| Objective:The objective of this study was to establish a rat model of immunological hepatic fibrosis, and comparing with control group of Daihuang Zhechong Pill, observe the effect of Supplemental Guizhi Fuling Pill on TGF-β1 and CTGF in liver organition of immune hepatic fibrosis rats, and analyze the effect of Supplemental Guizhi Fuling Pill on the prevention and cure of hepatic fibrosis, thus provide the scientific experimental basis for the clinical application and promotion of the drug.Methods:Sixty SPF Wistar male rats,weighted 180-200 g,were randomly divide into six groups including negative control group, model group, Daihuang hechong Pill control group, higher-, medium- and lower-dose of Supplemental Guizhi Fuling Pill treatment groups.Every group were 10 rats.In model group,porcine serum 0.5ml was injected into abdominal cavity,twice per week,incessantly for ten weeks.The rest five groups,except for normal control group which was through intraperitoneal injection of normal saline,were given the same as model group and gastric perfusion with Daihuang Zhechong Pill,lower-dose of Supplemental Guizhi Fuling Pill,medium-dose of Supplemental Guizhi Fuling Pill,higher--dose of Supplemental Guizhi Fuling Pill, espectively for once per day.For model and normal control groups, they were administered with distilled water 12.6ml/Kg by gavage.Adjustment of the amount of dose was based on weight of rats each week.At the end of 10 weeks,stop modeling. Weighing rats after fasting 24 hours,then fixing in supine position after nesthetization of 2% pentobarbital.Opening abdominal cavity to weigh liver and spleen,btaining 1.5cm*1cm*0.5cm size or so liver tissue in the same position in the right lobe of liver fixed by 10%formaldehyde solution,dehydrated by automatic dehydrator,and embedded sections by paraffin. By means of HE staining, observe liver pathomorphology change of rats in each group. The fibrotic staging standard was according to the pathological grading and staging standard of chronic hepatitis in the Consensus on evaluation of the diagnosis and efficacy of hepatic fibrosis passed by the Xi’an national conference of liver disease in 2000(Attached Table 1). Adopt immune histochemistry S-P method to observe the TGF-β1 and PDGF in rat liver tissue of each group.Results:1.Supplemental Guizhi Fuling Pill can significantly reduce the positive expression of TGF-β1 in rat liver tissue. The expression of TGF-β1 of lower-dose of Supplemental Guizhi Fuling Pill treatment group can be seen Inflammatory cells infiltration, liver cell edema, inflammatory cells and stromal cells in the cytoplasm and portal area, compared with model group. The expression of TGF-β1 of medium-dose of Supplemental Guizhi Fuling Pill treatment group Visible mesenchymal cells in the cytoplasm, a small amount of inflammatory cells, compared with model group significantly reduce. The expression of TGF-β1 of higher--dose of Supplemental Guizhi Fuling Pill treatment group at Expressed in liver tissue, close to normal liver tissue level, positive expression mainly portal area, inflammatory cells in the cytoplasm. Obviously lighter than the rest of the treatment group.There was significant statistical significance between Daihuang Zhechong Pill control group and Supplemental Guizhi Fuling Pill treatment groups(P<0.01). The positive expression of TGF-β1 of Daihuang Zhechong Pill control group was higher than medium-dose of Supplemental Guizhi Fuling Pill treatment group and higher-dose group(P<0.01). There was no statistical significance between Daihuang Zhechong Pill control group and lower-dose of Supplemental Guizhi Fuling Pill treatment group(P>0.05). The positive expression of TGF-β1 of higher-dose of Supplemental Guizhi Fuling Pill treatment group was lower than medium-dose and lower-dose groups,and there was a significant statistical significance between them(P<0.05).2.Supplemental Guizhi Fuling Pill can significantly reduce the positive expression of CTGF in rat liver tissue. The expression of CTGF of lower-dose of Supplemental Guizhi Fuling Pill treatment group can be seen Hepatic lobule structure damage, liver cell degeneration, portal area, the fiber spacing and the surrounding liver sinus wall, compared with model group.The expression of CTGF of medium-dose group was obviously lower than model group, can be seen Hepatic lobule structure damage loss, fiber interval, expressed portal area.There was a small amount of expression in liver cells of higher--dose of Supplemental Guizhi Fuling Pill treatment group can be seen Lobular structures damaged markedly improved, visible tiny collect abbacy and fiber interval expression,lighter than the other treatment groups. There was significant statistical significance between Daihuang Zhechong Pill control group and Supplemental Guizhi Fuling Pill treatment groups(P<0.01). The positive expression of CTGF of Daihuang Zhechong Pill control group was higher than medium-dose of Supplemental Guizhi Fuling Pill treatment group and higher-dose group(P<0.01). There was no statistical significance between Daihuang Zhechong Pill control group and lower-dose of Supplemental Guizhi Fuling Pill treatment group(P>0.05). The positive expression of CTGF of higher-dose of Supplemental Guizhi Fuling Pill treatment group was lower than medium-dose and lower-dose groups,and there was a significant statistical significance between them(P<0.05).Conclusion:Supplemental Guizhi Fuling Pill can significantly improve the pathomorphology of liver organization of immune hepatic fibrosis rats and reduce the TGF-β1 and CTGF in rat liver tissue, and there was a dose-effect relationship in this phenomenon. During the experiment, the rats in Supplemental Guizhi Fuling Pill groups has no obvious side effects and adverse reactions, suggested that it was safe and reliable. The experiment also showed that TGF- β1, CTGF has close relationship with liver fibrosis, and there was a positive correlation between them. It might be one of pathological mechanisms of hepatic fibrosis progression. |
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