Gynura Segetum Induced The Mouse Model Of HVOD And The Experimental Study On The Preventative Treatment Effect Of Salvia Miltiorrhiza On HVOD

Backgroud:Hepatic veno-occlusive(HVOD) another name is hepatic sinusoidal obstruction sydrome(HSOS).HVOD is characterized by tender hepatomegaly,fluid retention,weight gain,and jaundice.This condition is seen after hematopoietic stem cell transplantation(HSCT),high dose abdominal radiation therapy, use of certain chemotherapeutic agents,ingestion of pyrrolizidine alkaloids.Liver transplantation and lung transplantation can also lead to HVOD. HVOD is one of the three complications after HSCT.The incidence of HVOD varies from 10% to 50%, and the motality more than 90%.Preventing and treating HVOD plantation patients ealier are the key points for eusemia. Gynura segetum is one kind of Chinese materia medica which extensively planted. The reports about HVOD induced by Gynura segetum is increasing rencently years.Mechanisms for HVOD have not been identified, so the model for HVOD is the key target for research and internal and abroad have thrown much to it. Varies models have establised with advantages and disadvantages. Borowska et al induced HVOD model by nitrous acid amine which is not ideal because of the low fraction of animals with typical lesions,long period and high variability.Monocrotalinethe model establised by Deleve et al and Chen MY have made great progress in model cycle and achievement ratio,but they were limited to observe prostecdtive effiacy. Chinese researches have established mouse HVOD model by alkaloids of Sedum aizoon, but without evaluation. The treatments for HVOD are limited, and the drugs for HVOD are disputing. Some drugs effective for HVOD have been found to some extent (such as tPA, Low Molecular Heparin, prostaglandin E1, prednisone,N-acetyl cysteine,defibrotide et al). Nowadays,the reserarch about HVOD is popular with the prophylactic agent in international.For example,Ursode-oxycholic acid is good for early HVOD,but useless for late HVOD,and the administration route by oral is always limited for late HVOD patients with nausea,vomito or oral disease. Prednisone, low molecular heparin have some effect to early HVOD,but the side effect of hemorrhea is more graveness. Defibrotide is one kinds of effective drugs,but haven't taken to clinic extensivly in China. Salvia miltiorrhiza is one kind of drug good for circulation,especially for injury cardia,liver, lung and brain ischemia-reperfusion injury. It also found that Salvia miltiorrhiza is used for hepatocellular injury,hepatic fibrosis,hepatic cirrhosis, liver cancer, immune response of rugulation, anti-infection and anti-tumor. Tanshinol, the ingredient of salvia miltiorrhiza, can inhibit the cellular adhesion molecule express by platelet, leucocyte and endotheliocyte and the thromb. Clinic researches have found that Salvia miltiorrhiza have effect on preventing HVOD after transplantation.However, its preventive and therapeutic effects on HVOD induced by gynura segetum remain unclear. Constructing a mice model of HVOD induced by gynura segetum and establishing the control models by cyclophosphamide,Yunnan sanqi,mon-ocrotaline.The present study is to investigate the anti-HVOD effects of Salvia miltiorrhiza, the changes of adhesion molecule and the related molecular mechanisms. Objective:To establish the hepatic veno-occlusive disease reduced by gynura segetum. Phosphate buffer and Yunnan Sanqi as the nomal control groups. Comparing the mice models of Hepatic Veno-occlusive disease induced by cyclophosphamide, monocrotaline and gynura segetum to help advance research into this disease.Methods:110 female KunMing mice were randomly classified into five groups. PBS, Yunnan Sanqi, cyclophosphamide, monocrotaline or gynura segetum was administered. They were sacrificed on day 30,30,30,7,30. The weight of the liver and body were measured; blood samples were collected to determine 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.Result:The group of PBS and Yunnan Sanqi have no mouse with HVOD.5 of cyclophosphamide group,18 of monocrotaline group and 24 mice of gynura segetum group presented with clinical symptoms and the histopathological picture of HVOD. To compare with PBS group, the model groups had significant changes in the level of serum ALT, AST, ALB, TBIL and the weight of liver and body(P<0.05), and there were no obvious changes between the model groups. The model groups of liver had obvious pathological changes of HVOD. Most of gynura segetum and cyclophosphamide group with HVOD were in middle or late phases, while monocrotaline group were in early or middle phases.Conclusion:gynura segetum, monocrotaline and cyclophosphamide could induce the model of HVOD successfully. The model induced by gynura segetum and monocrotaline were higher than cyclophosphamide. The model induced by gynura segetum exhibited the clinical and histogical features of human HVOD, and this model may provide a new model experimental approach for the further study of the human HVOD. Objective:To investigate the preventive treatment effects of Salvia miltiorrhiza on mice hepatic veno-occlusive induced by gynura segetum.Methods:100 female KunMing mice were randomly classified into five groups. The control group were treated with PBS, model group induced by gynura segetum, gynura segetum groups preventively treated with Salvia miltiorrhiza at two dosages(gynura segetum+Salvia miltiorrhiza,100mg/kg,200mg/kg).The mice were sacrificed on day 30. The weight of the liver and body were measured; blood samples were collected to determine 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 protein expression of PAI-1 and VEGF in mice liver were detected by immunohistochemical staining and the mRNA expression by RT-PCR.Result:24 mice of gynura segetum group,8 of 100mg/kg Salvia miltiorrhiza group and 3 of 200mg/kg Salvia miltiorrhiza group presented with clinical symptoms and the histopathological picture of HVOD. To compare with gynura segetum group, the Salvia miltiorrhiza groups had significant decreased in the level of serum ALT, AST, ALB, TBIL and the weight of liver and body(P<0.05), and there were also significant changes between the Salvia miltiorrhiza groups(P<0.05). most of gynura segetum and cyclophosphamide group with HVOD were in middle or late phases, while 100mg/kg Salvia miltiorrhiza group were in early or middle phases and only one mouse presented with late HVOD. Most of 200mg/kg Salvia miltiorrhiza group mice with HVOD were presented early and no one was severe. The immunohistochemical staining demonsrated that the protein expression levels of PAI-1 and VEGF were singnificantly lower in 100mg/kg Salvia miltiorrhiza group and 200mg/kg Salvia miltiorrhiza group than that in gynura segetum group(P<0.05,respectively).The mRNA expression detection results by RT-PCR were consistent with that of protein expression levels measured by immunohistochemical staining(P<0.05,respectively). Compared with 100mg/kg Salvia miltiorr-hiza group, PAI-1 and VEGF of protein and mRNA expression in 200mg/kgSalvia miltiorrhiza group had decreased significantly(P<0.05, respectively).Conclusion:Salvia miltiorrhiza had significant biological function of sinusoidals and hepatocyte protection indicated by decreasing serum ALT,AST and TBIL levels,increasing serum ALB level, attenuating the modified Deleve scores of mice liver tissue induced by gynura segetum. Salvia miltiorrhiza with different dosages had therapeutic effect on HVOD induced by gynura segetum, and the Salvia miltiorrhiza 200mg/kg group did better than Salvia miltiorrhiza 100mg/kg group. The decrease of PAI-1 and VEGF protein and mRNA expression indicated better prognosis of HVOD, and detecting the expression of PAI-1 and VEGF may be used as monitors of the prognosis of HVOD. Objective:To explore the molecular mechanisms of the preventive treatment of salvia miltiorrhiza on mice HVOD induced by gynura segetum through the investigation of mRNA and protein expression of tumor necrosis factor-α(TNF-α), vascular endothelial growth factor(VEGF), vascular cellular adhesion molecule-1(VC AM-1), intercellular adhesion molecule-1(ICAM-1),nuclear trascription factor P65(NF-κBp65) and the mRNA expression of vascular endothelial growth factor recptor-1(flt-1), vascular endothelial growth factor recptor-2(KDR) in mice liver tissue.Methods:The liver tissue specimens were randomly collected from four mice in each group for RT-PCR and five mice in each group for western bloting.RT-PCR was applied to detect the mRNA expression of TNF-α, VCAM-1, ICAM-1, VEGF, flt-1, KDR,NF-KBp65.Western blot was employed to measure the protein expression of TNF-α, VCAM-1, ICAM-1, VEGF, NF-KBp65 in liver tissue.Result.The RT-PCR demonstrated that the mRNA expression levels of TNF-α, VCAM-1, ICAM-1, VEGF, flt,KDR,and NF-κBp65 were singnificantly lower in salvia miltiorrhiza groups than that in gynura segetum group (P<0.05,respectively).The protein expression detection results by western bloting were consist with that of mRNA expression levels by RT-PCR(P<0.05,respectively).Conclusion:Salvia miltiorrhiza may attenuate mice HVOD via down-regulating mRNA and protein expression of TNF-α,VCAM-1, ICAM-1 and NF-κBp65 and inhibiting the expression of inflammatory factor and thrombogenesis.Meanwhile, salvia miltiorrhiza may prevent the HOVD by inhibiting the migration and necrosis of sinusoidal endothielial cell through down-regulating the mRNA and protein expression VEGF and the mRNA expression of flt,KDR in liver tissue. Salvia miltiorrhiza is one kind of chinese patent medicine.they have effect on promoting blood flow and preventing the cardial and cerebral vascular thrombosis.Clinic researches demonstrated that salvia miltiorrhiza had significant effect on preventing HVOD after transplantation. However, wether it has preventive and therapeutic effects on HVOD induced by gynura segetum and by what way remain unclearGynura segetum juice was applied for the first time to Construct a mice model of HVOD in the present study and to assess the model by pathological assay. Salvia miltiorrhiza was first applied for preventively treat HVOD mice model and the mechanism was analyzed. The preventive treatment effects of salvia miltiorrhiza were assessed by both pathological and serological assays,such as HE tissue staining, Masson collagen staining.The mechanisms were explored respectively at histology, mRNA, RT-PCR and Western bloting.The results demonstrated that mice HVOD can be induced by gynura segetum and the pathological changes was similar to human HVOD. Salvia miltiorrhiza significantly decreased mice serum ALT,AST and TBIL,elevated serum ALB level, decreased sinusoidal endothielial cell necrosis and sinusoidal congestion, and inhibited the degeneration and necrosis of hepatic cell. Salvia miltiorrhiza can down-regulated the mRNA and protein expression of PAI-1 and VEGF,which had significant point to assess the prognosis of HVOD. Meanwhile, salvia miltiorrhiza attenuated mice HVOD via down-regulating mRNA and protein expression of TNF-a, VCAM-1, ICAM-1 and NF-κBp65 and inhibiting the expression of inflammatory factor and thrombogenesis. What's more, salvia miltiorrhiza also down-regulated the protein expression of VEGF, and the mRNA expression of VEGF, flt and KDR.The present study demonstrates the gynura segetum can induced mice HVOD and the preventative treatment effects of salvia miltiorrhiza on mice HVOD induced by gynura segetum. The molecular mechanisms were supposed to be associated with the attenuation of liver inflammatory factor,the inhibition on the expression of VCAM-1, ICAM-1, NF-KBp65, VEGF, VEGF-R1 and VEGF-R2,as well as prevention of thrombogenesis and migration and necrosis of sinusoidal endothielial cell.