The Study Of The Liver Toxicity And Mitochondrial Damage Mechanism On Aqueous Extract Of Evodiae Fructus In Rats

ObjectiveEvodiae Fructus (EF) as a traditional Chinese medicine (TCM), commonly used water decoction dosage forms in clinical. Recent years, more and more reports about toxicity or adverse reactions in clinical when patients are taking EF. Some experimental researches have confirmed that the EF aqueous extraction components can lead to acute liver injury in mice and chronic liver toxicity in rats, but the study of toxicity mechanism is relatively lacking, therefore, it is necessary to research the mechanism of liver toxicity in depth in order to clarify the liver toxicity mechanism and provide the basic experimental information for clinical rational use of drugs.Methods1. The toxicity of EF aqueous extract(1) The acute toxicity evaluation of EF aqueous extract30KM mice were divided into3groups and10mice each group, sex in half. The mice were oraled EF aqueous extract at6,12,24g/kg once with20mL/kg body weight. Observe last7days.(2) Dose-toxicity relationship study on hepatotoxicity caused by EF to mice60KM mice were divided into3groups and10mice each group, sex in half. The mice were oraled EFaqueous extract at6,12,24g/kg once with20mL/kg body weight, to separate the serum60min later and determine the level of AST and ALT.(3)The chronic toxicity of EF aqueous extract40SD rats were divided into4groups and10mice each group, sex in half. The rats were oraled EF aqueous extract at6,12,24g/kg once with20mL/kg body weight after4weeks, collecting blood by abdominal aortic method and separating the serum to determine the serum biochemical indices, isolating the heart, liver, spleen, kidney, brain, testis and epididymis, uterus, ovaries and weighing to calculate the organ coefficients, then making the pathological section to observe the changes of the organs.2. The study of liver toxicity mechanism about EF aqueous extract in SD rats(1)Assessment of ATPThe content of ATP in liver was determined with an ATP commercial Assay Kit(2)Electron microscopy findings40male SD rats were divided into4groups and10mice each group, The rats were oraled EF aqueous extract at6,12,24g/kg last15days with20mL/kg body weight, to make the liver into the thin sections and observe using a transmission electron microscope.(3) Assessment of Mitochondrial Permeability Transition40male SD rats were divided into4groups and10mice each group, the rats were oraled EF aqueous extract at6,12,24g/kg last15days with20mL/kg body weight, isolated the mitochondrial from the liver. Mitochondrial swelling as the indicator of MPT was estimated from the decrease in absorbance at540nm.(4) Assessment of Mitochondrial Membrane PotentialThe electrical transmembrane potential of mitochondria was monitored with a mitochondrial membrane potential assay kit with JC-1using a Fluorescence microscope and a fluorescence spectrophotometer.(5)Assessment of CytC ReleaseThe concentratios of CytC in the endochylema were determined with a commercial ELISA Kit(6)Assessment of oxidative stress related indicatorsThe contents of GSH, Mn-SOD and MDA in mitochondria were determined with commercial Assay KitsResults1. The toxicity of EF aqueous extract(1)The result of acute toxicity evaluationThree groups of mice were all survive, also do not have any abnormal reaction after gavageing with EF aqueous extract.(2)The result of dose-toxicity relationship study The serum AST level of mice which were oraled EF aqueous extract at6,12,24,48g/kg had elevated compared with control group, the ALT level had no change.(3)The result of the chronic toxicity evaluationThe cardiac index, spleen index, liver index and the contents of triglyceride (TG), total bilirubin (TBIL), creatinine (CRE), total cholesterol (CHO) of EF groups have obvious changed comparing with control group and Liver and kidney tissues were increased cell apoptosis.2. The result of liver toxicity mechanism study(1) Effect of ATP contentThe ATP content of EF in rat liver tissue was significantly higher than the control group.(2) Electron Microscopy FindingsIn the liver cells there were lesions in the mitochondria of aqueous extract of EF treated rats compared to distilled water treated animals. The effect of aqueous extract of EF was apparent with vacuolate mitochondria.(3) Effect on Mitochondrial Permeability Transition△OD value of EF groups reduced to some extent at different times.(4) Effect on Mitochondrial Transmembrane PotentialThe fluorescence intensity of EF groups was obviously higher in a dose dependent manner than that of control group, according to the results of fluorescence microscopy, the green fluorescent area significantly increased and red fluorescence decreased in the EF groups.(5) Effect on CytC ReleaseThe concentration of CytC in the High-dose group of EF was obvious higher than other groups.(6) Effect of oxidative stressThe content of GSH was obvious lower and MDA was higher, the activity of Mn-SOD reduced in the group of EF comparing with control group.ConclusionEF aqueous extract have obvious influence on liver, long-term medication can result in rat liver tissue ATP content reduce, and cause mitochondria vacuoles, membrane potential drop, membrane permeability increased and the release of cytochrome C, and induce increase of lipid peroxidationreaction and decrease of antioxidant ability, and lead to increase liver cell apoptosis in rats...