The Therapeutic Effect And Mechanism Of Ursodeoxycholic Acid On Mice Model Of Hepatic Sinusoidal Obstruction Syndrome Induced By Gynura Segetum

Part1Establishment and evaluation of mice model of Hepatic sinusoidal obstruction syndrome induced by Gynura segetumObjective:Mice models of Hepatic sinusoidal obstruction syndrome (HSOS) induced by Gynura segetum, Yunan Sanqi, monocrotaline (MCT) and cyclophosphamide (CTX). Phosphate buffer (PBS) was used as the control group. To evaluate and choose a more favourable mice model of HSOS for further research.Methods:One hundred and thirty-five mice were randomly divided into5groups:Gynura segetum group, Yunnan Sanqi group, Monocrotaline group, cyclophosphamide group and PBS control group. Monocrotaline group were sacrificed at seventh day and other4groups were sacrificed at thirtieth day. Blood samples were collected to test blood routine and liver function.Liver tissue specimens stained with HE and Masson staining and scored by the modified Deleve scoring criteria. Results:1. Twenty-four mice in Gynura segetum group,23in MCT group and5in CTX group developed HSOS. Mice in Yunan Sanqi group and PBS control group failed to show any manifestations of HSOS.2. Compared with the PBS control group, the Gynura segetum group and the MCT group had a higher WBC and lower RBC, PLT (P<0.05) CTX group had a lower WBC, RBC and PLT compared with the PBS control group (P<0.05). Mice developed HSOS showed higher liver ratio, ALT, AST, TBIL and DBIL but lower ALB (P<0.05) than the normal mice.3. According to the modified Deleve score scoring system, most of the molded mice in Gynura segetum group and CTX group developed more severe HSOS, while molded mice in MCT group developed more mild to moderate HSOS.Conclusion:Gynura segetum, MCT and CTX all can induce HSOS mice model. Yunnan Sanqi failed to induce HSOS mice model. Gynura segetum can induce HSOS model in80%mice, and include more chronic HSOS model. The HSOS mice model induced by Gynura segetum were fit for further study the pathogenesis and prevention measures for HSOS. Part2Therapeutic effect of ursodeoxycholic acid on mice model of Hepatic sinusoidal obstruction syndrome induced by Gynura segetumObjective:To explore the possible therapeutic effect of ursodeoxycholic acid (UDCA) with different doses on HSOS mice model induced by Gynura segetum.Methods:One hundred and fifteen mice were randomly divided into PBS control group, Gynura segetum group, low-dose UDCA group (15mg/kg/d) and high-dose UDCA group (30mg/kg/d). All mice were sacrificed after administered30days. Blood samples and livers were harvested. Specimens of serum and liver tissue. Ratio of liver and liver function were measured. Liver tissue specimens stained with HE and Masson staining and scored by the modified Deleve scoring criteria. Liver tissue fibers plasminogen activator inhibitor-1(PAI-1) expression levels was determined by immunohistochemical staining.Results:1. Gynura segetum group, low-dose UDCA group and high-dose UDCA group have24,7,4mice developed HSOS respectively. Compared with Gynura segetum group, two intervention groups had increased liver ratio, ALT, AST, TBIL, DBIL decline and decreased ALB level. The difference was statistically significant (P<0.05). with high-dose intervention group improved more significantly. Administration of UDCA improved the clinical signs and biochemistry parameters in a dose-dependent manner.2. According to the revised Deleve liver pathology score, Gynura segetum group showed more severe HSOS, low-dose UDCA group showed more mild to moderate HSOS and only one mice was severe HSOS, high dose UDCA group4molded mice showed mild to moderate HSOS.3. Gynura segetum group had a significantly higher liver tissue levels of PAI-1than PBS control group. Two UDCA groups had significantly lower PAI-1expression levels than Gynura segetum group (P<0.05). High-dose UDCA group had a lower PAI-1expression levels than low-dose UDCA group (P<0.05)Conclusion:1. UDCA has a preventive effect on HSOS mice model induced by Gynura segetum. UDCA can reduce symptoms, improve liver function, reduce pathological damage in HSOS mice model. High-dose UDCA group had better preventive effect than low-dose group.2. The expression of PAI-1is associated with the severity of HSOS. PAI-1can be used as a indicator for HSOS severity and efficacy. Part3The possible mechanism of the effect of UDCA on mice models of HSOS induced by Gynura segetumObjective:To investigate the possible mechanism of the protective effect of UDCA on HSOS mice by detecting and comparing liver tissue Nrf2/ARE path express.Methods:One hundred and fifteen mice were randomly divided into PBS control group, Gynura segetum group, low-dose UDCA group (15mg/kg/d) and high-dose UDCA group (30mg/kg/d). All mice were sacrificed after administered30days. We detected of mice liver tissue8-OHGD, MDA, GSH, SOD levels and detected the Nrf2, SOD, y-GCS mRNA levels of the liver tissue by RT-PCR method. Then liver tissue y-GCS and the cytoplasm/nucleus Nrf2protein levels were detected by Western blot and compared each group of Nrf2DNA binding activity.Results:1. Compared with the PBS control group, three drug intervention group had significantly higher8-OHGD, MDA levels (P<0.01) and significantly lower GSH, SOD levels (P<0.01or P<0.05). Two UDCA groups had significantly lower8-OHGD, MDA levels and significantly higher GSH, SOD levels than the Gynura segetum group (P<0.01or P＜0.05).2. Compared with the PBS control group, three drug intervention group SOD, y-GCS mRNA levels were significantly increased (P<0.01). Nrf2mRNA levels was no significant difference among each group. Two UDCA groups had higher SOD, y-GCS mRNA levels than the Gynura segetum group (P<0.01or P<0.05). There was no significant difference between the two UDCA intervention groups.3. Compared with the PBS control group, three drug intervention group cytoplasmic y-GCS, Nrf2protein levels were significantly increased (P<0.01), two UDCA intervention group y-GCS protein levels higher than the Gynura segetum group (P<0.01or P<0.05), while Nrf2protein level was lower than the Gynura segetum group. Three drug intervention group nucleus Nrf2protein level were higher than the PBS control group and the two UDCA intervention groups nucleus Nrf2protein level were higher than the Gynura segetum group.4. Compared with the PBS control group, three drug intervention groupS Nrf2DNA binding activity were significantly increased (P <0.01). Two UDCA group had higher Nrf2DNA binding activity than the Gynura segetum group (P<0.01or P<0.05). High-dose UDCA group had higher Nrf2DNA binding activity than the low dose group.Conclusion:1. UDCA intervention can reduce liver oxidative stress injury, increased liver endogenous antioxidant GSH and SOD levels in HSOS mice induced by Gynura segetum. 2. UDCA may stimulate the generation and nuclear translocation of Nrf2then activate Nrf2/ARE pathway increase the endogenous antioxidant enzymes SOD and y-GCS level to reduce oxidative stress damage of HSOS. The effect of high-dose UDCA group was better than the low dose group.