Evaluation On Protecting Damaged Liver Cells Induced By Acetaminophen By Using Exogenous Mitochondria

Acetaminophen(APAP)is a widely used analgesic and antipyretic drug.The curative effect of APAP with therapeutic dose is definite,but excessive APAP use can cause severe liver damage which characterized by liver cells necrosis.Mitochondria is an important target of APAP induced hepatocellular injury.Interference with the biosynthesis of mitochondria may lead to hepatocellular toxicity induced by APAP,resulting in abnormal mitochondrial structure and function and disrupting the function of liver cells.Although acetylcysteine is a standard drug in clinical treatment,the methods of administration,dosage,and time are still controversial.It is important to find new methods to reduce liver toxicity induced by APAP.We constructed liver injury model in cells and evaluated the effects of exogenous mitochondria on damaged liver cells induced by APAP.Firstly we constructed HepG2 cells which expressed cyan fluorescent protein in mitochondria.We extracted mitochondria from cells expressed cyan fluorescent protein.At the same time,we extracted mitochondria from normal HepG2 cells,used MitoTracker Red CMXRos for staining normal mitochondria and obtained mitochondria marked with red fluorescence.After Hep G2 cells incubated with fluorescent mitochondria for 24 h,the results showed that exogenous mitochondria uptake occurred within 10 min.Then pretreatment of cells with macropinocytosis inhibitor(amiloride hydrochloride)and clathrin inhibitor(hypertonic sucrose)for 30 min,we detected uptake rates of exogenous mitochondria.The results showed the uptake rates reduced significantly in amiloride hydrochloride group.In addition,we dyed the isolated mitochondria extracted from mice liver by Janus green B,identified the diameter of mitochondria and detected mitochondrial membrane permeability.The detection of mitochondrial membrane permeability results indicated that diameter of mitochondria distributes between 531.2 to 955.4 nm.The absorbance of 540 nm of mitochondrial solution is stable.Finally we determined that the appropriate concentration was 10 m M,the damaged time was 24 h and established liver injury model in cells induced by APAP.We made comments on protecting damaged cells induced by APAP by using exogenous mitochondria preliminarily.Cells were divided into three groups,normal control group,model group,exogenous mitochondrial protection group respectively.We detected apoptosis on cells,the contents of alanine aminotransferase(ALT)and aspertate aminotransferase(AST)in cell cultures,the contents of glutathione(GSH)and adenosine triphosphate(ATP)in cells,mitochondrial membrane potential changes and mitochondrial morphology changes in each group.Compared with normal control group,the apoptosis on cells increased,the contents of ALT and AST in cell cultures of model group increased significantly,the contents of ATP and GSH in cells decreased significantly,mitochondrial membrane potential also decreased and mitochondria became cavitate.Compared with model group,the apoptosis on cells reduced,the contents of ALT and AST in cell cultures in exogenous mitochondrial protection group decreased significantly,the contents of ATP and GSH in cells increased,mitochondrial membrane potential increased markedly and the degrees of mitochondrial cavitation reduced.In conclusion,exogenous mitochondria uptake occurred within 10 min in HepG2 cells.It is possiable that the mechanism of HepG2 cells uptake exogenous mitochondria was macropinocytosis.Exogenous mitochondria showed normal structure which can be used for functional detection.Exogenous mitochondria can reduce liver cells injury induced by APAP.