Study On Early Hepatotoxicity Of Drugs Based On Multi-parameter High Content Screening

Drug-induced liver injury（DILI）is a common adverse drug reaction,which is the main reason for drug development failure and withdrawal from the market.According to research,traditional liver toxicity evaluation of drugs was mainly achieved through animal experiments.However,due to extensive use of animals,long time-consuming,high cost,low sensitivity,and species diversity,the existing animal models do not evaluate human liver damage well.With the deep understanding of drug hepatotoxicity mechanism,Toxicological alternatives based on"toxicity pathway"and"toxic mechanism"has emerged.Using in vitro cell experiments instead of animal experiments has become an important direction of liver toxicity prediction.The prediction of drug toxicity based on the"toxicity pathway"was proposed in the2007 by the US National Research Council（NRC）,entitled“Toxicity Testing in the21st Century:A Vision and a Strategy”,the report mentioned that:Exogenous chemicals entering the body will initiate molecular initiating events（MIE）and disturbing toxic pathways,resulting in a series of biological tissues（including cells,sub-cells,tissues and organs,etc.）which cause adverse reactions.By in vitro cell-level experiments,the dose and toxic effects of the drug-perturbed toxic pathways are linked to provide a more comprehensive assessment of drug safety.The application of high-content cell imaging analysis technology in alternative toxicology provides a great help for this idea.High-content cell imaging analysis technology is take advangtage of fluorescence microscope imaging automation to automatic quantification and positioning analysis of the cell morphology and its fluorescence intensity and distribution,the obtained drug toxicity test data can make the dose-effect curve of drug toxicity and analyze the dose of early cytotoxicity caused by the drug,so as to realize more accurate prediction of the potential toxicity of the drug.In this study,HepG2 cells as a model,according to the mechanism of anti-tumor drugs from a variety of drugs screened four representative drugs:paclitaxel,podophyllotoxin,cisplatin,5-fluorouracil,SDS as the study object,using MTT to measure the IC₅₀ value,and take the IC₅₀ value as a reference（CCl₄ 100%saturated solution for reference）,treat cells with take the concentration gradients of 100%,75%,50%,25%,and 10%for 1 h,2 h,4 h,and 24 h.HCS was then used in conjunction with fluorescent labeling to detect changes in related parameters（eg,cell number,DNA content,calcium homeostasis,ROS,mitochondrial membrane potential,cell membrane permeability,etc.）caused by direct action of the drug,and comprehensive analysis of various indicators to establish analytical methods,the results showed that the hepatotoxicity of paclitaxel,cisplatin,and 5-fluorouracil on HepG2 cells was caused by impaired of cell membrane integrity;Podophyllotoxin is caused by imbalance of calcium homeostasis due to elevated calcium concentration;CCl₄cytotoxicity is caused by ROS,and establishing an HCS method for detecting early drug hepatotoxicity.Based on the anti-tumor mechanisms of paclitaxel and podophyllotoxin,the present study screened two drugs as colchicine and etoposide from multiple drugs with the same mechanism.The same HCS method was used to detect the changes of related indicators caused by direct action of drugs and verify the established HCS analysis method.In this study,early hepatotoxicity of traditional Chinese medicines was studied by using established HCS analysis method.From the hepatotoxic Chinese herbs,two different components of traditional Chinese medicine were Dioscin and Alisaniol B.The results of early hepatotoxicity detected by HCS showed that the cytotoxicity of Alisma alcohol B and dioscin on HepG2 cells was caused by oxidative stress,and it was speculated that oxidative stress was related to iron apoptosis.In this experimental study,the total ginsenosides and ginsenoside monomers from processed Panax notoginseng as research subjects to measure their cytotoxicity and to study the effect of processing time on cytotoxicity.The results showed that the color and composition of ginsenosides after processing were changed,and the processing time was positively related to the content of components and cytotoxicity（P<0.001）.It is indicated that raw panax pseudoginseng cannot be over-concocted,if the processing time exceeds 9 hours,it will show some toxic side effects.Ginseng total saponins and ginsenosides monomer show some cytotoxicity and have potential hepatotoxicity.In summary,using the HCS detection method can detect early hepatotoxicity of the drug with multiple parameters,multiple indexes,rapid and high throughput,which has higher sensitivity and specificity than the existing methods,increases safety,and reduces experimental costs,and fill in the gaps in the detection of natural compounds by HCS and expand the application of HCS.