Expressions Of Osteopontin In Schistosoma Japonicum-infected Mice Liver And Efficacy Of Its Neutralizing Antibody Against Schistosomiasis Hepatopathologic Changes

Background:The prevention and control situation of schistosomiasis japonica in China is still serious. Schistosomal liver fibrosis will continue even after effective insecticide treatment and caused some severe complications. So far there were few effective therapies to prevent or reverse schistosomal liver fibrosis due to lack of detailed and accurate research on pathological mechanisms and appropriate intervention target. Osteopontin (OPN) is a multifunctional glycophosphoprotein which play roles of chemokine, inflammatory factor and immunoregulatory factor in inflammatory liver diseases such as steatohepatitis and toxic hepatitis. In the present study, we sought to investigate the dynamic changes of expressions of OPN in Schistosoma japonica (S. japonicum)-infected mice liver and the relationship of OPN to hepatopathologic changes and various potential promoters of fibrosis progression, as well as the therapeutic effects of OPN neutralization treatment on schistosomal liver injury. We hope to obtain a further understanding of the mechanisms involved in schistosomal liver injury and provide theoretical basis for clinical prophylaxis and treatment of schistosomiasis.Chapter I Construction and evaluation of mice model of S. japonicum induced liver injuryObjective:To construct mice model of S. japonicum induced liver injury, stable and resemble to human schistosomiasis japonica, and to observe progression of hepatopathologic changes, then provide reliable foundation for further investigation on expression pattern and action mechanism of OPN as well as timing choice and efficacy evaluation of drug intervention. Methods:Mice were percutaneously infected with S. japonicum by placing a glass slide carrying15±1cercariae in non-chlorine water on its abdomen for20min. Mice stool were collected daily after infection, made into smears and observed under optical microscopy in order to identify eggs. The day on which "stool-eggs-positive" first appeared was defined as "onset day". From onset day, mice were randomly chosen to be sacrificed every2weeks till12weeks. Liver samples were extracted and histopathological changes were observed and evaluated by haematoxylin-eosin (HE) and Masson trichrome staining.Results:"Stool-eggs-positive" first appeared at42days after infection. As HE staining showed, schistosome eggs deposited and egg granulomas formed at week6, degree of actue granulomatous inflammation reached peak at week10, inflammatory cells disappeared and fibrocytes and collagen fibers formed chronic granulomas at week14, schistosome eggs degenerated and disintegrated at week18. As Masson trichrome staining showed, collagen fibers were interspersed among the periphery of the granulomas at week6, degree of collagen deposition reached peak at week10and classic pathological characteristic of liver fibrosis appeared at this moment, fibrosis was reduced gradually but more stable after week14. Success rates of S. japonicum infection and schistosomiasis liver injury model construction were all100%.Conclusion:Abdominal infection with cercaria of S. japonicum constructed a stable model which can show reliably the natural progress of human schistosomiasis japonica. Schistosomal liver injury mainly included two aspects of actue granulomatous inflammation and collagen fibers deposition. Chapter Ⅱ Associations between OPN expressions and hepatopathologic changes in S. japonicum infected miceObjective:To investigate osteopontin expressions and its association with hepatopathologic changes in BALB/C mice infected with S. japonicum.Methods:One hundred BALB/C mice were randomly divided into control group and model group (n=50each). The schistosomal hepatopathologic model was established by abdominal infection with schistosomal cercaria. Mice in control group were treated with nonchlorine water containing no cercariae. Liver samples were extracted from mice sacrificed at6,8,10,14, and18weeks after infection. Liver histopathological changes were observed with HE and Masson trichrome staining. The expressions of osteopontin were determined by immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting. The expressions of a-smooth muscle actin (a-SMA) and transforming growth factor-β1(TGF-β1) were determined by immunohistochemistry. Correlations of osteopontin expressions with other variables (a-SMA, TGF-β1, hepatopathologic features including granuloma formation and degree of collagen deposition) were analyzed.Results:The expressions of OPN, a-SMA and TGF-β1in control group had no significant changes (P>0.05) and were lower than those in model group at all different time points (P<0.01). The expressions of OPN, a-SMA and TGF-β1in model group were observed at week6. The expressions increased, peaked at week10(P<0.01), and then gradually decreased. Positive correlations between OPN expressions and granuloma formation (r=0.875, P<0.01), degree of collagen deposition (r=0.858, P<0.01), a-SMA (r=0.720, P<0.01), and TGF-β1(r=0.905, P<0.01) were also observed. Conclusion:OPN may play an important role in schistosomal hepato—pathology and may promote granulomas formation and liver fibrosis through an unexplored mechanism. Chapter Ⅲ Efficacy of combined regimen of OPN immunoneutralization and praziquantel treatment on liver injury in S. japonicum infected miceObjective:To evaluate the therapeutic effects of OPN neutralization plus praziquantel treatment on schistosome-induced liver injury in BALB/C mice.Methods:One hundred BALB/C mice were randomly divided into group A, B, C, D and group E(n=20each). Mice in all groups except group A were abdominally infected with schistosomal cercariae to induce schistosomal hepatopathological model. Mice in group C, D and group E were respectively administered with praziquantel, praziquantel plus colchicines and praziquantel plus neutralizing osteopontin antibody. Liver samples were extracted from mice sacrificed at9and15weeks after infection. Liver histopathological changes were observed with HE and Masson trichrome staining. The expressions of OPN, a-SMA and TGF-β1were determined by immunohistochemistry, RT-PCR, and Western blotting.Results:Praziquantel plus neutralizing osteopontin antibody treatment significantly decreased the granuloma dimension, the deposition of collagen fibers and the expressions of OPN, a-SMA and TGF-β1compared to praziquantel treatment and praziquantel plus colchicine treatment in both the acute and chronic stage of schistosomal liver injury (P<0.05).Conclusion:The combined regimen of OPN immunoneutralization and anti-helminthic treatment can reduce the granulomatous response and liver fibrosis during the schistosomal hepatopathologic course.