Therapeutic Effect And Mechanisms Of Prednisone Or Hepatic Sinusoidal Obstruction Syndrome Induced By Gynura Segetum

Part1Establishment and evaluation of mice model of hepatic sinusoidal obstruction syndrome induced by Gynura segetumObjective:To establish and evaluate mice model of hepatic sinusoidal obstruction syndrome (HSOS) induced by Gynura segetum. Phosphate buffer (PBS) was used as the control group. Mice models of HSOS induced by Gynura segetum, monocrotaline, cyclophophamide and mycophenolate mofetil were evaluated in order to choose a more favourable model for further research about the disease.Methods:160female KunMing mice were randomly divided into six groups. PBS, monocrotaline, cyclophosphamide, mycophenolate mofetil (MMF), Yunnan Sanqi, and gynura segetum was intragastric administered. The mice in cyclophosphamide group were administered every other day, meanwhile other groups were administered every day. They were sacrificed on day30,7,30,30,30,30. The weight of the liver and body were measured; blood samples were collected to determine cytometry, alanine aminotransferase (ALT), aspartate aminotransferase (AST),albumin (ALB), total bilirubin(TBIL). Liver were sectioned and stained using HE and Masson to observe the pathological changes, and the changes on light microscopy were assessed by a modified Deleve scoring system.Results:1.23mice in group monocrotaline,5in group cyclophosphamide, and24in group gynura segetum developed HSOS. Mice in group PBS, Yunnan Sanqi and mycophenolate mofetil failed to show any manifestations of HSOS.2. Compared with the controls, mice in group monocrotaline and group gynura segetum showed increased the weight of body (P<0.05) especially in group gynura segetum. Meanwhile, the body weight were significantly decreased in group cyclophosphamide (P<0.05). There were no significant differences between the MMF and Yunnan Sanqi group compared with the controls (P>0.05)3. Compared with the controls, the value of WBC increased in group monocrotaline and gynura segetum, while the values of RBC and PLT reduced significantly (P<0.05). the value of RBC slightly increased in group Yunnan Sanqi, while the values of WBC and PLT reduced moderatly(P>0.05)without any statistical significance. Hypocytosis was found in mice gavaged with cyclophosphamide and mycophenolate mofetil, with more significant reduction in mice gavaged with cyclophosphamide (P<0.05)4. Compare with PBS group, the model groups had significant increased in the level of liver index, serum ALT, AST, TBIL and decreased in the level of serum ALB (P<0.05). Although, the liver function in group MMF had injured at a certain level compare with PBS group, obviously better than in group cyclophosphamide.5. Most of gynura segetum and cyclophosphamide group were in moderate or severe HSOS, while monocrotaline group were in mild or moderate HSOS by a modified Deleve scoring system.Conclusion:1. Monocrotaline, cyclophosphamide and gynura segetum could induce the model of HSOS successfully. The model induced by gynura segetum and monocrotaline were higher than cyclophosphamide. Yunnan Sanqi and MMF failed to induce the model of HSOS.2. The model induced by gynura segetum exhibited the clinical and histogical features of human HSOS, and this model may provide a new animal model experimental approach for the further study of the human HSOS. Monocrotaline could induce the acute HSOS model, while gynura segetum could induce the chronic ones.3. Compared with monocrotaline, gynura segetum induced more reliable animal models of HSOS in research of mechanism and effects of medicine.4. Compared with gynura segetum, Yunnan Sanqi had no hepatotoxicity, and more safe in clinical use.5. Compared with cyclophosphamide, MMF is considered to be a immunosuppressive safe drugs without obvious hepatotoxicity and bone marrow suppression.Part2Therapeutic effect of prednisone on mice model of hepatic sinusoidal obstruction syndrome induced by Gynura segetumObjective:To investigate the possible therapeutic effect of prednisone with different doses on HSOS induced by Gynura segetum.Methods:115female KM mice were randomly divided into4groups, they were gavaged with30ml·kg-1·d-1PBS (group A),30g·kg-1·d-1Gynura segetum (group B),30g·kg-1·d-1Gynura segetum+5mg·kg-1·d-1prednisone (groupC), or30g·kg-1·d-1Gynura segetum+10mg·kg-1·d-1prednisone (groupD). All the mice were sacrificed on the30day. Blood samples were collected to determine cytometry, alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), total bilirubin(TBIL). Liver were sectioned and stained using HE and Masson to observe the pathological changes, and the changes on light microscopy were assessed by a modified Deleve scoring system. The expression of TGF-β1and CTGF was determined by immunohistochemical staining and RT-PCR.Results:1.24mice in group B,5in group C and7in group D developed HSOS. Compared with the controls, they had increased WBC,liver ratio, TBIL,DBIL,ALT,AST and decreased RBC,PLT,ALB (P<0.05). Administration of prednisone improved the clinical signs and biochemistry parameters without dose-dependent manner.2. Most mice of group B developed moderate or severe HSOS, with those in group C and D were in mild or moderate HSOS by a modified Deleve scoring system. The value of group C was lower (P<0.05).3. Compared with group B, the expression of TGF-β1and CTGF decreased in groups C and D (P<0.05), and there was no significant difference between groups C and D (P>0.05). These changes were in accordance with the histopathologic changes.ConclusionPrednisone with different doses has therapeutic effect on HSOS, improving clinical signs and liver function and independent of dose. The expression of TGF-β1and CTGF is associated with the clinical course and severity of HSOS and response to treatment, so it can be an index for monitoring.Part3The possible mechanism of the effect of prednisone on mice models of HSOS induced by Gynura segetumObjective:To investigate the possible mechanism of the therapeutic effect of prednisone on HSOS induced by Gynura segetum.Methods:115female KM mice were randomly divided into4groups, they were gavaged with30ml·kg-1·d-1PBS (group A),30g·kg-1·d-1Gynura segetum (group B),30g·kg-1·d-1Gynura segetum+5mg·kg-1·d-1prednisone (groupC), or30g·kg-1·d-1Gynura segetum+10mg·kg-1·d-1prednisone (groupD). All the mice were sacrificed on the30day. The livers were harvested. The expression of MMP-1,TIMP-1,MCP-1, TNF-α,IL-1β and NF-κBp65mRNA were determined by RT-PCR and the expression of TNF-α,TGF-β1and CTGF were determined by western blot.Results:RT-PCR:Compared with the controls, the expression of MMP-1, TIMP-1, MCP-1, TNF-a, IL-1β and NF-KBp65mRNA increased significantly in group B (P<0.01). Prednisone reduced the mRNA expression of MMP-1,TIMP-1,MCP-1,TNF-α,IL-1β and NF-κBP65in group C and D (P<0.05). Compared with the controls, the expression of MMP-1/TIMP-1decreased significantly in group B (P<0.01) while MMP-1/TIMP-1significantly increased (P<0.05). No significant difference between group C and D (P>0.05)Western blot:Compared with the controls, the expression of TNF-α, TGF-β1and CTGF protein increased significantly in group B (P<0.01) Prednisone reduced the protein expression of TNF-α,TGF-β1and CTGF in group C and D (P<0.05). No significant difference between group C and D (P>0.05)Conclusion:Prednisone has therapeutic effect on HSOS, the possible mechanism may be that prednisone could reduce inflammation and fobrosis by down regulate the expression of NF-κB, reduce MCP-1, TNF-a and IL-1(3, thereby reducing the activation of HSC, inhibit synthesis of TGF-β, CTGF, TIMP-1, promote MMP-1degradating ECM, and reducing SEC damage and ECM deposition.