Salvianolic Acid B Exerts Anti-hepatic Fibrosis-carcinoma Effect Via The Mediation Of PSmad3C/pSmad3L

Aims1.To simulate the development of liver fibrosis and hepatocellular carcinoma by building diethylnitrosamine（DEN）induced hepatic fibrosis-carcinoma dynamic model.2.To observe the effect of Salvianolic-aid B（Sal B）on the progression of hepatic fibrosis-carcinoma in mice induced by diethylnitrosamin（DEN）.3.To investigate the mechanism of Sal B involved in the shift beteween pSmad3C/p21-mediated tumor suppressive signaling and pSmad3L/PAI-/c-Myc-mediated pro-fibrogenic/oncogenic signaling.4.To observe the effect of Sal B on the migration of HSC-T₆ cells induced by TGF-beta 1（TGF-β₁）,and to investigate how Sal B regulates the shift of pSmad3C/p21 and pSmad3L/PAI-1/c-Myc signaling in HSC-T₆ cells.Methods1.To establish an animal model and observe the effect of Sal B on the progression of liver fibrosis-hepatocellular carcinomaMale Kunming mice were randomly divided into 5 groups:normal control group,model group,different doses of Sal B（15,30 mg·L-1）group,and positive drug Col（0.2 mg·L-1）group,20 mice in each group.In addition to the normal control group,the rest groups received intraperitoneal injection of 1%DEN solution（16 weeks,one time in a week）according to body weight of 10 mL·L-1 to induce liver fibrosis-hepatocellular carcinoma.The control group was given the corresponding solvent.The mice in the two Sal B groups were intragastrically treated by Sal B with two dosages(15,30 mg·kg^-1),respectively,for 16 weeks.And the mice in the positive control drug treatment group were administered colchicine at the dose of 0.2 mg·kg^-1.The normal control group was given corresponding solvent to gavage.The mice were sacrificed at 12^th week or 16^th week after the start of DEN administration.Pathological changes of livers in each group were assessed by liver biopsy,hematoxylin-eosin（HE）staining and Van Gieson（VG）staining.2.To assess the effect of Sal B on the migrating ability of HSC-T₆ cells by cell scratch assayExperimental group:control group,TGF-beta 1（60 pmol·L-1）stimulation group,salvianolic acid B（25,50,100 μmol· L-1）+TGF-beta 1（60 pmol·L-1）group.HSC-T₆ cells at the log phase of growth with the cell density adjusted to 5.O×106·ml-1 were incubated in a 6-well plate.After HSC-T₆ cells covered the growth plate surface,the cells were incubated in serum-free medium to be starved for 2 hours.TGF-beta 1 stimulation group was treated with 60 pmol·L-1 TGF-beta 1 and Sal B intervention group were treated with Sal B（25,50,100 μmol· L-1）and 60 pmol·L-1 TGF-beta 1 after scratching.At the same time,the control volume was treated with the solvent.Pictures were taken at 0,12,24 h by inverted optical microscope after wounded.3.Western blot method was used to detect the expression of pSmad3C,pSmad3L,p21,PAI-1 and c-Myc protein in hepatic tissue and HSC-T₆ cells.Total protein from liver tissue cells or HSC-T₆ was extrated in routine methods.pSmad3C,pSmad3L,p21,PAI-1 and c-Myc protein were measured by western blot using corresponding antibody.The GAPDH was monitored as the reference.Results1.Sal B improved the general status of DEN induced liver fibrosis-hepatocarcinoma mice.After DEN induced liver fibrosis-hepatocarcinoma 12 weeks,mice in the model group show withered and dull fur,loss of appetite,decreased activity performance,and gradually lose weight.Compared with the model group,the symptoms of mice in the Sal B group were improved,the appetite was increased,and the mental condition was better.2.Effect of Sal B on DEN induced liver fibrosis in mice2.1 There was no significant effect of Sal B on body mass,liver weight and liver index in mice.At the 12^th week,compared to the normal group,the body mass,liver weight and liver index of the mice in the model group decreased.Compared with the model group,the body mass and liver weight of the mice in the Sal B group increased,but there was no significant difference.And there was no significant change in liver index.2.2 Sal B improved liver fibrosis in miceAt the end of the 12^th week,in the model group,the liver surface was rough,the edge was blunt,the inflammatory cells infiltrated in the liver tissue,the collagen fibers proliferated,and a large number of collagen fibers separated the hepatic lobules and formed the pseudo leaflet structure.Compared with the model group,the liver surface of mice in Sal B treatment group was smooth and soft in texture.The proliferation ofcollagen fibers in the liver tissue was lighter than that of the model group,and there was no false lobule structure.So the degree of pathological changes of mice in the Sal B group was reduced.2.3 Effect of Sal B on the expression of pSmad3C,pSmad3L,p21 and PAI-1 proteins in liver tissues of mice with DEN induced liver fibrosisAt the 12^th week,compared with the normal group,the expression of pSmad3L,PAI-1,p21 in the model group increased significantly,and there was no significant change in the expression of pSmad3C.Sal B treatment markedly increased the pSmad3C,p21 expression and decreased the elevated pSmad3L,PAI-1 expression.3.Effect of Sal B on DEN induced precancerous lesion of hepatocellular carcinoma in mice3.1 Sal B increased body mass,liver weight and liver index in miceAt the 16^th week,the body mass,liver weight and liver index of the mice in the model group were less than that the normal group.Compared with the model group,the body mass,liver weight and liver index of the mice in the Sal B group significantly increased.3.2 Sal B improved the precancerous lesion of liver in miceAt the end of the 16^th week,in the model group,the liver had a large number of diffuse nodules,hard texture and disorder of hepatic lobule.The nucleus volume of the liver cell became larger,the staining became deeper and the nucleus division phase increases.Compared with the model group,the mice in the Sal B group had less liver nodules and softer texture.Liver cell nuclear heteromorphity in the Sal B group is less than that in the model group.3.3 Effect of Sal B on the expression of pSmad3C,pSmad3L,p21,PAI-1 and c-Myc proteins in liver tissues of mice with DEN induced HCC precancerous lesionsAt the 16^th week,compared with the normal group,the expression of pSmad3C and p21 in the liver tissues of the model mice increased,and the levels of pSmad3L,PAI-1 and c-Myc protein were significantly higher.Sal B treatment markedly increased the expression of pSmad3C and decreased the expression of pSmad3L,PAI-1 and c-Myc.But the expression of p21 was almost unchanged.4.Sal B inhibited the migration of HSC-T₆ cells induced by TGF-beta 1In HSC-T₆ cells,compared with the control group,the width of scratched wound in the TGF-beta 1 stimulation group gradually narrowed.Compared with the TGF-beta 1 stimulation group,the wound healing speed was slower and the width of the wound was bigger in the Sal B intervention group.5.The effect of Sal B on the expression of pSmad3C,pSmad3L,PAI-1 and p21 in HSC-T₆ cells induced by TGF-beta 1In HSC-T₆ cells,compared with the TGF-beta 1 stimulation group,the expression of pSmad3C and p21 in the Sal B intervention group increased significantly,while the expression of pSmad3L and PAI-1 markedly decreased.Conclusions1.DEN administration successfully induced fibrosis at 12 weeks and DEN induced liver precancerous lesion at 16 weeks in mice.2.Sal B could delay the progression of hepatic fibrosis-carcinoma in mice induced by DEN.3.Sal B inhibited the migration of HSC-T₆ cells induced by TGF-beta 1 and exerts anti-hepatic fibrosis effect.4.Sal B inhibited the development of hepatic fibrosis-hepatocarcinoma,which may be related to the regulation of the signal conversion of pSmad3C/p21 and pSmad3L/PAI-1/c-Myc.