| Hepatic fibrosis (HF)is a pathologic process that every kind of disease cause liver injure and inflammation. Its essense is excessive deposition of extracellular matrix(ECM) components in liver,which occurs due to an imbalance between the production and degradation of matrix. It is the hallmark of most chronic liver diseases. The United States 3th liver fibrosis conferences in 2006 indicated that HF is reversible. Professor Hans Popper says:"Who can prevent or delay liver fibrosis, who will be able to cure most chronic liver disease."The activation and proliferation of hepatic stellate cell (HSC), which are the major source of ECM, are the crucial event to liver fibrosis. It is advantageous in HF treatment from the cell factor and its signal pathways to study mechanism of drug intervention.Transforming growth factor-beta1-1 (TGF-β1)is one of the most important Hepatic Fibrosis cell factors, can inhibition of hepatocyte proliferation, induce hepatocyte apoptosis, activate HSC,promote formation of ECM.TGF-β1 exerts biological effect mainly through TGF-β/samd signal pathway.At present clinical still lackes highly effective and no obvious poisonous side effect medicine to treat HF.Traditional Chinese Medicine has seen significant advancement against liver fibrosis. After many years clinical practices and experiment research, combining with current disease pattern and disease spectrum changes,professor Li dian-gui advances the zhuodu theory , applies this theory to treat Hepatic Fibrosis patients ,which obtaining the good curative effect.Ganfujian formula based on zhuodu theory can improve patients'clinical findings, inhibiting inflammatory reaction and fibroplasias to promote the reversion of hepatic fibrosis. The experimental study indicated that,it can obviously reduce HF rat blood serum HA,LN,Ⅳ-C,PCⅢ,reduces the liver inflammation and the fibrosis degree.We still used Hepatic Fibrosis rat's model, taking the colchicine as the comparison, to determine if Ganfujian formula could inhibit the expression of TGFβ1,TβRI,TβRII,smad2,smad3 and promote the expression of smad7 and then intercepts TGF-β/Samd signal pathway,.This provides scientific evidence for clinic to apply Ganfujian formula in treating cirrhosis. This experiment was separated into five parts:Part one ZhuoDu theory and Hepatic FibrosisObjective: The aim was to study the effect of The ZhuoDu theory in the position of therapy for Hepatic Fibrosis in order to proposes the new viewpoint and the treatment method for HF.Methods: Reviewed zhuodu's source and course in the Chinese medicine theory,put forward that the main pathogenesis of Hepatic Fibrosis was zhuodu,and made a systematic summarization on the characteristics of Hepatic Fibrosis,symptoms,general therapies and the treatment of combined syndromes.Results: We compiled the history of zhuodu ,discussed the relations of Hepatic Fibrosis and zhuodu, searched the treatment of huozhuo jiedu tiaogan theoretical basis,put forward that Hepatic Fibrosis's main pathogenesis was zhuodu. Clinical curative effect proved that zhuodu theory has a positive position in treatment methods of Hepatic Fibrosis.Conclusion: Zhuodu closely links with Hepatic Fibrosis in etiology and pathogenesis, also is it's main pathogenesis.The therapy of huazhuo jiedu used in clinic can not only relieve the symptoms,but also restore the liver physiology characteristic and reversal liver pathology changes.Part two Effect of Ganfujian formula on liver histopathology in HF ratsObjective: The aim was to observe Ganfujian formula's effect on rats'ordinary circumstances and change of liver histopathology in HF rats.Methods:Sixty male Sprague-Dauley rats were selected in this experiment.The animals were randomly divided into normal group(10 rats)and HF model group(50 rats).Besides the normal group, pig's serum(0.5ml per mouse,twice a week)was established in rats by intraperitoneal injection for eight weeks. The rats of HF model were randomly divided into five groups: the model group,colchicines group,Ganfujian formula low dose group(GFJL), Ganfujian formula of medium dose group(GFJM) and Ganfujian formula of high dose group(GFJH)with 10 rats for each group. After HF models were made successfully,the drugs started be given to the rats according to adult dosage 5,15,20 times,respectively,once daily.The rats were respectively treated with Ganfujian low dose(1.5g crude-drug/ml),Ganfujian middle dose(2.25g crude-drug/ml),Ganfujian high dose(3.0g crude-drug/ml) and colchicine(0.154g/kg·d) intragastrically,the normal group and the model group were given physiological saline by gavage.Each group was given the medicine for 12 weeks. Observed rats'ordinary circumstances.All animals were killed ,the specimens were taken and the degree of hepatic fibrosis was judged by routine haematoxylin-eosin staining and Masson staining. Fibrosis score was calculated by semi-quantitative analysis of liver histopathology.Results:1 Rat ordinary circumstances observationThe rat ordinary circumstances observation demonstrated that,The strong 1iver fries by the stasis may obviously improve the HF rat's state of mind,the diet, the superficial knowledge luster,increases its body weight.2 Gross findingsLivers of control group were tender and smooth.The ones of model group were hard,coarse and tarnish,with some observable small nodules, whereas the ones of drug groups were smooth and tender.3 Effect of Ganfujian formula to pathomorphology in hepatic fibrosis ratsThe structure of hepatic lobule were integrated hepatic cord radially ranged as the center of navel vein. There was not degeneration and necrosis in liver tissue.There was not collagen proliferation.Compared with normal group,the model control rats had obvious liver inflammation including degeneration,necrosis,fibrosis and collagen proliferation.Pseudolobule proliferation could be seen in individual animal. Compared with model group,in various drug groups,the destructions were lightened,proliferatio ns of collagen fibers were also lightened,fiber cords were loosened and narrowed.Swelling of liver cells and degeneration alleviated,infiltrating cells decreased.These improvements were more significant in high and medium-dose GFJ groups.4 Semi-quantitative analysis of liver histopathology in HF ratsAccording to HF semi-quantitative analysis standard,fibrosis score was evaluated on the liver tissue sections performing with HE and Reticulation's stain.The fibrosis score of normal group was 0,model group was 15.76±0.33, colchicines group was 5.46±0.20, GFJL was 11.54±0.36, GFJM was 9.6±0.16, GFJH was 5.2±0.12. Compared with model group, the fibrosis score all decreased in colchicines group, GFJL, GFJM and GFJH(p<0.05).Conclusion:Ganfujian formula can improve the HF rat's ordinary circumstances and obviously reduce the rat liver organization inflammation activity and the fibrosis degree,presents the good local constable liver,the anti-HF function.Part three Effect of Ganfujian formula on TGF-β1 in HF ratsObjective: The aim was to explore Ganfujian formula's effect on expression of TGF-β1 in HF rats.Methods: TGFβ1 protein and mRNA expression were determined by using immunohistochemical technique and RT-PCR.Results:1 Experimental study on Ganfujian Formula effect on TGF-β1 protein of the hepatic fibrosis ratsLess positive expression of TGF-β1 cell can be seen in the liver tissue of rats form normal group.The expression of TGF-β1 was strongly positive in model group. The cell proportion of TGF-β1 cell expression in the liver tissue of rats form model group were significantly higer than that of the normal group,which were manifested by the unsymmetric brown yellow particulates.The protein level of TGF-β1 of hepatic fibrosis rats could be decreased from Ganfujian group and colchicines group significantly.The effect of GFJH group was better than that others group. 2 Experimental study on Ganfujian formula effect on TGF-β1 mRNA of the hepatic fibrosis ratsCompared with normal group, the level of TGF-β1 mRNA all increased in model group ,Ganfujian group and colchicines group(p<0.05).But the level of TGF-β1 mRNA decreased in Ganfujian group and colchicines group as compared with that in model group(p<0.05),in which,the reduction in medium dose group and high dose group were more than colchicines group(p<0.05).Conclusion: Ganfujian formula possibly reduce or prevent hepatic fibrosis through decreasing the expression of TGF-β1 protein and mRNA. Part four Effect of Ganfujian formula on TβRI,TβRII in HF ratsObjective: The aim was to explore Ganfujian formula's effect on expression of TβRI,TβRII in HF rats.Methods: TβRI,TβRII protein and mRNA expression were determined by using immunohistochemical technique and RT-PCR, respectively.Results:1 Expression of TβRI,TβRII protein in liver tissueTβRI,TβRII protein expressed in hepatocellular and interstitial cells. The expression of TβRI,TβRII protein in model group was stronger than which in normal group(P<0.05), TβRI,TβRII protein especially expressed around fibre hyperplasia and inflammatory cells.Compared with normal group,the expression of TβRI,TβRII protein in other groups was stronger(P<0.05).Compared with model group,the expression of TβRI,TβRII protein in Ganfujian groups and colchicines group was significantly lighter(P<0.05).The expression of TβRI,TβRII protein in GFJH group and GFJM group were lighter than that of colchicines group, there were significantly difference among those groups(P<0.05).2 Expression of TβRI,TβRII mRNA in liver tissueCompared with normal group,the expression of TβRI,TβRII mRNA in other groups was stronger(P<0.05).Compared with model group,the expression of TβRI,TβRII mRNA in Ganfujian groups and colchicines group was significantly lighter(P<0.05).The expression of TβRI,TβRII mRNA in GFJH group and GFJM group were lighter than that of colchicines group, there were significantly difference among those groups(P<0.05).Conclusion: Ganfujian formula can significantly inhibits the expression of TGFβ1 receptors'protein and mRNA.Part five Effect of Ganfujian formula on Smads in HF ratsObjective: The aim was to explore Ganfujian formula's effect on expression of Smads'protein in HF rats.Combined with part two,three and four, Our study is to determine if Ganfujian formula intercepts TGF-β/Samd signal pathway.Methods: Western blotting was used for detecting the protein expression level of Smad2/3,Smad4,Smad7, respectively.Results:1 Expression of Smad2/3, Smad4 protein in liver tissueCompared with normal group,the expression of Smad2/3, Smad4 in other groups was stronger(P<0.05).Compared with model group,the expression of Smad2/3, Smad4 in Ganfujian groups and colchicines group was significantly lighter(P<0.05).Compared with colchicines group, the expression of Smad2/3, Smad4 in GFJM group and GFJH group was significantly lighter(P<0.05),and there was no difference between colchicines group and GFJL group(P>0.05). The expression of Smad2/3, Smad4 in GFJH group was lighter than that of GFJM group,but there was no difference between two groups(P>0.05).2 Expression of Smad7 protein in liver tissueCompared with normal group,the expression of Smad7 in other groups was lower(P<0.05).Compared with model group,the expression of Smad7 in Ganfujian groups and colchicines group was significantly stronger(P<0.05).Compared with colchicines group, the expression of Smad7 in GFJM group and GFJH group was significantly stronger(P<0.05),and there was no difference between colchicines group and GFJL group(P>0.05). The expression of Smad7 in GFJH group was stronger than that of GFJM group,but there was no difference between two groups(P>0.05).Conclusion: Ganfujian could inhibit the expression of smad2,smad3 and promote the expression of smad7 which could intercepts TGF-β/Samd signal pathway, then delay the process of HF and become an effective therapy for HF.
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