Fuzheng Huayu Recipe Ameliorates Nonalcoholic Steatohepatic Fibrosis By Regulating Angiogenic Growth Related Genes In Mice

Objective: Non-alcoholic fatty liver disease(NAFLD) is a clinical and pathological syndrome, that is characterized by diffuse hepatocellular fatty degeneration, but the patients suffer from it without excessive alcohol intake and other specific damage of liver. NAFLD includes a spectrum of non-alcoholic simple steatosis, non-alcoholic steatohepatitis, fibrosing steatohepatitis, cirrhosis and hepatocellular carcinoma(HCC). non-alcoholic fibrosing steatohepatitis is the crucial stage of the disease, But its pathogenesis is not yet clear and definitive therapy for these patients are lacking. Fuzheng Huayu recipe(FZHY), a traditional Chinese medicine compound, is based on the theory that blood stasis and deficiency of healthy energy are the basic pathological changes of hepatic fibrosis. It consists of Radix Salvia Miltiorrhizae, Cordyceps, Semen Persicae, Fructus Schisandrae Chinensis, Gynostemma Pentaphyllammak and Pollen Pini. We have demonstrated that FZHY improved nutritional fibrosing steatohepatitis by suppressing oxidative stress, regulating inflammation and fibrosis related genes. Recent studies indicated that hepatic stellate cell(HSC) activation is associated with hepatic microcirculation and the angiogenesis, But the effect of angiogenic growth related genes in the development of non-alcoholic fibrosing steatohepatitis is still unclear. In this study, The nonalcoholic fibrotic steatohepatitis was induced by feeding mice methionine-choline deficient(MCD) diet for 8 weeks. Meanwhile, the mice were administrated with FZHY or/and heme oxygenase-1(HO-1) chemical inducer(hemin). The aim of this study was to explore the potential roles of α-smooth muscle actin(α-SMA) hypoxia- inducible factor-1α(HIF-1α) its downstream molecules vascular endothelial growth factor(VEGF), inducible NO synthase(i NOs) and macrophage inflammatory protein-1α(MIP-1α) and effects of FZHY or/and hemin on these genes regulation in nutritional fibrosing steatohepatitis induced by MCD diet.Methods: C57BL/6J mice were fed with methionine-choline deficient(MCD) diet for 8 weeks to induce nonalcoholic fibrotic steatohepatitis. Meanwhile, the mice were administrated with FZHY, hemin or the combination of FZHY and hemin, respectively. Control animals were fed with choline-methionine supplemented diet(Control group). Experimental animal mental status, hair and body weight were observed. Hepatic steatosis, inflammation and fibrosis of mice were observed with hematoxylin & eosin(H&E) and Masson trichrome staining. The m RNA and protein expression of α-SMA, HIF-1α and its downstream molecules VEGF, i NOs and MIP-1α was detected by quantitative real-time PCR, western blot and immune- ohistochemistry, respectively. Quantity one analysis system software was used for semiquantitative analysis of the Western blots results, appling the integral optical density ratio of HIF-1α/β-actin、VEGF/β-actin、i NOs/β-actin. All data was presented as mean±SD. Statistical analysis was performed by one-way analysis of variance(ANOVA) and Student-Newman-Keuls test for evaluating differences between groups using SPSS 16.0. A P-value of less than 0.05 was considered statistically significance. We analyzed the relationship between the expressions of genes and histopathological changes of liver and clarified the relationship between different genes expressions and progress of liver fibrosis.Results:1 The general situation of Mice: Control mice looked spirited, swift with their weight increased gradually, while the mice fed with MCD diet for 8 weeks looked exhausted, less dynamic and lost weight significantly. Mice administered with FZHY, hemin or the combination of hemin and FZHY presented better general condition compared to MCD feeding mice.2 Liver histopathology: In control mice, the liver sections was almost normal. Mice fed with MCD diet for 8 weeks exhibited hepatocyte macrosteatosis, spot or focal hepatocyte necrosis and inflammatory infiltration, perisinusoidal and portal fibrosis. The degree of hepatic steatosis, inflammation and fibrosis in mice treated with FZHY and/or hemin was alleviated, especially in the mice treated with FZHY combinated hemin.3 The hepatic m RNA expression of α-SMA, HIF-1α, VEGF, i NOs �'�MIP-1α: Compared to the control mice, the m RNA expression of α-SMA, HIF-1α, VEGF, i NOs and MIP-1α were up-regulated in mice fed with MCD diet(8.33±2.32 vs 1.01±0.19, 4.50±0.78 vs 1.03±0.28, 7.40±1.54 vs 0.92±0.20,9.94±1.60 vs 0.93±0.20,7.65±1.87 vs 1.01±0.11, respectively, P<0.001);whereas the levels of α-SMA, HIF-1α, VEGF, i NOs and MIP-1α in mice treated with FZHY were descended compared with MCD group(3.83±0.53,3.02±0.55,3.23±0.94,3.60±0.96,3.70±1.14,P<0.01); the levels of these genes in mice administrated with Hemin were also descended(3.94±0.83,2.29±0.78,3.17±1.06,2.81±0.41,4.16±1.31,P<0.00);The combination of FZHY and hemin led to a further down-regulations of the levels of these genes(2.33±0.76, 1.61±0.20, 1.58±0.23, 2.09±0.62,2.07±0.10).4 The hepatic protein expression of α-SMA, HIF-1α, VEGF, i NOs and MIP-1α: Compared to the control mice, the protein expression of α-SMA, HIF-1α, VEGF, i NOs and MIP-1α were up-regulated in mice fed with MCD diet [(1.08±0.20)% vs(0.29±0.07)%, 0.55±0.08 vs 0.24±0.07, 1.08±0.034 vs 0.36±0.05, 3.30±0.88 vs 1.08±0.09,(0.71±0.09)% vs(0.11±0.03)%, respe- ctively, P<0.001]; whereas the protein levels of α-SMA, HIF-1α, VEGF, i NOs and MIP-1α in mice treated with FZHY were descended compared with MCD group[(0.53±0.04)%, 0.44±0.02, 0.54±0.07, 1.61±0.21,(0.41±0.07)%, P< 0.05]; the protein expression of these genes in mice administrated with Hem- in were also descended [(0.49±0.11)%, 0.36±0.11, 0.46±0.05, 2.31±0.39,(0.43±0.11)%, P<0.01); The combination of FZHY and hemin led to a further down-regulations of the levels of these genes[(0.29±0.07)%,0.28± 0.02, 0.31±0.06, 1.26±0.38,(0.25±0.07)%)].Conclusions:1 α-SMA, HIF-1α, VEGF, i NOs and MIP-1α played a crucial role in the process of nonalcoholic fibrotic steatohepatitis induced by feeding mice MCD diet for 8 weeks.2 FZHY could improve fibrotic steatohepatitis induced by MCD diet in mice, which is associated with the suppression of HSCs activation and hepatic angiogenic growth factors.3 FZHY combined with hemin played a synergistic effect on amel- iorating nonalcoholic fibrotic steatohepatitis by suppressing the activation of HSCs and hepatic angiogenic growth related genes.