,the amount of Drugs used,the cost of research costs larger,the experimental results can not fully reflect the characteristics of human liver toxicity.Therefore,in recent years,in vitro hepatocytes in the evaluation of Drug safety has become a common liver cytotoxicity evaluation of in vitro test tools are widely used in Drug cytotoxicity research.HCA(High Content Analysis)is an efficient new Drug screening technique based on cell phenotype analysis.It can detect the morphology,growth,cycle,migration,and migration of cells to be tested under the premise of maintaining cell structure and function integrity.Apoptosis,metabolic pathway and signal transduction,and obtain a large amount of relevant information from a single experiment to determine the biological activity and potential toxicity of the compound.At present,HCA can monitor the number of important markers,such as the morphology and number of nuclei,mitochondrial membrane potential and oxidative stress,apoptosis and early DNA damage,in vitro.It is an in vitro evaluation of the potential of compounds in vitro.Liver toxicity,from the mechanism to predict the safety of candidate Drugs and efficient means.This method can make up the low sensitivity of animal experiment,long test period and can not quantify the shortcomings.Therefore,high-throughput toxicity evaluation technology has become a new technical trend of Drug quality risk and safety risk assessment.This topic will be the laboratory has established a high content of liver screening method for further study,and high content of liver screening method in the application of injection to explore.The study was divided into three parts:The first part,the factors that influence the screening method of high-content hepatectomy;The second part,the methodological method of high-content liver screening method;The third part,application of High Content Hepatic Hepatitis Screening Method in Injection.The First Part,the factors that in:fluence the screening method of high-content hepatectomyFirst:Effect of initial cell density on the results of screening methods with high content of liver The results show:the positive Drug ticlopidine of initial inoculation density that contains1×104 per·mL-1?1×105per·mL-1 and 2×105per·mL-1 in the number of cells,DNA content,GSH reduction level,ROS content and MMP test results were positive.The number of cells,DNA content,GSH reduction level,ROS content and MMP in the negative Drug aspirin was negative This indicates that aspirin has no toxic effect on Hep G2 hepatocytes.The results of the five indicators of comprehensive analysis,2×105per·mL-1 the density of the indicators of the results are relatively good.Second:Effects of Different Cell Models on the results of Highly Connected Hepatitis B Virus ScreeningThe results show:Drugs in Hep G2 cells and L-02 cells,the positive Drug ticlopidine in the number of cells,DNA content,GSH levels,ROS content and MMP five indicators were positive,indicating that the positive Drug ticlopidine on Hep G2 hepatocytes and L-02 cells had significant toxic effects.The results of negative aspirin were negative in the number of cells,DNA content,GSH reduction level,ROS content and MMP five indexes.This indicates that aspirin has no toxic effect on Hep G2 hepatocytes and L-02 cells.Since Hep G2 cells are considered to be suitable liver toxicity test models,and their biotransductive metabolic enzymes are homologous to human normal liver parenchymal cells,the degree of differentiation is high and a more complete and stable biotransformation Metabolic enzymes,so the next experiment will choose Hep G2 cells for liver toxicity test model.The Second Part,the methodological method of high-content liver screening methodFirst:Repetitive study of screening methods for high-content liverThe results show:After the error analysis was performed between the three parallel wells of each Drug concentration,the errors of the three indexes were the smaller of the number of cells,the DNA content and the GSH reduction level,and the error of the ROS content and the two indexes of MMP Larger.Hep G2 hepatocellular toxicity three times high content analysis of the repeatability of the results showed that the number of cells,DNA content and GSH reduction of the level of repeatability is better,and ROS content and MMP repeat reproducibility is poor.Second:A Study on the Reproducibility of Highly Connected LiverThe results show:The results showed that the cell number,DNA content and GSH reduction level of the repeatability is better,and ROS content and MMP repeat reproducibility is poor.The reproducibility and reproducibility of the high-content analysis method showed good reproducibility and reproducibility of the number of cells,DNA content and GSH reduction level,and the repeatability of ROS and MMP-2 Reproducibility is relatively poor,the reason for further study.The Third Part,application of High Content Hepatic Hepatitis Screening Method in InjectionFirst:High Content Analysis results of Multi-batch Fluconazole InjectionThe results show:In this test system,fluconazole injection of more than 90%will appear a certain degree of liver toxicity,but the detection of "toxic warning" indicators and the degree of toxicity there are some differences.Suggesting that fluconazole injection clinical application may occur in varying degrees of liver damage or liver biochemical abnormalities.Analysis of 74 batches of fluconazole injection samples showed that 70 batches of fluconazole injection glutathione(GSH)indicators s?ggested "toxicity warning";43 batches of sample peroxide(ROS)indicators suggested "toxicity warning";18 batches of sample cell nuclear DNA indicators suggest "toxicity warning";14 batches of sample cell volume reduction(Cell Loss)indicators suggest "toxicity warning";12 sample mitochondrial membrane potential(MMP)indicators suggest "toxicity warning." Suggesting that more than 90%of fluconazole injection of liver toxicity mechanism may be related to its interference with liver GSH production.Second:Analysis of High Intensity of Ten Traditional Chinese Medicine InjectionThe results show:In addition to the diclofenac hydrochloride injection,the other nine kinds of traditional Chinese medicine injection were at risk of hepatotoxicity,in which the risk of hepatotoxicity was the highest,and the risk of hepatotoxicity was the least.The results showed that the four indexes of glutathione(GSH)in traditional Chinese medicine were suggested as "toxic warning" by eight kinds of traditional Chinese medicine injection.The Cell Loss indicator indicates "Toxicity warning".Suggesting that the mechanism of liver toxicity caused by traditional Chinese medicine injection may be a direct cytotoxic effect.