| Backgrounds and Aim:Previous experiments have concluded that the mutation of Smad3 gene C-terminal phosphorylation site promotes signal conversion between p Smad3C/p Smad3L and promotes hepatic fibrosis-carcinoma process.In addition,Sal B has been shown to delay the hepatic fibrosis-carcinoma process.The purpose of this project is to study the effect of Salvianolic acid B?Sal B?on the against Diethylinitrosamine?DEN?induced hepatic fibrosis-carcinoma in mice when the Smad3C phosphorylation site is mutated,so as to further clarify the mechanism of Sal B against hepatic fibrosis-carcinoma process.Method:Using C57BL/6J mice as the genetic background,successfully generated the Smad3 C-terminal phosphorylation site mutant mice(heterozygous,p Smad3C+/-),and then the mice were reared,bred,and identified in the SPF environment.When the mice were 6 weeks old,selecting male healthy p Smad3C+/-mice and Wild-type(p Smad3C+/+)mice for experiments.Experimental grouping:p Smad3C+/-+control;p Smad3C+/-+DEN;p Smad3C+/-+Sal B;p Smad3C+/++control;p Smad3C+/++DEN;p Smad3C+/++Sal B,20/group.Except for the control group,the other groups of mice induced hepatic fibrosis and hepatic carcinoma with DEN.Sal B group was administered with the corresponding dose of Sal B?15 mg/kg/d?at the same time from the day of modeling.Serum samples were collected before sacrifice and used for transaminase kit assays.After sacrifice,the liver was isolated,and a portion was fixed in 4%paraformaldehyde.Samples were embedded in paraffin,sections were cut at 4-5?m,and either stained with Hematoxylin&eosin?HE?or Van Gieson?VG?used for immunofluorescence.The remaining liver tissue was stored at-80°C for western-blot analysis.Western-blot to detect liver fibrosis lesion markers Alpha smooth muscle actin??-SMA?,precancerous markers Glutathione S-transferase placenta?GST-P?,p Smad3C,p21,p Smad3L,Plasminogen activator inhibitor-1?PAI-1?,c-Myc protein levels to explore the mechanism of Sal B anti-DEN-induced hepatic fibrosis and hepatic carcinoma.Results1. Smad3 gene C-terminal phosphorylation site mutation could decrease the action of Sal B against hepatic fibrosis.After 18 weeks of DEN modeling,the control groups of p Smad3C+/+and p Smad3C+/-mice were normal in liver index,serum transaminase,liver appearance,pathology,and there was no significant difference between the p Smad3C+/+and p Smad3C+/-mice.In the DEN group of p Smad3C+/+and p Smad3C+/-,the body weight and liver weight of the mice were reduced;the liver surface was granular,the appearance was dark yellow,and the texture was hard;the serum transaminase?Alanine transaminase,ALT;Aspartate aminotransferase,AST?was significantly increased;the HE staining and VG staining showed that Inflammatory cells aggregated and collagen fibers increased;western-blot results showed that?-SMA and GST-P protein expressions were significantly increased;and the degree of lesions in p Smad3C+/-mice was higher than that of p Smad3C+/+mice.Sal B group in p Smad3C+/+and p Smad3C+/-mice was lighter than DEN group,and p Smad3C+/+mice had lighter symptoms than p Smad3C+/-mice.2. Smad3 gene C-terminal phosphorylation site mutation could inhibit upregulation of p Smad3C and p21 proteins during Sal B against hepatic fibrosis.After 18 weeks of DEN modeling,the expression of p Smad3C and p21 protein were significantly down-regulation in the liver tissue,and the protein levels were higher in p Smad3C+/+mice than in p Smad3C+/-mice.In the Sal B group,the expression of p Smad3C and p21 protein were significantly upregulation compared with their DEN group;and the increase in protein level was higher in p Smad3C+/+mice than in p Smad3C+/-mice.3. Smad3 gene C-terminal phosphorylation site mutation could inhibit down-regulation of p Smad3L,PAI-1,c-Myc proteins during Sal B against hepatic fibrosis.After 18 weeks of DEN modeling,the expression of p Smad3L,PAI-1,c-Myc protein were significantly upregulation in the liver tissue,and the protein levels were higher in p Smad3C+/-mice than in p Smad3C+/+mice.In the Sal B group,the expression of p Smad3L,PAI-1,c-Myc protein were significantly down-regulation compared with their DEN group;and the reduction in protein level was higher in p Smad3C+/+mice than in p Smad3C+/-mice.4. Smad3 gene C-terminal phosphorylation site mutation could decrease the action of Sal B against hepatic carcinoma.After 23 weeks of DEN modeling,the control groups of p Smad3C+/+and p Smad3C+/-mice were normal in liver index,serum transaminase,liver appearance,pathology,and there was no significant difference between the p Smad3C+/+and p Smad3C+/-mice.In the DEN group of p Smad3C+/+and p Smad3C+/-,the body weight and liver weight decreased,and the liver index increased compared with the control group;the liver surface was rough,the liver appearance was dark yellow,the liver texture was hard,with the white granular nodules,and heterozygous mice liver also accompanied by hemorrhagic necrotic tumor lesions;serum transaminase is significantly increased;HE staining shows localized cell necrosis of the liver tissue with obvious nuclear aberrations;western-blot show significant increase in GST-P protein expression and the degree of lesions in p Smad3C+/-mice was higher than that of p Smad3C+/+mice.Sal B group in p Smad3C+/+and p Smad3C+/-mice was lighter than DEN group,and p Smad3C+/+mice had lighter symptoms than p Smad3C+/-mice.5. Smad3 gene C-terminal phosphorylation site mutation could inhibit upregulation of p Smad3C and p21 proteins during Sal B against hepatic carcinoma.After 23 weeks of DEN modeling,the expression of p Smad3C and p21 protein were significantly down-regulation in the liver tissue,and the protein levels were higher in p Smad3C+/+mice than in p Smad3C+/-mice.In the Sal B group,the expression of p Smad3C and p21 protein were significantly upregulation compared with their DEN group;and the increase in protein level was higher in p Smad3C+/+mice than in p Smad3C+/-mice.6. Smad3 gene C-terminal phosphorylation site mutation could inhibit down-regulation of p Smad3L,PAI-1,c-Myc proteins during Sal B against hepatic carcinoma.After 23 weeks of DEN modeling,the expression of p Smad3L,c-Myc protein were significantly upregulation in the liver tissue,and the protein levels were higher in p Smad3C+/-mice than in p Smad3C+/+mice;but the expression of PAI-1 protein is low which may be related to necrosis and liquefaction of liver cancer cells.In the Sal B group,the expression of p Smad3L,c-Myc protein were significantly down-regulation compared with their DEN group;the reduction in protein level was higher in p Smad3C+/+mice than in p Smad3C+/-mice;and the expression of PAI-1 protein was increased,which may be caused by Sal B treatment leading to reduced hepatocyte necrosis.Conclusions1.Smad3 gene C-terminal phosphorylation site mutation could decrease the action of Sal B against hepatic fibrosis-carcinoma process.2.Smad3 gene C-terminal phosphorylation site mutation could inhibit upregulation of p Smad3C and p21 proteins during Sal B against hepatic fibrosis-carcinoma process.6. Smad3 gene C-terminal phosphorylation site mutation could inhibit down-regulation of p Smad3L,PAI-1,c-Myc proteins during Sal B against hepatic fibrosis-carcinoma process. |
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