Study On Hepatotoxicity Of Myrobin Liver And Screening Method Of High - Content Hepatotoxicity

Myrobalan is the ripe fruit of Terminalia chebula Retz. and Terminalia chebula Retz. Var.Tomentella Kurt. It is a traditional Tibetan medicine that is in great need in clinic.It has many efficacy and definite functional mechanisms. However, so far there is little studies concening the toxicology both at home and abroad, so there is not enough research data to evaluate its toxicity. High Content Screening (HCS) is a new efficient screening technology based on the analysis. HCS can dynamic screens toxic substances through a multi-system, multi-channel and multi-target way at a cellular level. HCS not only understand the change in the cell biology which is associated with the test drug, and can illustrate the interaction relationship between test drug and targets, but also can predict the test drug toxicity by observed the change of morphology of cell. Therefore HCS can be used to predict toxicity early, rapid and high throughput.The aims of the the study subject are as follows: ①Studied the hepatotoxicity of Myrobalan with acut toxicity test, subacute toxicity test, dose-toxicity relationship test and time-toxicity relationship test.②Taking Myrobalan for research object, attempting to establish a hign cotent screening method in vitro for hepatotoxicity.The First Part:Studied the hepatotoxicity of MyrobalanFirst:Acute toxicity in ICR mice of Terminalia chebula Retz. Var.Tomentella Kurt, water decoctionObjective:To study the Terminalia chebula Retz. Var.Tomentella Kurt, toxicity level. Methods:Observe Terminalia chebula Retz. Var.Tomentella Kurt, water decoction on acute toxicity in mice and minimal lethal dose. Experimental animals were divided into 6 groups, oral give different concentration of solution, continuous observation for 14 d after the treatment, record the type toxic symptoms and deaths. Results:In the Terminalia chebula Retz. Var.Tomentella Kurt, group, some animals showed retardation, piloerection phenomenon after dosing. Severe animals can appear ventral decubitus. Animals began to die after 24 hour of dosing, and autopsied dead animals showed no abnormal organs. The minimal lethal dose (MLD) is 28.4 g·kg-1BW (170.1 times the dose of adult). Conclusion:The result showed that Terminalia chebula Retz. Var. Tomentella Kurt.has toxicity, but its taget maybe not liver.Second:Acute toxicity in ICR mice and subacute toxicity in SD rats of Terminalia chebula Retz. water decoctionObjective:To investigate Terminalia chebula Retz. water decoction in rodents and to confirm the mainly toxic target organs, character and degree of toxicological effects. Methods: ①Observe Terminalia chebula Retz. water decoction on acute toxicity reaction of mice and median lethal dose (LD50). Experimental animals were divided into 6 groups, oral give different concentration of solution, continuous observation for 14 d after the treatment, record the type toxic symptoms and deaths. ②Observe Terminalia chebula Retz. water decoction on subacute toxicity reaction of rats. SD rats were ig given myrobalan water decoction 16.0,8.0, 4.0g crude drugs·kg-1,once daily for 28 days. Rats were sacrificed on d 29. Blood biochemistry and hematological indexes were measured. The major organ indexes were determined and histopathology was detected. Results:①In the Terminalia chebula Retz. group, the animals showed retardation, piloerection phenomenon within 0.5 hour of dosing. After 24 h, the symptom such as prone, drowsiness, tremor, convusion were observed, some animials or even meet with death. Autopsied dead animals showed abnormal liver. The median lethal dose (LD50) is 9.7583 g·kg-1BW (58.4 times the dose of adult). ②The hematological indexes and the biochemical indexes of drug groups have significant difference when compared with that in blank group, but they were not biologically.Conclusion: Terminalia chebula Retz. water decoction maybe induce liver injury.Third:Study Myrobalan induce hepatotoxicity in ICR miceObjective:To study on the hepatotoxicity caused by single dose water extraction components of Myrobalan to mice. Methods:Mice were grouped according to different dose, to observe the death condition and toxicity of mice. The changes of the activity of ALT, AST, Urea,Cre and ALP were detected, and the morphological changes of liver tissue were observed under light microscope. Results:①In animals show lose weight, the liver weight and the liver index were decrease after treated.② The levels of ALT、AST、Urea、Cre and ALP changed as compared with control group, and with dose-effect relationship.③ Pathology results show that different degrees of liver damage were found. Conclusion: Myrobalan can induce hepatotoxicity in ICR mice by administrating a lager dose of Myrobalan with dose-effect relationship.Fourth:Time-toxicity relationship analysis about Myrobalan induce hepatotoxicity in ICR miceObjective:To study on the time-toxicity relationships caused by single dose water extraction components of Myrobalan to mice. Methods:Mice were grouped according to different time, to observe the death condition and toxicity of mice. The changes of the activity of ALT, AST were detected, and the morphological changes of liver tissue were observed under light microscope. Results: ①At 9g/kg dose level, the leve of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased with time prolonging. After lh of medication, the leve of ALT and AST started to rise, and increased to its peak in 24h. Pathology results confirme there were large necrosis area in liver in 24h. ②At 6g/kg dose level, the leve of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased with time prolonging for a while. After 1h of medication, the leve of ALT and AST started to rise, and recovered in 24h. Conclusion:Myrobalan can induce hepatotoxicity in ICR mice by administrating a lager dose of Myrobalan.The Second Part:To establish a method for predicting hepatotoxicity by High Content Screening analysis technologyFirst:The study of Hepatotoxicity of different polar parts extracted from Myrobalan by High Content Screening AnalysisObjective:To observe the hepatotoxicity of different polar parts extracted from Myrobalan on HepG2 cells using High Content Screening analysis technology, and to explore the characteristics of toxicity. Methods:Multi-parameter analysis of cytotoxicity (cells number, nuclear intensity, GSH reduce level, ROS level, mitochondrial membrane potentia) was carried out on the human hepatoma cell line HepG2 cells by using HCS. Results show: ①The precipitate of Myrobalan treated HepaG2 cells 24h, four targets (cells number, nuclear intensity, ROS level, mitochondrial membrane potential) test results are negative, the only one targe of GSH reduce level test results is positive. Beginning the precipitate at 62.5μg·ml-1 GSH reduce level exceed the toxic threshold. Therefor the precipitate induce liver injury because of cell membrane damage. ②The supernatant of Myrobalan treated HepaG2 cells 24h, five targets (cells number, nuclear intensity, GSH reduce level, ROS level, mitochondrial membrane potential) test results are positive. The supernatant at 1000μg·ml-1, The targets of cells number and mitochondrial membrane potential exceed the toxic threshold. The rest of tagets exceed the toxic threshold begin at 31.25μg·ml-1. Conclusion:The precipitate and the supernatant all can cause liver damage, and the supernatant are at much greater risk. The precipitate induces liver injury because of a decrease of GSH level, reduced ability for scavenging of free radicals. The causes of early stage damages of the supernatant are including chromatic agglutination, ROS over produce and GSH levels reduction. As the dose increased, the supernatant can reduced the number of cells andchanged the MMP.Second:The MTT methods examined whether the congruence of HCS on cellproliferation rates.Objective:To prove the validity of HCS by MTT methods. Methods:MTT assay was used to examine cell proliferation under the different polar parts treated 24h, and to explore the consistency between MTT assay and HCS assay. Result:The precipitate and the supernatant can’t inhibit the proliferation of HepG2. The ratio of the relative growth rate of HCS assay to the relative growth rate of MTT assay, were 1.11±0.08 and 1.01±0.06, respectively. The RSD were 7.59% and 5.83% respectively. It turns out that HCS assay and MTT assay had a good correlationship. Conclusion:the result of cell proliferation tested by HCS assay is effective and reliable, and HCS assay can get the quantitative relation of the cell number, which makes the result more intuitional and credible.Third:The animal experiment examined whether the congruence of HCS on toxicity determine.Objective:To prove the validity of HCS by animal experiment. Methods:To investigate the effect of different polar parts extracted from Myrobalan on AST and ALT in serum of mice, and to explore the consistency between animal experiment and HCS assay. Results:The precipitate can increase the level of ALT as compared with control group, and the supernatant can both increase the level of ALT and AST as compared with control group. The results sugges that the precipitate and the supernatant all can cause liver damage, and the precipitate was milder than the supernatant. It turns out that HCS assay had a good accuracy.Fourth:The proteomics technology examined whether the congruence of HCS on toxicological mechanisms.Objective:To prove the validity of HCS by proteomics technology. Methods:To identify the hepatic protein targets involved in the supernatant toxicity in mice to study the toxic mechanism, and to explore the consistency between the proteomics technology assay and HCS assay on toxic mechanism area. Results:the supernatant can induce the changes of the expression of proteins in liver. There were 20 abnormal protein spots expression in medicated group than those of blank group. Such as malate dehydrogenase (Mdhl), fumarylacetoacetase (FAA), ATP Synthase subunit alpha, NADH dehydrogenase [ubiquinone] iron-sulfure protein 2, lactoylglutathionelyase, Thiosulfate sulfurtransferase (TST), Betaine-homocysteine S-methyltransferase 1(BHMT1), Cytochrome b5, Carbamoyl-phosphste synthase [ammonia](CPS), Nicotinate phosphoribosyltransferase (PncB), voltage-dependent anion-selective channel protein 1(VDAC), Acetyl-CoA acetyltransferase, mitochondrial (ACAT), Calreticulin, Regucalcin, Dihydropteridine reductase (DHPR),and so on. The expression of those proteins was correlated with oxidative stress, amino acid metabolism and energy metabolism. It turns out that the toxic mechanism was consistent between the proteomics technology assay and HCS assay. Fifth:The study of Hepatotoxicity of C, D, E, F polar parts extracted from Myrobalan by High Content Screening AnalysisObjective:To observe the hepatotoxicity of different polar parts extracted from Myrobalan on HepG2 cells using High Content Screening analysis technology (HCS), and to explore the characteristics of toxicity. Methods:Multi-parameter analysis of cytotoxicity (cells number, nuclear intensity, GSH reduce level, ROS level, mitochondrial membrane potentia) was carried out on the human hepatoma cell line HepG2 cells by using HCS. Results ① C polar parts treated HepaG2 cells 24h, two targets (cells number, ROS level) test results are negative, but the rest targets (nuclear intensity, GSH reduce level, mitochondrial membrane potential) test results are positive. Beginning C polar parts at 15.6μg·ml-1, GSH reduce level and nuclear intensity exceed the toxic threshold. And the value of the MMP target belowe the toxic threshold at the concentrations between 15.6～500μg·ml-1.②D polar parts treated HepaG2 cells 24h, two targets (cells number, ROS level) test results are negative, but the rest targets (nuclear intensity, GSH reduce level, mitochondrial membrane potential) test results are positive. The value of GSH reduce level and nuclear intensity exceed the toxic threshold only at 500μg·ml-1, and the MMP target value belowe the toxic threshold at the concentrations between 31.25～500μg·ml-1. ③E polar parts treated HepaG2 cells 24h, four targets (cells number, nuclear intensity, ROS level, mitochondrial membrane potential) test results are negative, the only one targe of GSH reduce level test results is positive. and the value belowe the toxic threshold at the concentrations between 62.50～500μg·ml-1.④F polar parts treated HepaG2 cells 24h, five targets (cells number, nuclear intensity, GSH reduce level, ROS level, mitochondrial membrane potential) test results are positive.