| IntroductionAcetaminophen (APAP)is one of the most widely used of many antipyretic and analgesic drugs. And the poisoning by acetaminophen is the common intoxication by drugs .The clinical situation is hepatitis by drugs and fulminant hepatitis .The main organ to be involved in the acute poisoning by acetaminophen is the hepar. But it is unknown that the mechanism of hepatic injury induced by acetaminophen . There are some scholars' investigations to make clear that hepato-cyte apoptosis is the important mechanism .IL-18 is one of the Thl immunolore-gulating cytokine ,and it's action was paid close attention to hepatic injury by virus . It is scarcely reported that the role of IL-18 in acetaminophen-induced hepatic injury .Our research is to investigate the relationship between level of IL-18 and dose of acetaminophen, and to try to find out the relationship between dose of acetaminophen and number of apoptotic cells.Experiment materials and methods1.Experiment objects:32 Wistar Rats, 16 female rats and 16 male rates, weight 230~270g. 2.Experiment material and instruments:acetaminophen,TUNEL Kit,Protease K,cleantable,Light microscope, Electronic balance ,buffer,Beaker ,dropper,Straws ,10% formaldehyde solution, Dimethylbenzene, IL-18 ELISA Kit.3.Experiment procedures:(l)Randomize 32 rats into 4 groups,one is control,the others are experiment groups.(2)Inject 150, 300, 450mg/kg acetaminophen into abdominal cavities of experiment rats, inject saline into abdominal cavities of control rats.(3)After 8 hours of injection ,we anaesthesiaed all the animals , exposed liver , separated in inferior vena cava and took 3~6 ml blood .Serum was separated and stored at -20 ℃ . At once ,we separated hepatic portal vein and insert catheter and fixation , cut open inferior vena cava to put blood , then , used PBS buffer from hepatic portal vein to inferior vena cava , via Liver circulation . After liver color turn bright red into grey white , we stop filling . Stabilize liver , section 10 leaves for sample , 5 for paraffin embedding , the other for HE staining.(4)IL-18 was detected in blood of rat by ELISA kit.(5)Apoptosis was detected by terminal deoxynucleotidyl trasferase mediated dUTP nick end - labeling technique (TUNEL test) . We treat sample by 3% H2O2 and wash 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 blocking fluid and biotic anti-DIG-antibody , reach for 30 minutes, wash it by TBS. We add SABC for 30 minutes reaction ,wash it by TBS ,deal it by DAB.(6)Detect level of ALT in serum..4.Result determination standard: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 × 100% .5.Statistical analysis :Database was set up in SPSS for windows 12.0. ANOVA and Pearson correlation analysis were used to solve the data .Results1.Levels of IL-18 and ALT in serum:Level of IL-18 in serum is elevated with the increasing dosage of acetaminophen. So did the level of ALT.2.Detection of hepatocyte apoptosis:Hepatocyte of various kinds of dose of acetaminophen sample almost all show hepatocyte apoptosis. 32 samples was detected, 30 samples was found to show hepatocyte apoptosis. There is significant positive correction between dose of acetaminophen and number of apoptotic cells . the more dose of acetaminophen was, the more numer of apoptoic cells was.3.Relationship between level IL-18 and hepato cellular apoptosis index:Level of IL-18 and hepato cellular apoptosis index is evaluated with the increasing dosage of acetaminophen.The correlation between IL-18 and hepato cellular apoptosis was positive.DiscussionInterleukin (IL)-18 is a kind of cytokine which it was to be detected formerly called interferon γ (IFN-γ)-inducing factor, belong to members of the IL-1 family. IL-18 is a novel modulator of Thl Cytokine Response , augment IFN-γ production by inducing Thl cell .In addition, The efficiency of processing may produce a sufficient number of active molecules (such as TNF α, FasL, ICAM) for a biological response but at the same time also limit secretion to control excessive activity of the cytokine.It has been reported that IL-18 acts an important role in many hepatic injury . On activation with LPS, there is slight increase in mRNA levels for many proinflammatory cytokine, such as IL-18, IL-12, IFN- γ, TNF α and FasL. And the anti-IL-18 antibodies can prevent the expression of IFN- γ , TNF α, FasLmRNA. It is suggested that IL-18 is involved in this hepatic injury animal model and IL-18 maybe the upper stream cytokine of those .There is an investigation to cue that the syste of Fas /FasL is the important... |
PDF Full Text Request