Therapeutic Effect And Mechanisms Of G-CSF On Hepatic Veno-occlusive Disease Induced By Monocrotaline

Hepatic veno-occlusive disease(HVOD)belongs to uncommon and severe liver damage induced by drugs.There are still few effective therapies nowadays because of obscure pathogenesis.In this study,we investigated the possible therapeutic effect of granulocyte colony stimulating factor(G-CSF)on HVOD after establishing and evaluating HVOD model induced by monocrotaline(MCT).Part oneExperimental model of HVOD induced by MCTObjective:To evaluate an experimental model of hepatic veno-occlusive disease(HVOD)raised by Deleve and make some minor changes if necessary in order to do further research about this disease.Methods:Forty-two male Sprague-Dawley(SD)rats were randomly classified into 5 groups. They were gavaged with MCT,100mg/kg(group D and E)or 160mg/kg(group B and C),except group A on day 0 and treated on day 7(group A,B and D)or day 10 (group C and E).Blood samples and liver were harvested.Weight ratio of liver to body,ascites amount,serum biochemistry parameters(including TBil,ALT and AST), and blood cell count were calculated or measured,and the percentage of proliferating cell nuclear antigen(PCNA)positive cells were calculated by immunohistochemistry. Sections were evaluated by light microscopy with Deleve's scoring system or the modified one.Results:1.Rats in group B,C and D manifested ascites,increased weight ratio and TBil.One of rats in group B and D died of heavy HVOD.The values of WBC,Neu%,ALT and AST were elevated,but the platelet count reduced in group B(P＜0.05).Compared with those in group B,the values of WBC,Neu%,ALT and AST reduced in group C (P＜0.05),the same as group E compared with those in group D.When evaluated with modified scoring system,all rats in group B belonged to heavy HVOD,8/9 in group C was heavy,5/8 in group D was modest and 6/8 in group E were mild.2.The percentage of PCNA positive hepatocytes in group A was 18.9±6.1%,which was enhanced after the liver damage induced by MCT(P＜0.05).on the other hand, the percentage of sinusoidal endothelial cells(SEC)with positive PCNA was 2.0±0.8%in group A,which had no significant enhancement after damage(P＞0.05).Conclusion:1.MCT can induce rats to suffer HVOD with dose dependence which has the reversal tendency especially at low doses,but MCT of 160mg/kg can make a relative stable severe HVOD.2.The modified scoring system seems to more accurate in reflecting the pathology because the sinusoidal fibrosis and coagulative necrosis exists in the liver of HVOD.3.Enhancement in the percentage of positive PCNA cells may be related to the reversal tendency.Part twoTherapeutic effect and mechanisms of G-CSF on HVOD Chapter 1 Therapeutic effect of G-CSF on HVODObjective:To investigate the possible therapeutic effect of G-CSF with different doses to HVOD rats.Methods:Thirty-six male SD rats were randomly classified into 4 groups.Group A was control, rats in group B,C and D were gavaged with MCT 160mg/kg,injected subcutaneously G-CSF 10μg/kg(group C),100μg/kg(group D)or normal sodium(group A)every day till day 9,and treated on day 10.Blood samples and liver were harvested.Weight ratio of liver to body,ascites amount,serum biochemistry parameters(including TBil, ALT and AST),and blood cell count were calculated or measured.Sections were evaluated by light microscopy with modified scoring system,and the percentage of PCNA positive cells were calculated by immunohistochemistry.The level of TNF-αand PAI-1 was determined by ELISA.Results:1.Compared with group A,rats in group C or D still had ascites,increased weight ratio and TBil,but they improved in contrast to group B(P＜0.05)and transaminases almost restored.WBC was higher in group D compared with C.The value of TNF-αand PAI-1 was low in group A,which increased in group B and reduced in group C and D(P＜0.05).2.Compared with group B,the value of scoring in microscopy decreased in group C and D(P＜0.05),but there were no significant difference between them(P＞0.05).3.Hepatocytes and SEC reduced in group B,C and D,but the percentage of PCNA positive cells increased,especially in group C or D(P＜0.05).Conclusion1.G-CSF with different doses has therapeutic effect on HVOD,improving clinical manifestations and liver function.But proper attention to WBC is essential when G-CSF with higher dose is applied. 2.The value of PAI-1 changes with pathogenetic condition of HVOD,so it can be used as an index for monitoring.3.The percentage of PCNA positive cells' enhancement may associate with the role of G-CSF to contribute regeneration.The decrease of TNF-αmay be connected with the function of G-CSF to inhibit the secretion of TNF-α.Chapter 2 Expression of G-CSFR and STAT3 on HVOD rats after G-CSF treatmentObjectiveTo investigate possible molecular mechanism of G-CSF's therapeutic effect on HVOD by determining the expression of G-CSFR and STAT3 in rats' livers.MethodsDetermining the expression of G-CSFR,STAT3 and p-STAT3 in rats' livers with the methods of RT-PCR,Western blot and immunohistochemistry analysis.ResultsThe expression of G-CSFR,STAT3 and p-STAT3 existed in all rats.The relative expression was slightly increased in group B(P＞0.05)compared with that in group A, and significantly elevated in group C and D(P＜0.05).ConclusionThe existence and increased expression of G-CSFR,STAT3 and p-STAT3 in the livers of G-CSF treated rats suggests the molecular mechanism of G-CSF on HVOD probably is JAK-STAT3 signaling pathway mediated by G-CSFR.