The Role Of Inflammation In Acetaminophen-induced Hepatotoxicity And The Effect Of Recombinant Human Interleukin-11 In Acetaminophen-induced Hepatotoxicity And Its Mechanism

ObjectiveAcetaminophen (AAP) is widely used as an analgesic and antipyretic agent in the world, but overdosage can induce severe hepatotoxicity. In therapeutic doses,more than 90% of AAP undergoen glucuronidation and sulfation are excreted in the urine, while only less than 5% of that are transformed into electrophilic toxic intermediate metabolite N—acetyl —P — benzoquinone imine(NAPQI) by cytochrome P450 system,then NAPQI combined with glutathione are eliminated rapidly. However, overdosage of AAP induces saturation of the pathways of glucuronidation and sulfation. Then a large amount of AAP are changed into oxidation by cytochrome P450 system,which leads to the depletion of glutathione and excess NAPQI without combination with glutathione,NAPQI binding cova-lently to hepatocyte protein induces hepatotoxicity. Recently, the role of inflammation in AAP — induced hepatotoxicity has attracted our attention. AAP—induced hepatotoxicity can activate hepatic endothelial cells and Kupffer cells to produce various proinflammation factors. Those factors induce the infiltration and activation of polymorphonuclear leukocyte, macrophage and monocyte in liver which keep inflammatory reaction continuing and magnifying. Finally oxidation stress and formation of per-oxynitrite induced by the inflammatory reaction play an important magnifying and promoting role on hepatotoxicity. Trepicchio WL reports thatpretreating mice with recombinant human interleukin—lKrhIL—11) may alleviate AAP—induced inflammatory reaction and reduce hepatotoxicity. Those indicate that rhIL—11 maybe play a role on alleviating AAP —induced hepatotoxicity. Our trial's object: To establish a rat model of AAP — induced hepatotoxicity. To discuss the role of inflammation in rat model of AAP —induced hepatotoxicity. To discuss the effect of rhIL—11 in rat model of AAP—induced hepatotoxicity and its mechanism.METHODS1 Experiment designEighty female SD rats are divided into four large groups and sixteen small groups in random. The four large groups are control group, AAP group,rhIL—11 group, AAP+rhIL—11 group. Each large group contains four small groups,and there are five rats in every small group. The treatment of each group is-.Control group:sterile water for injection is administered by subcutaneous injection firstly,then PBS is administered by intraperitoneal injection two hours later.AAP group .-sterile water for injection is administered by subcutaneous injection firstly,then AAP(lg/ kg)is administered by intraperitoneal injection two hours later.RhIL—11 group:rhIL —11 (lmg/ kg) is administered by subcutaneous injection firstly,then PBS is administered by intraperitoneal injection two hours later.AAP+rhIL—11 group:rhIL—11 (lmg/kg)is administered by subcutaneous injection firstly,then AAP(lg/kg)is administered by intraperitoneal injection two hours later.On the time of 3h,6h,12h,24h after intraperitoneal injection,a small group rats from each lagrge group are anesthetized with 10 % chloral hydrate by intraperitoneal injection. The blood is obtained by cardiac puncture, then the serum is suspended to be conserved at — 20°C. Two piecesof liver tissues from liver middle lobe are separately fixed in 10% formaldehyde and 2.5% glutaral,then the liver remainder are conserved in liquid nitrogen at — 80"Cat once.2 Detection methodDetermination of serum ALT level, observation of liver pathological change by HE stain, determination of liver cell apoptosis by TdT—mediated dUTP—biotin nick end labeling (TUNED and transmission electron microscope,determination of serum tumour necrosis factor—a(TNF — a) level by radioimmunoassay, determination of expression of TNF — a and Inducible nitric oxide synthase(iNOS) in the liver tissue by immunohisto-chemical method and Western—blot method,determination of mRNA expression of intercellular adhesion molecule—1 (ICAM— 1) , cytokine—induced neutrophil chemoattractant —1 (CINC—1) and interleukin—10 (IL — 10) by reverse transcription—polymerase chain reaction (RT —PCR) technique.Results1. The change of serum ALT.- Serum ALT of AAP group is apparently higher than that of control group (p<0. 01), its peak is at 24h. Serum ALT of AAP+rhIL —11 group is apparently lower than that of AAP group(3h,6h,12h P<0. 05,24h P<0. 01),but is still apparently higher than that of control group(p<0. 01). There is no apparent differentiation between control group and rhIL—llgroup(P>0. 05).2. Pathological changes of liver tissue .-The liver of AAP group is obviously intumescent and congestive. The lighter is spotty necrosis of hepa-tocytes,the severer is extensive band necrosis of hepatocytes in the centri-lobular region which is progressively aggravating and peaking at 24 hours. The pathological lesion of AAP+rhIL—11 group is apparently reduced than AAP group (Comparison of the necrotic areas between the two groups, 12h P0. 05). There are no abnor-malities in control group and rhIL—11 group.3. Apoptosis index (AI): There are scarce apoptotic hepatocytes in control group and rhIL—11 group. There are a large amount of apoptotic hepatocytes near the central vein in AAP group, which peaks at 12h. AI of AAP group is apparently higher than that of control group(P0. 05),and is still apparently higher than that of control group(P<0. 01).4. Observation of liver tissue ultrastructures: There is almostly normal in control group and rhIL—11 group. There is obvious phenomenon of hepatocyte apoptosis in AAP group, which is most easy to find at 12h. There is scarce phenomenon of hepatocyte apoptosis in AAP + rhIL — llgroup.5. The change of serum TNF—a level: At 24h,Serum TNF — a level of AAP group is apparently higher than that of control group(P 0. 05). There are no apparent differentiations in each groups at other times(P!>0. 05).6. Determination of expression of TNF—a and iNOS in the liver tissue by immunohistochemical method:There are small amounts expression of TNF — a and iNOS in hepatocytes near the central veins in control group and rhIL—11 group,pale—stained,small area. There are expression of TNF —a and iNOS in many hepatocytes and a few endotheliocytes, Kupffefs cells near the central vein in AAP group,deep —stained,large area. The peaks appear at 6 hour and 12 hour respectively . The expressive site of AAP+rhIL—11 group is amolstly the same as that of AAP group, but the expressive intension of AAP+rhIL—11 group is apparently lower than that of AAP group and is still higher than that of control group(P 0. 05).8. Expressions of ICAM— 1,CINC— 1 and IL —lOmRNA in liver tissue: Expression of ICAM—1 ,CINC— lmRNA of AAP group is obviously higher than that of control group(P0. 05). Expression of IL—lOmRNA of AAP group is obviously higher than control group(P0. 05). There are no apparent differentiations between control group and rhIL—11 group(P>0. 05).Conclusions1. SD rat model of AAP —induced hepatotoxicity is successfully established by intraperitoneal injection of AAPdg/ kg).2. There exist necrosis and apoptosis of hepatocytes at the same time in the course of AAP — induced hepatotoxicity. Apoptosis is the major change in early stage and necrosis is major in late stage.3. The proinflamination factors such as TNF —a, iNOS, CINC —1, ICAM—1 play a key role in occurrence and development of AAP —in-duced hepatotoxicity, which indicates that inflammation is important in AAP—induced hepatotoxicity.4. IL—10 suppresses the expression of above proinflammation factors to protect the liver.5. RhIL—11 can effectively reduce AAP — induced hepatotoxicity by suppressing the expression of above proinflammation factors in the liver .6. RhIL—11 and endogenous IL —10 have an effect of inflammation suppression independently, there are no synergism between them.7. RhIL—11 itself has no harm to the liver.