Mechanisms Of Compound Astragalus And Salvia Miltiorrhiza Extract’s Anti-Hepatic Fibrosis-carcinoma Via TGF-β/TβR/Smad2/3

Background Hepatic fibrosis caused by chronic liver inflammation progressively leads to hepatic carcinoma ultimately, which is one of the most intractable diseases of mankind. It is a novel strategy for inhibiting or blocking the progress of liver disease that further investigate the pathogenesis of liver disease, especially clarify the mechanism on cell and molecular level from fibrogenesis to hepatocarcinogenesis, and then intervening these specific cells and molecules. Hepatic stellate cell(HSC) is regarded as the direct target cell of various pro-fibrogenic factors. The activated HSCs excessively producing collagen protein, the most ingredient of extracellular matric(ECM), and then trans-differentiating into myofibroblasts, which leads to reconstruction of liver, is recognized as a central event in liver fibrogenesis. Transforming growth factor beta-1(TGF-β1) has been verified as the strongest profibrotic cytokine in liver fibrogenesis, and aberrant activation of intracellular signaling molecules Smads is closely related with hepatic fibrogenesis and hepatocarcinogenesis. Previous reports from our research team have shown that compound Astragalus and Salvia miltiorrhiza extract(CASE), a Chinese herbal formula, has a protective effect against acute and chronic injuries induced by carbon tetrachloride(CCl4). Moreover, a recent study shows that CASE exhibits anti-cancer effects in rats with hepatocellular carcinoma(HCC) induced by DEN. The present study was performed to dynamically observe the effect of CASE on the progress of hepatic fibrosis-carcinoma in rats induced by diethylinitrosamine(DEN) and investigate the possible mechanism via in vivo(DEN-induced HCC in rats) and in vitro(TGF-β1-stimulated HSCs and HepG2 cells). Aims 1. To assess the effect of CASE on the development of hepatic fibrosis-carcinoma in rats induced by DEN via observing pathologic change(hematoxylin-eosin staining) based on previous studies which had illustrated improved serological indictors of liver function and decreased incidence and multiplicity of hepatoma by CASE. 2. To observe the effect of CASE on TGF-β1, TβRI, TβRII and phosphorylated Smad2/3 at both C-terminal region and linker region protein expression in the development of hepatic fibrosis-carcinoma induced by DEN, analyze the relation between hepatic fibrogenesis-carcinogenesis and CASE intervene effect and TGF-β/TβR/Smad2/3 signaling transduction. 3. To investigate the effect of CASE on TβRI and TβRII protein expression in HSCs and HepG2 cells stimulated by TGF-β1, analyze the possible target of CASE. Methods 1. To investigate the effect and molecular mechanism of CASE on the development of hepatic fibrosis-carcinoma in rats induced by DEN(in vivo) Male Sprague-Dawley(SD) rats were randomly divided into six groups: the normal control group, the DEN alone group(10 mg/kg), three CASE treatment groups(60, 120, 240 mg/kg) and the positive drug(Liver-aid tablet) group(921 mg/kg). Rats in the DEN group, CASE groups and Liver-aid tablet were treated with 0.2% DEN dissolved in 0.5% CMC-Na by gavage 5 times a week for 14 weeks to induce hepatocarcinogenesis, and concomitantly administered corresponding drugs(CASE or Liver-aid tablet) per day respectively for 16 weeks. Rats in the control group were administered with 0.5% CMC-Na for 16 weeks. Rats were sacrificed at 12 th week or 16 th week after the start of DEN administration. One lobe from each rat liver was harvested and fixed in 10% formalin, embedded in paraffin and manufactured tissue microarray. Others were preserved in liquid nitrogen for protein detection. Pathological changes of livers in each group were assessed by hematoxylin and eosin(H&E) staining; the protein level and distribution of TGF-β1, TβRI, pSmad2C/L and pSmad3C/L in livers was detected by immunohistochemical methods. The protein expression of TGF-β1, TβRI and TβRII was measured by Western blot, and GAPDH regarded as a internal control; the protein level of phosphorylated(p)-Smad3C/L was measured by immunoprecipitation and immunoblot analysis, and Smad3 regarded as a internal control. 2. To investigate the effect of CASE on TβRI and TβRII protein expression in HSCs and HepG2 cells stimulated by exogenous TGF-β1(in vitro) HSCs and HepG2 cells at the log phase of growth were used for the experiment, which includes five groups: the control group, TGF-β1 group(40 pmol/L) and CASE groups(20, 40 and 80 μg/ml group). The cells were starved in the absence of presence of the indicated doge of CASE for 24 h, and treated with 40 pmol/L TGF-β1 for 1 h except for the equal volume serum-free medium in the control group. The level of TβRI and TβRII protein expression was measured by western blot. Results 1 CASE delayed the progression of hepatic fibrosis-carcinoma in rats induced by DEN in pathological changes and mediated TGF-β/TβR/Smad2/3 signaling transduction 1.1 CASE withstood pathological changes in rats with hepatic fibrosis-carcinoma induced by DEN In the normal control group, the surface of rat’s liver was smooth and sheeny with soft texture and sharp edge; the structure of the hepatic lobule was intact. In the DEN alone group, at 12 th week, the surface of rat’s liver was rough with granular hard-plaques and blunt edge; the hepatic lobule was encysted and/or separated by collagen bundles, and pseudolobules emerged. At 16 th week, the surface of rat’s liver in DEN group was rough with some nodules preliminarily identified as cancerous nodes; pathological examination showed that the hepatocytes of nodules with obvious atypia and hyperchromatic nuclei were verified as hepatocellular carcinoma. However, these pathological changes evidently improved in CASE treatment groups compared with the DEN group, which proved by reductive cirrhotic nodules and alleviative fibrosis at 12 th week, and decreasing cancerous nodes and ameliorative differentiation via CASE treatment. 1.2 CASE decreased the protein level of TGF-β1 in rats with hepatic fibrosis-carcinoma induced by DEN The protein expression of TGF-β1 persistently increased in the development of hepatic fibrosis-carcinoma induced by DEN. The high-level of TGF-β1 protein were observed both in hepatoma nodule areas and in adjacent relatively normal liver tissues in DEN-treated rats by using immunohistochemical methods. CASE treatment decreased the level of TGF-β1 elevated by DEN in a dose-dependent manner. 1.3 CASE down-regulated the protein expression of TβRI in rats with hepatic fibrosis-carcinoma induced by DEN At 12 th week, the protein expression of TβRI was up-regulated in the DEN alone group, while down-regulated by CASE treatment in a dose-dependent manner. At 16 th week, the protein expression of TβRΙ dramatically increased only in relatively normal liver tissues adjacent to hepatoma tissues, while CASE treatment markedly decreased the level of TβRΙ elevated by DEN. 1.4 CASE down-regulated the protein expression of TβRII in rats with hepatic fibrosis-carcinoma induced by DEN The protein expression of TβRII continuously up-regulated in the development of hepatic fibrosis-carcinoma induced by DEN. CASE treatment decreased the level of TβRII elevated by DEN in a dose-dependent manner. 1.5 CASE inhibited the phosphorylation of Smad2 at C-terminal and linker region(pSmad2C/L) in rats with hepatic fibrosis-carcinoma induced by DEN The protein level of pSmad2C/L observably increased in the development of hepatic fibrosis-carcinoma induced by DEN, particularly in the precancerous tissue, while all three doses of CASE significantly inhibited the phosphorylation of Smad2 at C-terminal and linker region. 1.6 CASE modulated the phosphorylation of Smad3 at C-terminal and linker region(pSmad3C/L) in rats with hepatic fibrosis-carcinoma induced by DEN The protein level of pSmad3 L markedly increased in the development of hepatic fibrosis-carcinoma induced by DEN, while pSmad3 C slightly changed. CASE treatment remarkably decreased the protein level of p Smad3 L, but increased pSmad3 C in a dose-development manner compared with the DEN alone group. 2 CASE down-regulated the protein expression of TβRI/TβRII in HSC/HepG2 cells stimulated by TGF-β1 Under TGF-β1 stimulation, the protein expression of TβRI and TβRII significantly increased in HSCs as well as HepG2, while pre-treatment by CASE inhibited or reversed up-regulated expression of TβRI and TβRII induced by TGF-β1 in the two cells. Conclusions 1. CASE observably delayed pathological process of hepatic fibrosis-carcinoma in rats induced by DEN. 2. CASE’s anti-hepatic fibrosis-carcinoma effects were involving in mediating the production of cytokine TGF-β1. 3. CASE could directly target TβRI and TβRII to retard the progression of hepatic fibrosis-carcinoma. 4. CASE decreased the protein level of pSmad2C/L, pSmad3 L and increased pSmad3 C in the development of hepatic fibrosis-carcinoma, which suggested that CASE’s anti-hepatic fibrosis-carcinoma effects were associated with modulating the phosphorylation of Smad2/3 at C-terminal and linker region.