Acetaminophen-induced Hepatocyte Apoptosis On The Liver Tissues In Rat

IntroductionApoptosis is a form of intiative cell death that permits the removal of damaged , senescent , or unwanted cells in multicellular organisms , without damage to the cellular microenvironment . Indeed , apopto-sis is increasingly being recognized as a key biological regulatory process. Fas is a cell surface protein that mediate apoptosis , Fas -mediated cell death may represent a major mechanism of cytotoxic T -lymphocyte ( CTL) cytotoxicity. Cell immune mechanisms provide more development of acetaminophen overdose . At present , acetaminophen - induced hepatocyte apoptosis is not reported . Our research is to investigate the relationship between expression of Fas/FasL and dose of acetaminophen , the relationship between dose of acetaminophen ; the relationship between dose of acetaminophen and number of apoptotic cells.Experiment materials and Methods1. Experiment objects: Male Wistar Rat , weight 280 - 320g.2. Expriment material and instruments:acetaminophen , Fas/FasL immunohistochemistry Kit , TUNEL Kit, Protease K , Clean table , Light microscope , Electronic balance, All kins of alcohol, TBS\PBS buffer , Beaker , dropper , Straws , 10% formaldehyde solution , Dimethylbenzene.3. Experiment procedures :(1) randomize 49 rats into 4 groups , one is control , the others are experiment groups .(2) Inject 150N30(K450mg/Kg acetaminophen into abdominal cavities of experiment rats, inject saline into abdominal cavities of control rats.(3) Put rats to death after 8 hours of injection.(4) Establish animal model : At once , we cut open thoracic cavity; abdominal cavity ;exposure heart ;shears open pericardium , left ventricle; insert catheter and fixation, at the same time , we use surgical scissors to cut open right auricle to put blood, then , use PBS buffer from left ventricle to right auricle , via systermic circulation. After liver color turn bright red into grey white , we stop fliiing . stabilize liver , section 10 leaves for sample , 5 for paraffin embedding , the othe for HE staining.(5 ) Fas/FasL were determined in liver tissue of rat by immuno-histochemistry.(6) Apoptosis was detected by terminal deoxynucleotidyl tras-ferase mediated dUTP nick end - labeling technique (TUNEL test) . We treat sample by 3% H202 and washi it by distilled water ,use protease K to digest the sample , wash it by distilled water , add tagging buffer to the sample , wash it by TBS , add blockingfluid and biotic anti - DIG - antibody , reach for 30 minutes, wash it by TBS . we add SABC for 30minutes reaction , wash it by TBS , deal it by DAB.4. Result determination standard;(1) Fas/FasL expression: Detect the Fas/FasL expression by Im-?7 ?munohistochemistry S - P method . We used a scoring system to evaluate semiquantitatively the immunoexpression , judging the cases with brown plasma staining as positive . The sum score of positive cell was classified into the following 3 grades ; 100 hepatocyte were counted in 10 field which were selected at random , ( + ) , less than 1/3 field ; ( ++ ) , less than 2/3 field ; ( +++ ) , more than 2/3 field.(2)TUNEL expression : We also used apoptosis index (AI) to evaluate semiquantitatively the immunoexpression , judging the cases with brown nuclei staining as positive . We counted 200 cell in 1 - 3 high time positive field at random, Calculation formula : AI = apoptosis cell/cell sum x 100%.5. Statistical analysis:SPSS for windows 10. 0 statistical software was used to solve the data . \ test was used to solve the enumeration data ; we use rank sum test ( Kruskal - wallis test) to analysis apoptotic cell data ; use rank correlation analysis ( spearman test) to express between expression of Fas/FasL and dose of acetaminophen . The significance level chosen was P value less than 0. 05 .Results1. Fas/FasL expression of hepatocyte:Hepatocyte of various kinds of dose, of acetaminophen sample almost all show Fas/FasL expression . 49 samples was detected , 43 samples was found to show Fas/FasL expression , po...