Therapeutic Effect And Mechanism Of Ligustrazine On Mice Model Of Hepatic Veno-occlusive Disease Induced By Gynura Segetum

Part 1 Establishment and evaluation of mice model of hepatic veno-occlusive disease induced by Gynura segetumObjective:To establish and evaluate mice model of hepatic veno-occlusive disease (HVOD) induced by Gynura segetum. Phosphate buffer (PBS) was used as the control group. Mice models of HVOD 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:135 female Kunming (KM) mice were randomly classified into 5 groups, they were gavaged with 30g·kg-1·d-1 Gynura segetum(group A),150mg·kg-1·d-1 monocrotaline(group B),30mg·kg-1·d-1 cyclophosphamide(group C),100mg·kg-1·d-1 mycophenolate mofetil(group D) or 30ml·kg-1·d-1 PBS (group E). Mice were sacrificed 7 days (group B) or 30 days (group A?C?D?E) later. Blood samples were collected, liver function and blood cell counts were measured. Histopathologic changes of liver tissues were assessed by a modified Deleve scoring system. The expression of vWF was detected by immunohistochemistry while the expression of ET-1?TNF-aand IL-1?mRNA were investigated by RT-PCR.Results:1.24 mice in group A,23 in group B and 5 in group C developed HVOD. Mice in group D and E failed to show any manifestations of HVOD.2.Compared with the controls, mice with HOVD showed increased liver ratio?serum total bilirubin(TBIL)?direct bilirubin(DBIL)?transaminase and decreased albumin(ALB) (P<0.05). There were no significant differences between the models. Compared with the controls, the value of WBC increased in group A and B, while the values of RBC and PLT reduced significantly. Cytopenia was found in mice gavaged with cyclophosphamide and mycophenolate mofetil, with more significant reduction in mice gavaged with cyclophosphamide (P<0.05).3.When evaluating with modified scoring system, most of the mice in group A and C belonged to moderate or severe HVOD, and most of the mice in group B belonged to mild or moderate HVOD.4.1mmunohistochemical staining demonstrated high dense deposition of vWF in liver tissues with HVOD. Moreover, when compared with the controls, the expression of ET-1?TNF-a and IL-1?mRNA were increased significantly in mice with HVOD (P<0.05)Conclusion:Compared with cyclophosphamide, Gynura segetum and monocrotaline induced more reliable models of HVOD. The increased expression of ET-1?TNF-a and IL-1?suggested that injury to sinusoidal endothelial cells and hepatocytes may be involved in the development of HVOD associated with pyrrolizidine alkaloids. As one of the most frequently used immunosuppressive drugs in solid organ transplant, mycophenolate mofetil is considered to be a safe agent without obvious toxicity to liver and myelosuppression. Part 2 Therapeutic effect of ligustrazine on mice model of hepatic veno-occlusive disease induced by Gynura segetumObjective:To investigate the possible therapeutic effect of ligustrazine with different doses on HVOD induced by Gynura segetum.Methods:115 female KM mice were randomly divided into 4 groups, they were gavaged with 30g·kg-1·d-1 Gynura segetum (group A), 30g·kg-1·d-1 Gynura segetum+100mg·kg-1·d-1 ligustrazine (group B), 30g·kg-1·d-1 Gynura segetum+200mg·kg-1·d-1 ligustrazine (groupC) or 30ml·kg-1·d-1 PBS (groupD).30 days later, all the mice were sacrificed. Blood samples and livers were harvested. Weight ratio of liver to body and liver function were measured. Liver sections were evaluated by light microscopy and the expression of PAI-1 was determined by immunohistochemical staining.Results:1.24 mice in group A,6 in group B and 2 in group C developed HVOD. Compared with the controls, they had increased liver ratio?TBIL?DBIL?ALT?AST and decreased ALB (P<0.05). Administration of ligustrazine improved the clinical signs and biochemistry parameters in a dose-dependent manner. Compared with group A, the value of scoring in microscopy decreased in groups B and C.2. Compared with group A, the expression of PAI-1 decreased in groups B and C (P<0.05), and there were significant differences between groups B and C (P<0.05). These changes were in accordance with the histopathologic changes.Conclusion Ligustrazine with different doses has therapeutic effect on HVOD, improving clinical signs and liver function in a dose -dependent manner. The expression of PAI-1 is associated with the clinical course and severity of HVOD and response to treatment, so it can be an index for monitoring.Part 3 The possible mechanism of the effect of ligustrazine on mice models of hepatic veno-occlusive disease induced by Gynura segetumObjective:To investigate the possible mechanism of the therapeutic effect of ligustrazine on HVOD induced by Gynura segetum.Methods:115 female KM mice were randomly divided into 4 groups, they were gavaged with 30g·kg-1·d-1 Gynura segetum (group A), 30g·kg-1·d-1 Gynura segetum+100mg·kg-1·d-1 ligustrazine (group B), 30g·kg-1·d-1 Gynura segetum+200mg·kg-1·d-1 ligustrazine (groupC) or 30ml·kg-1·d-1 PBS (groupD).30 days later, all the mice were sacrificed and livers were harvested. The expression of TF?Egr-1?NF-K Bp65 were determined by RT-PCR and western blot.Results:Compared with group D, the expression of TF?Egr-1?NF-KBp65 increased significantly in group A (P<0.05).Administration of ligustrazine reduced the expression of TF?Egr-1?NF-K Bp65 in a dose-dependant manner, these findings were confirmed by both RT-PCR and western blot.Conclusion:Ligustrazine has therapeutic effect on HVOD, the possible mechanism may be that ligustrazine could reduce the expression of TF by down regulate the expression of transcription factors:Egr-1 and NF-KBp65.