The Effect Of IL-18 And Anti-IL-18 To The Immune Hepatic Fibrosis Model Induced By Repeatedly Injection Of Concanavalin A In Mice And Its Mechanism

Objective Liver fibrosis is commonly observed in chronic liver disease. However, the immunological mechanisms underlying hepatic fibrosis due to chronic inflammation are not well defined, mainly because suitable experimental models have not been established. To explore the fibrosis mechanisms, we established immunological fibrosis model induced by repeatedly injection of concanavalin A(Con A) in kunming mice.Since it has been reported that IL-18 play critical roles in Con A-induced liver injury ,in order to reveal the role of IL-18 in hepatic fibrosis, we investigated the protective effects of passive immunization with IL-18 and anti-IL-18 Ab in this model and explored apoptosis and proliferation induced by blood serum of IL-18 or anti-IL-18 in mice hepatic stellate cells. Methods 35 Kunming mice were divided into four groups, control group, concanavalin A-(10mg/kg)- once- a -week group, twice -a —week group, 15mg/kg- once -a -week group. Twenty -four hours after concanavalin A challenged at 4, 8 and 12 weeks, 1-2 mice were killed by cervical dislocation randomly,respectively.The livers of the mice of different groups were excised and fixed in 10% formalin for HE staining and Masson staining or frozen in liquid nitrogen for immunohistochemical staining for IL-18. After establishing immunological fibrosis model induced by repeatedly injection of concanavalin A in kunming mice, we randomly selected 80 Kunming mice and divided them into five groups,control group,IgG-of-rabbit group,concanavalin A group,IL-18 group and anti-IL-18 group.The treated groups were treated with IL-18(10μg/mouse),and anti-IL-18(100μg/mouse) before 1/2 hour of treatment with concanavalin A respectively.Two hours after concanavalin A challenged at 12 weeks, 8 mice were killed by cervical dislocation, repectively.The blood serum were storaged 4℃ for checking up TNF-α by ELISA. The livers of different groups of mice were excised and fixed in 10% formalin for HE staining and Masson staining or frozen in liquid nitrogen for immunohistochemicalstaining for a-SMA. After total RNA were extracted from liver tissues, MMP-2 and TIMP-1A messenger RNA were amplified by reverse transcription poly-merase chain reaction(PCR) products and electrophoresed on agrose containing ethidium bromide and visualized under ultraviolet light. Densitometric RT-PCR date were standardized with P-actin signals. Blood serum were obtained afterl2 weeks from mice that were repeatedly injected with Normal Saline, IgG of rabbit, concanavalin A,IL-18 and anti-IL-18.Eighty mice were divided into five groups: Normal Saline group, IgG of rabbit group, concanavalin A group, IL-18 group and anti-IL-18 group. All blood serum of drug were diluted to 10% by DMEM. The proliferation effects of blood serum of Normal Saline, IgG of rabbit, concanavalin A,IL-18 and anti-IL-18 on mice hepatic stellate cells were observed by using MTT assay, cell apoptosis was analyzed by using flow cytometry, agrose gel electrophoresis of DNA and fluorescent microscopy. MMP-2mRNA and TIMP-1 mRNA were measured by quantitative reverse transcripation polymerase chain reaction (RT-PCR). Result At the beginning of pro-experiment(4th week), hepatocellular necrosis had become widespread throughout the lobule. No evidence of hepatic fibrosis was observed at this period. However, at the middle of experimemt(8th week), small fibrosis strips had been found in mice of ConA group, bridging fibrosis and large fibrosis strips in the parenchyma with hepatocellular necrosis were detectable in these mice at 12th week by HE staining and Masson staining. Immunohistochemical staining for IL-18 indicated that the semi-quantu scores in concanavalin A group were more than control group. At the formal experiment, the dead mice of ConA group, IL-18 group and anti-IL-18Ab group were3, 7 and 1 respectively. There were significant difference among each group (P<0.05) .At the fouth week of experiment in IL-18 group mice, hepatocellular necrosis had become widespread throughout the lobule. Evidence of hepatic fibrosis was observed at this period. However, at the twelfth week of the experimemt , bridging fibrosis and large fibrosis strips in the parenchyma with hepatocellular necrosis were detectable in the IL-18 treated group, but at the twentieth week, only small fibrosis strips could be found in anti-IL-18Ab treated mice by HE staining and Masson staining.The serum levels of TNF-a in IL-18 group were higher than concanavalin A treated mice and anti-IL-18 treated mice (/><0.05. Moreover, immunohistochemical staining for a-SMA indecated that the semi-quantu scores in IL-18 group were more than concanavalin A group and anti-IL-18 group(P<0.05). MMP-2-mRNA, TIMP-1- mRNA expression levels increased significantly comparing withconcanavalin A group and anti-IL-18 group (PO.05) . The proliferation of mice hepatic stellate cells in blood serum of IL-18 group were significant higher than other groups and the proliferation of mice hepatic stellate cells in blood serum of concanavalin A group were also higher than that of anti-IL-18 group(p<0.05,). Using fluorescent microscopy, the dense and compact fluorescence particles and alterated nucleus were observed in the hepatic stellate cells of the mice which were treated by Normal Saline and IgG of rabbit. And a tipical subdiploid peak before Phase GO/Gl was detected by flow cytometry.The rates of apoptosis were 8.38±1.23, 8.45±1.31, 5.29±0.87, 3.46±0.56 and 4.67±0.76 in Normal Saline group, IgG of rabbit group,concanavalin A group, IL-18 group and anti-IL-18 group. MMP-2-mRNA and TIMP-1-mRNA expression levels of IL-18 group increased signifigantly comparing with concanavalin A group and anti-IL-18 group(P<0.05). Conclusion Polyclonal antibody of IL-18 were prepared by immunizing New Zealand rabbits and purified . Immuno fibrosis model were established by injecting concanavalin A in kunming mice and the mechanism was correlated with IL-18. Moreover, the formation of liver fibrosis could be deteriorated by IL-18 and relieved by anti-IL-18.This phenomenon may be related with the proliferation or apotosis of mice hepatic stellate cells .