Multi-index Cytotoxicity Inspection Methods And Exploratory Research On Drug Hepatotoxicity

PurposeThis study uses the representative method of drug safety assessment,namely acute toxicity test、cytotoxicity test and multi-index cytotoxicity test,and selects drugs with hepatotoxicity(sodium valproate、paclitaxel、isoniazid、cyclophosphamide,tripterygium glycosides),and conducts safety evaluation with detection of hepatotoxicity.The results were compared and analyzed to explore the feasibility of the multi-index cytotoxicity test as a comprehensive evaluation method and standard quality control method for drug hepatotoxicity.Method1.Acute toxicity testLDso value of the drugs was determined.Totally 70 mice in half genders were randomly divided into 7 groups,each had 10 mice.According to the results of the pre-test,set the dose for each group of one-time administration.After the end of the experiment,the LDso value of the drugs was calculated based on the death of the mouse.2.Pathological section and proteomic analysisAfter acute toxicity test,the liver of tripterygium glycosides group was taken for pathological section analysis(HE staining)and proteomics analysis.3.Detecting cytotoxicity of L929 cells and HepG2 cells by MTT assayThe IC50 value of the drug was mainly determined by MTT assay,and the cultured cells were mouse fibroblast L929 cells and human liver cancer HepG2 cells,respectively.MTT assay was used to measure the optical density of living cells and the relative proliferation rate of cells.The two methods of cell manipulation are the same.Test operation method:5×104 · ml-1 L929 cell and 1×105 · ml-1 HepG2 cells in logarithmic growth phase were planked separately.After 24 hours incubation,add different concentrations of drugs(set the negative control group).Then 24 hours incubation,add 20μl of MTT solution to each well,and then incubation for 4 hours.After that,add 150μl of DMSO solution to each well,and then 5 minutes of oscillation,and then the optical density of each well was measured by double wavelength(570 nm,630 nm)of enzyme labeling instrument.Finally,the IC50 value was calculated.4.Evaluation of hepatotoxicity by HCS assayMulti-index cytotoxicity test uses high-content screening(HCS)assay.Four fluorescent dyeing reagents(Hoechst 33342、mBC1、Mito、ROS)were used for HCS staining,and corresponding to five indicators(DNA,cell loss,GSH,MMP,ROS),multi-target,multi-channel analysis,and the automatic dynamic analysis system integrates microscope,image processing technology and quantitative analysis results into one.After comprehensive analysis of multi-indicator hepatotoxicity,the hepatotoxicity of the test drug is predicted.Test operation method:2×105 · ml-1 HepG2 cells in logarithmic growth phase are planked,incubator for 24 hours,100ul of different concentration of the tested drug was added into each well.After 24 hours incubation,add the prepared mixed solution of mBC1,Hoechst 33342,Mito and ROS at 37 ℃,incubator for 45 minutes.When the time is up,discard the liquid,and wash once with PBS,and then add 100μl of PBS into each well,affix transparent microplate sealing film to plate for testing.The results were used to calculate ICso value and toxicity threshold concentration with a new calculation method,and compared with the LDso value and the ICso value measured by acute toxicity test and MTT assay.Results1.The results show that the poisoning phenomenon and the time of death on the day of administration of different drugs are different.The mice in the high-dose group administered by tail vein injection had shorter and faster abnormal reaction time than the high-dose group administered by intragastric administration,and the death time was relatively fast.The death of paclitaxel and isoniazid acute test animals occurred within 24 hours.In addition to the death of mice on the day of administration,sodium valproate showed a small number of mice deaths a few days after administration.The latest time of death was 11 and 12 days after intravenous administration of cyclophosphamide.The death time of mice administered with tripterygium glycosides by intragastric administration was 24 hours after administration.2.The results show that liver pathological sections of tripterygium glycosides showed severe liver injury.Proteomics analysis showed 112 differentially expressed proteins,of which 67 were down-regulated and 45 were up-regulated,involving biological processes,cellular components,KEGG pathway,and molecular functions.It is related to many metabolic pathways such as cholesterol and lipid,and active components in mitochondria and endoplasmic reticulum are active.The differentially expressed proteins Npcll1 and Hmgcsl were more studied,and the up-regulation of Cyp2a5 expression has potential research value.3.The results show that MTT assay showed no difference in the ICso value results of L929 and HepG2 cells.4.The results show that GSH,ROS,MMP results speculated that the drug dose did not show apoptosis,but hepatotoxicity has early warning,indicating that the HCS assay can alert liver toxicity in advance,and the hepatotoxicity warning is consistent with the results of the acute toxicity test.The trajectories of the five indicators of three batches of tripterygium glycosides were slightly different.The analysis of the test results of tripterygium glycosides was consistent with proteomics analysis.HCS analysis is sensitive,but the accuracy is not enough.The drug toxicity threshold concentration obtained by HCS analysis has a good correlation with the ICso value determined by MTT assay and HCS assay,and the correlation coefficient is>0.9,which is worthy of further study.ConclusionIn summary,it is feasible to establish the safety evaluation and diagnose the hepatic toxicity of tripterygium glycosides by using HCS multi-index cytotoxicity assay.